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Sample records for bovine insulin human

  1. Bovine and human insulin adsorption at lipid monolayers: a comparison

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    Mauri, Sergio; Pandey, Ravindra; Rzeznicka, Izabela; Lu, Hao; Bonn, Mischa; Weidner, Tobias

    2015-07-01

    Insulin is a widely used peptide in protein research and it is utilised as a model peptide to understand the mechanics of fibril formation, which is believed to be the cause of diseases such as Alzheimer and Creutzfeld-Jakob syndrome. Insulin has been used as a model system due to its biomedical relevance, small size and relatively simple tertiary structure. The adsorption of insu lin on a variety of surfaces has become the focus of numerous studies lately. These works have helped in elucidating the consequence of surface/protein hydrophilic/hydrophobic interaction in terms of protein refolding and aggregation. Unfortunately, such model surfaces differ significantly from physiological surfaces. Here we spectroscopically investigate the adsorption of insulin at lipid monolayers, to further our understanding of the interaction of insulin with biological surfaces. In particular we study the effect of minor mutations of insulin’s primary amino acid sequence on its interaction with 1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) model lipid layers. We probe the structure of bovine and human insulin at the lipid/water interface using sum frequency generation spectroscopy (SFG). The SFG experiments are complemented with XPS analysis of Langmuir-Schaefer deposited lipid/insulin films. We find that bovine and human insulin, even though very similar in sequence, show a substantially different behavior when interacting with lipid films.

  2. A protein with amino acid sequence homology to bovine insulin is present in the legume Vigna unguiculata (cowpea

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    Venâncio T.M.

    2003-01-01

    Full Text Available Since the discovery of bovine insulin in plants, much effort has been devoted to the characterization of these proteins and elucidation of their functions. We report here the isolation of a protein with similar molecular mass and same amino acid sequence to bovine insulin from developing fruits of cowpea (Vigna unguiculata genotype Epace 10. Insulin was measured by ELISA using an anti-human insulin antibody and was detected both in empty pods and seed coats but not in the embryo. The highest concentrations (about 0.5 ng/µg of protein of the protein were detected in seed coats at 16 and 18 days after pollination, and the values were 1.6 to 4.0 times higher than those found for isolated pods tested on any day. N-terminal amino acid sequencing of insulin was performed on the protein purified by C4-HPLC. The significance of the presence of insulin in these plant tissues is not fully understood but we speculate that it may be involved in the transport of carbohydrate to the fruit.

  3. Specific insulin binding in bovine chromaffin cells; demonstration of preferential binding to adrenalin-storing cells

    International Nuclear Information System (INIS)

    Serck-Hanssen, G.; Soevik, O.

    1987-01-01

    Insulin binding was studied in subpopulations of bovine chromaffin cells enriched in adrenalin-producing cells (A-cells) or noradrenalin-producing cells (NA-cells). Binding of 125 I-insulin was carried out at 15 0 C for 3 hrs in the absence or presence of excess unlabeled hormone. Four fractions of cells were obtained by centrifugation on a stepwise bovine serum albumin gradient. The four fractions were all shown to bind insulin in a specific manner and the highest binding was measured in the cell layers of higher densities, containing mainly A-cells. The difference in binding of insulin to the four subpopulations of chromaffin cells seemed to be related to differences in numbers of receptors as opposed to receptor affinities. The authors conclude that bovine chromaffin cells possess high affinity binding sites for insulin and that these binding sites are mainly confined to A-cells. 24 references, 2 figures, 1 table

  4. Interaction of PAMAM dendrimers with bovine insulin depends on nanoparticle end-groups

    International Nuclear Information System (INIS)

    Nowacka, Olga; Milowska, Katarzyna; Bryszewska, Maria

    2015-01-01

    We have looked at the interactions between polyamidoamine (PAMAM) dendrimers with different terminal groups (−COOH, −NH 2 , −OH) and bovine insulin. The influence of PAMAM dendrimers on insulin was tested by measuring zeta potential and fluorescence quenching. The secondary structure of insulin in the presence of dendrimers was examined by circular dichroism. The effect of dendrimers on dithiotreitol-induced aggregation of insulin was investigated by spectrophotometry. Dendrimers quenched the fluorescence of insulin, but did not change its secondary structure. Thus dendrimers neither induce hormone aggregation nor inhibit the aggregation process induced by dithiotreitol (DTT), except at 0.01 µmol/l. Dendrimers–insulin interactions are mainly electrostatic. - Highlight: • The interactions between PAMAM dendrimers and insulin were investigated. • The PAMAM dendrimers can quench the fluorescence of insulin. • The PAMAM dendrimers did not change the secondary structure of insulin. • Dendrimers did not induce aggregation of hormone. • Dendrimers–insulin interaction is mainly electrostatic

  5. Bovine α-Lactalbumin Hydrolysates (α-LAH Ameliorate Adipose Insulin Resistance and Inflammation in High-Fat Diet-Fed C57BL/6J Mice

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    Jing Gao

    2018-02-01

    Full Text Available Obesity-induced adipose inflammation has been demonstrated to be a key cause of insulin resistance. Peptides derived from bovine α-lactalbumin have been shown to inhibit the activities of dipeptidyl peptidase IV (DPP-IV and angiotensin converting enzyme (ACE, scavenge 2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonate] (ABTS+ radical and stimulate glucagon-like peptide-2 secretion. In the present study, the effects of bovine α-lactalbumin hydrolysates (α-LAH on adipose insulin resistance and inflammation induced by high-fat diet (HFD were investigated. The insulin resistance model was established by feeding C57BL/6J mice with HFD (60% kcal from fat for eight weeks. Then, the mice were fed with HFD and bovine α-LAH of different doses (100 mg/kg b.w., 200 mg/kg b.w. and 400 mg/kg b.w. for another 12 weeks to evaluate its protective effects against HFD-induced insulin resistance. The oral glucose tolerance test (OGTT and intraperitoneal insulin tolerance test (ipITT were conducted after intervention with α-LAH for 10 weeks and 11 weeks, respectively. Results showed that bovine α-LAH significantly reduced body weight, blood glucose, serum insulin, and HOMA-IR (homeostatic model assessment of insulin resistance levels, lowered the area-under-the-curve (AUC during OGTT and ipITT, and downregulated inflammation-related gene [tumor necrosis factor (TNF-α, interleukin (IL-6, monocyte chemoattractant protein (MCP-1] expression in adipose tissues of HFD-fed C57BL/6J mice. Furthermore, bovine α-LAH also suppressed insulin receptor substrate 1 (IRS-1 serine phosphorylation (Ser307, Ser612, enhanced protein kinase B (known as Akt phosphorylation, and inhibited the activation of inhibitor of kappaB kinase (IKK and mitogen activated protein kinase (MAPK signaling pathways in adipose tissues of HFD-fed C57BL/6J mice. These results suggested that bovine α-LAH could ameliorate adipose insulin resistance and inflammation through IKK and MAPK signaling

  6. Berberine Protects against NEFA-Induced Impairment of Mitochondrial Respiratory Chain Function and Insulin Signaling in Bovine Hepatocytes

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    Zhen Shi

    2018-06-01

    Full Text Available Fatty liver is a major lipid metabolic disease in perinatal dairy cows and is characterized by high blood levels of non-esterified fatty acid (NEFA and insulin resistance. Berberine (BBR has been reported to improve insulin sensitivity in mice with hepatic steatosis. Mitochondrial dysfunction is considered a causal factor that induces insulin resistance. This study investigates the underlying mechanism and the beneficial effects of BBR on mitochondrial and insulin signaling in bovine hepatocytes. Revised quantitative insulin sensitivity check index (RQUICKI of cows with fatty liver was significantly lower than that of healthy cows. Importantly, the Akt and GSK3β phosphorylation levels, protein levels of PGC-1α and four of the five representative subunits of oxidative phosphorylation (OXPHOS were significantly decreased in cows with fatty liver using Western Blot analysis. In bovine hepatocytes, 1.2 mmol/L NEFA reduced insulin signaling and mitochondrial respiratory chain function, and 10 and 20 umol/L BBR restored these changes. Furthermore, activation of PGC-1α played the same beneficial effects of BBR on hepatocytes treated with NEFA. BBR treatment improves NEFA-impaired mitochondrial respiratory chain function and insulin signaling by increasing PGC-1α expression in hepatocytes, which provides a potential new strategy for the prevention and treatment of fatty liver in dairy cows.

  7. STAT6-Dependent Collagen Synthesis in Human Fibroblasts Is Induced by Bovine Milk.

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    Stefan Kippenberger

    Full Text Available Since the domestication of the urus, 10.000 years ago, mankind utilizes bovine milk for different purposes. Besides usage as a nutrient also the external application of milk on skin has a long tradition going back to at least the ancient Aegypt with Cleopatra VII as a great exponent. In order to test whether milk has impact on skin physiology, cultures of human skin fibroblasts were exposed to commercial bovine milk. Our data show significant induction of proliferation by milk (max. 2,3-fold, EC50: 2,5% milk without toxic effects. Surprisingly, bovine milk was identified as strong inducer of collagen 1A1 synthesis at both, the protein (4-fold, EC50: 0,09% milk and promoter level. Regarding the underlying molecular pathways, we show functional activation of STAT6 in a p44/42 and p38-dependent manner. More upstream, we identified IGF-1 and insulin as key factors responsible for milk-induced collagen synthesis. These findings show that bovine milk contains bioactive molecules that act on human skin cells. Therefore, it is tempting to test the herein introduced concept in treatment of atrophic skin conditions induced e.g. by UV light or corticosteroids.

  8. Insulin during in vitro oocyte maturation has an impact on development, mitochondria, and cytoskeleton in bovine day 8 blastocysts.

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    Laskowski, Denise; Båge, Renée; Humblot, Patrice; Andersson, Göran; Sirard, Marc-André; Sjunnesson, Ylva

    2017-10-01

    Insulin is a key metabolic hormone that controls energy homeostasis in the body, including playing a specific role in regulating reproductive functions. Conditions associated with hyperinsulinemia can lower developmental rates in bovine in vitro embryo production and are linked to decreased fertility in humans, as in cases of obesity or type 2 diabetes. Embryo quality is important for fertility outcome and it can be assessed by choosing scoring standards for various characteristics, such as developmental stage, quality grade, cell number, mitochondrial pattern or actin cytoskeleton structure. Changes in the embryo's gene expression can reflect environmental impacts during maturation and may explain morphological differences. Together with morphological evaluation, this could enable better assessment and possibly prediction of the developmental potential of the embryo. The aim of this study was to use a bovine model to identify potential gene signatures of insulin-induced changes in the embryo by combining gene expression data and confocal microscopy evaluation. Bovine embryos were derived from oocytes matured in two different insulin concentrations (10 µg mL - 1 and 0.1 µg mL - 1 ), then stained to distinguish f-Actin, DNA and active mitochondria. The total cell number of the embryo, quality of the actin cytoskeleton and mitochondrial distribution were assessed and compared to an insulin-free control group. A microarray-based transcriptome analysis was used to investigate key genes involved in cell structure, mitochondrial function and cell division. Our results indicate that insulin supplementation during oocyte maturation leads to lower blastocyst rates and a different phenotype, characterised by an increased cell number and different actin and mitochondrial distribution patterns. These changes were reflected by an up-regulation of genes involved in cell division (MAP2K2; DHCR7), cell structure (LMNA; VIM; TUBB2B; TUBB3; TUBB4B) and mitochondrial activation

  9. Insulin receptors in the mammary gland

    International Nuclear Information System (INIS)

    Smith, D.H.

    1986-01-01

    Insulin binding studies were conducted using mammary membrane preparations to further the authors understanding of insulin's role in regulating mammary metabolism, particularly ruminant mammary metabolism. Specific objectives were to: (1) characterize insulin binding to bovine mammary microsomes and determine if the specificity and kinetics of binding indicate the presence of insulin receptors in bovine mammary gland; (2) examine and compare insulin binding by liver and mammary microsomes of the pig and dairy cow; (3) examine insulin binding to bovine milk fat globule membranes (MFGM) and evaluate this model's usefulness in assessing insulin receptor regulation in the mammary gland of the cow; (4) examine the effect of dietary fat in insulin binding by rat mammary and liver microsomes. The specificity and kinetics of 125 I-insulin binding of bovine mammary microsomes indicated the presence of insulin receptors in bovine mammary gland. Bovine liver and mammary microsomes specifically bound less 125 I-insulin than did the corresponding porcine microsomes, and mammary microsomes, regardless of species, specifically bound less 125 I-insulin than did liver microsomes. These differences in binding suggest differences in insulin responsiveness between pigs and cattle, as well as between the liver and mammary glands

  10. Subcutaneous insulin absorption explained by insulin's physicochemical properties. Evidence from absorption studies of soluble human insulin and insulin analogues in humans.

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    Kang, S; Brange, J; Burch, A; Vølund, A; Owens, D R

    1991-11-01

    To study the influence of molecular aggregation on rates of subcutaneous insulin absorption and to attempt to elucidate the mechanism of absorption of conventional soluble human insulin in humans. Seven healthy male volunteers aged 22-43 yr and not receiving any drugs comprised the study. This study consisted of a single-blind randomized comparison of equimolar dosages of 125I-labeled forms of soluble hexameric 2 Zn2+ human insulin and human insulin analogues with differing association states at pharmaceutical concentrations (AspB10, dimeric; AspB28, mixture of monomers and dimers; AspB9, GluB27, monomeric). After an overnight fast and a basal period of 1 h, 0.6 nmol/kg of either 125I-labeled human soluble insulin (Actrapid HM U-100) or 125I-labeled analogue was injected subcutaneously on 4 separate days 1 wk apart. Absorption was assessed by measurement of residual radioactivity at the injection site by external gamma-counting. The mean +/- SE initial fractional disappearance rates for the four preparations were 20.7 +/- 1.9 (hexameric soluble human insulin), 44.4 +/- 2.5 (dimeric analogue AspB10), 50.6 +/- 3.9 (analogue AspB28), and 67.4 +/- 7.4%/h (monomeric analogue AspB9, GluB27). Absorption of the dimeric analogue was significantly faster than that of hexameric human insulin (P less than 0.001); absorption of monomeric insulin analogue AspB9, GluB27 was significantly faster than that of dimeric analogue AspB10 (P less than 0.01). There was an inverse linear correlation between association state and the initial fractional disappearance rates (r = -0.98, P less than 0.02). Analysis of the disappearance data on a log linear scale showed that only the monomeric analogue had a monoexponential course throughout. Two phases in the rates of absorption were identified for the dimer and three for hexameric human insulin. The fractional disappearance rates (%/h) calculated by log linear regression analysis were monomer 73.3 +/- 6.8; dimer 44.4 +/- 2.5 from 0 to 2 h and

  11. Procedure for the preparation of tritium-labelled insulins

    International Nuclear Information System (INIS)

    Bienert, M.; Haensicke, A.; Beyermann, M.; Kaufmann, K.D.; Oehlke, J.; Klauschenz, E.; Bespalowa, S.; Titov, M.; Pleiss, U.

    1986-01-01

    This invention is concerned with a procedure for the preparation of specific 3 H-labelled insulins with sequences of human, bovine or porcine insulins and without simultaneous chemical modifications of the insulin. On the basis of this procedure a 3 H 2 -Typ (B26)-insulin can be obtained in good yield and purity with a specific radioactivity appropriate to biopharmaceutical and pharmacokinetic purposes in medicine and pharmaceutical industry, resp

  12. Comparative analysis of human and bovine teeth: radiographic density

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    Jefferson Luis Oshiro Tanaka

    2008-12-01

    Full Text Available Since bovine teeth have been used as substitutes for human teeth in in vitro dental studies, the aim of this study was to compare the radiographic density of bovine teeth with that of human teeth to evaluate their usability for radiographic studies. Thirty bovine and twenty human teeth were cut transversally in 1 millimeter-thick slices. The slices were X-rayed using a digital radiographic system and an intraoral X-ray machine at 65 kVp and 7 mA. The exposure time (0.08 s and the target-sensor distance (40 cm were standardized for all the radiographs. The radiographic densities of the enamel, coronal dentin and radicular dentin of each slice were obtained separately using the "histogram" tool of Adobe Photoshop 7.0 software. The mean radiographic densities of the enamel, coronal dentin and radicular dentin were calculated by the arithmetic mean of the slices of each tooth. One-way ANOVA demonstrated statistically significant differences for the densities of bovine and human enamel (p 0.05. Based on the results, the authors concluded that: a the radiographic density of bovine enamel is significantly higher than that of human enamel; b the radiodensity of bovine coronal dentin is statistically lower than the radiodensity of human coronal dentin; bovine radicular dentin is also less radiodense than human radicular dentin, although this difference was not statistically significant; c bovine teeth should be used with care in radiographic in vitro studies.

  13. The effect of mutations on the structure of insulin fibrils studied by Fourier transform infrared (FTIR) spectroscopy and electron microscopy.

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    Garriques, Liza Nielsen; Frokjaer, Sven; Carpenter, John F; Brange, Jens

    2002-12-01

    Fibril formation (aggregation) of human and bovine insulin and six human insulin mutants in hydrochloric acid were investigated by visual inspection, Thioflavin T fluorescence spectroscopy, transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopy. The fibrillation tendencies of the wild-type insulins and the insulin mutants were (in order of decreasing fibrillation tendencies): Glu(B1) + Glu(B27) = bovine < human < des-(B1,B2)-insulin < Ser(B2) + Asp(B10) < Glu(A13) + Glu(B10) = Gln(B17) < Asp(B10). Transmission electron micrographs showed that the protofibrils of the mutants were similar to those of wild-type insulins and had a diameter of 5-10 nm and lengths varying from 50 nm to several microns. The fibrils of human insulin mutants exhibited varying degrees of lateral aggregation. The Asp(B10) mutant and human insulin had greater tendency to form laterally aggregated fibrils arranged in parallel bundles, whereas fibrils of the other mutants and bovine insulin were mainly arranged in helical filaments. FTIR spectroscopy showed that the native secondary structure of the wild-type insulins and the human insulin mutants in hydrochloric acid were identical, whereas the secondary structure of the fibrils formed by heating at 50 degrees C depended on the amino acid substitution. FTIR spectra of fibrils of the human insulin mutants exhibited different beta-sheet bands at 1,620-1,640 cm(-1), indicating that the beta-sheet interactions in the fibrils depended on variations in the primary structure of insulin. Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2473-2480, 2002

  14. High-fat diet based on dried bovine brain: an effective animal model of dyslipidemia and insulin resistance.

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    Araújo, Tiago Gomes; Leite, Ana Catarina Rezende; Martins da Fonseca, Caíque Silveira; Carvalho, Bruno Melo; Schuler, Alexandre Ricardo Pereira; Lima, Vera Lúcia de Menezes

    2011-09-01

    Currently, there are no reports in the literature demonstrating any animal model that ingests one of the fattiest animal food source, the bovine brain. We hypothesized that a high-fat diet (HFD), based on dried bovine brain, could be used to develop an animal model possessing a spectrum of insulin resistance-related features. The HFD was formulated with 40% dried bovine brain plus 16.4% butter fat, prepared in-house. Furthermore, the diet contained 52% calories as fat and 73% of total fatty acids were saturated. Swiss mice weighing about 40 g were assigned to two dietary groups (n=6/group), one group received a standard chow diet and the other was given HFD for 3 months. The body weight and biochemical parameters of the animals were measured initially and at monthly intervals until the end of the experiment. Animals fed on a HFD showed a significant increase in the body and adipose tissue weight, serum total cholesterol and triglyceride levels, when compared with mice fed on the control diet. Additionally, the HFD group showed higher circulating levels of liver transaminases, such as alanine aminotransferase and aspartate aminotransferase, compared with the control group. Finally, to illustrate the usefulness of this model, we report that the HFD induced mild hyperglycemia, fasting hyperinsulinemia, and increased the homeostasis model of assessment (HOMA-IR), in comparison with the control group. In conclusion, our results show that HFD, based on dried bovine brain, causes insulin resistance-related metabolic disturbances. Thus, this may be a suitable model to study disturbances in energy metabolism and their consequences.

  15. Bovine brain ribonuclease is the functional homolog of human ribonuclease 1.

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    Eller, Chelcie H; Lomax, Jo E; Raines, Ronald T

    2014-09-19

    Mounting evidence suggests that human pancreatic ribonuclease (RNase 1) plays important roles in vivo, ranging from regulating blood clotting and inflammation to directly counteracting tumorigenic cells. Understanding these putative roles has been pursued with continual comparisons of human RNase 1 to bovine RNase A, an enzyme that appears to function primarily in the ruminant gut. Our results imply a different physiology for human RNase 1. We demonstrate distinct functional differences between human RNase 1 and bovine RNase A. Moreover, we characterize another RNase 1 homolog, bovine brain ribonuclease, and find pronounced similarities between that enzyme and human RNase 1. We report that human RNase 1 and bovine brain ribonuclease share high catalytic activity against double-stranded RNA substrates, a rare quality among ribonucleases. Both human RNase 1 and bovine brain RNase are readily endocytosed by mammalian cells, aided by tight interactions with cell surface glycans. Finally, we show that both human RNase 1 and bovine brain RNase are secreted from endothelial cells in a regulated manner, implying a potential role in vascular homeostasis. Our results suggest that brain ribonuclease, not RNase A, is the true bovine homolog of human RNase 1, and provide fundamental insight into the ancestral roles and functional adaptations of RNase 1 in mammals. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Protein Crystal Recombinant Human Insulin

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    1994-01-01

    The comparison of protein crystal, Recombiant Human Insulin; space-grown (left) and earth-grown (right). On STS-60, Spacehab II indicated that space-grown crystals are larger and of greater optical clarity than their earth-grown counterparts. Recombiant Human Insulin facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  17. Quantum-mechanical DFT calculation supported Raman spectroscopic study of some amino acids in bovine insulin.

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    Tah, Bidisha; Pal, Prabir; Roy, Sourav; Dutta, Debodyuti; Mishra, Sabyashachi; Ghosh, Manash; Talapatra, G B

    2014-08-14

    In this article Quantum mechanical (QM) calculations by Density Functional Theory (DFT) have been performed of all amino acids present in bovine insulin. Simulated Raman spectra of those amino acids are compared with their experimental spectra and the major bands are assigned. The results are in good agreement with experiment. We have also verified the DFT results with Quantum mechanical molecular mechanics (QM/MM) results for some amino acids. QM/MM results are very similar with the DFT results. Although the theoretical calculation of individual amino acids are feasible, but the calculated Raman spectrum of whole protein molecule is difficult or even quite impossible task, since it relies on lengthy and costly quantum-chemical computation. However, we have tried to simulate the Raman spectrum of whole protein by adding the proportionate contribution of the Raman spectra of each amino acid present in this protein. In DFT calculations, only the contributions of disulphide bonds between cysteines are included but the contribution of the peptide and hydrogen bonds have not been considered. We have recorded the Raman spectra of bovine insulin using micro-Raman set up. The experimental spectrum is found to be very similar with the resultant simulated Raman spectrum with some exceptions. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

    International Nuclear Information System (INIS)

    Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

    1987-01-01

    In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10 6 receptors per cell. The cell line with the highest 125 I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10 6 receptors with a K/sub d/ of 10 -9 M. This level was not dependent on exposure to metals but could be increased further to 2 x 10 7 receptors per cell by addition of sodium butyrate to the culture medium. The α and β subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the α and β subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

  19. Procedure for the preparation of tritium-labelled insulins. Verfahren zur Herstellung von Tritium-markierten Insulinen

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    Bienert, M; Haensicke, A; Beyermann, M; Kaufmann, K D; Oehlke, J; Klauschenz, E; Bespalowa, S; Titov, M; Pleiss, U

    1986-12-17

    This invention is concerned with a procedure for the preparation of specific /sup 3/H-labelled insulins with sequences of human, bovine or porcine insulins and without simultaneous chemical modifications of the insulin. On the basis of this procedure a /sup 3/H/sub 2/-Typ (B26)-insulin can be obtained in good yield and purity with a specific radioactivity appropriate to biopharmaceutical and pharmacokinetic purposes in medicine and pharmaceutical industry, resp.

  20. Shear bond strength and fracture analysis of human vs. bovine teeth.

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    Stefan Rüttermann

    Full Text Available PURPOSE: To evaluate if bovine enamel and dentin are appropriate substitutes for the respective human hard tooth tissues to test shear bond strength (SBS and fracture analysis. MATERIALS AND METHODS: 80 sound and caries-free human erupted third molars and 80 freshly extracted bovine permanent central incisors (10 specimens for each group were used to investigate enamel and dentine adhesion of one 2-step self-etch (SE and one 3-step etch and rinse (E&R product. To test SBS the buccal or labial areas were ground plane to obtain appropriate enamel or dentine areas. SE and E&R were applied and SBS was measured prior to and after 500 thermocycles between +5 and +55°C. Fracture analysis was performed for all debonded areas. RESULTS: ANOVA revealed significant differences of enamel and dentin SBS prior to and after thermocycling for both of the adhesives. SBS- of E&R-bonded human enamel increased after thermocycling but SE-bonded did not. Bovine enamel SE-bonded showed higher SBS after TC but E&R-bonded had lower SBS. No differences were found for human dentin SE- or E&R-bonded prior to or after thermocycling but bovine dentin SE-bonded increased whereas bovine dentine E&R-bonded decreased. Considering the totalized and adhesive failures, fracture analysis did not show significances between the adhesives or the respective tooth tissues prior to or after thermocycling. CONCLUSION: Although SBS was different on human and bovine teeth, no differences were found for fracture analysis. This indicates that solely conducted SBS on bovine substrate are not sufficient to judge the perfomance of adhesives, thus bovine teeth are questionnable as a substrate for shear bond testing.

  1. Characterization of insulin-like growth factor I and insulin receptors on cultured bovine adrenal fasciculata cells. Role of these peptides on adrenal cell function

    International Nuclear Information System (INIS)

    Penhoat, A.; Chatelain, P.G.; Jaillard, C.; Saez, J.M.

    1988-01-01

    We have characterized insulin-like growth factor I (IGF-I) and insulin receptors in cultured bovine adrenal cells by binding and cross-linking affinity experiments. At equilibrium the dissociation constant and the number of binding sites per cell for IGF-I were 1.4 +/- (SE) 0.3 x 10(-9) M and 19,200 +/- 2,100, respectively. Under reduction conditions, disuccinimidyl suberate cross-linked [ 125 I]iodo-IGF-I to one receptor complex with an Mr of 125,000. Adrenal cells also contain specific insulin receptors with an apparent dissociation constant (Kd) of 10(-9) M. Under reduction conditions [ 125 I]iodo-insulin binds to one band with an approximate Mr of 125,000. IGF-I and insulin at micromolar concentrations, but not at nanomolar concentrations, slightly stimulated DNA synthesis, but markedly potentiated the mitogenic action of fibroblast growth factor. Adrenal cells cultured in a serum-free medium containing transferrin, ascorbic acid, and insulin (5 micrograms/ml) maintained fairly constant angiotensin-II (A-II) receptor concentration per cell and increased cAMP release on response to ACTH and their steroidogenic response to both ACTH and A-II. When the cells were cultured in the same medium without insulin, the number of A-II receptors significantly decreased to 65% and the increased responsiveness was blunted. Treatment of such cells for 3 days with increasing concentrations of IGF-I (1-100 ng/ml) produced a 2- to 3-fold increase in A-II receptors and enhanced the cAMP response (3- to 4-fold) to ACTH and the steroidogenic response (4- to 6-fold) to ACTH and A-II. These effects were time and dose dependent (ED50 approximately equal to 10(-9) M). Insulin at micromolar concentrations produced an effect similar to that of IGF-I, but at nanomolar concentrations the effect was far less

  2. Evidence for altered transport of insulin across the blood-brain barrier in insulin-resistant humans.

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    Heni, Martin; Schöpfer, Patricia; Peter, Andreas; Sartorius, Tina; Fritsche, Andreas; Synofzik, Matthis; Häring, Hans-Ulrich; Maetzler, Walter; Hennige, Anita M

    2014-08-01

    Eating behavior, body weight regulation, peripheral glucose metabolism, and cognitive function depend on adequate insulin action in the brain, and recent studies in humans suggested that impaired insulin action in the brain emerges upon fat intake, obesity, and genetic variants. As insulin enters into the brain in a receptor-mediated fashion, we hypothesized that whole-body insulin sensitivity might affect the transport of insulin into the brain and contribute to the aversive effect of insulin resistance in the central nervous system. In this study, we aimed to determine the ratio of insulin in the cerebrospinal fluid and serum to whole-body insulin sensitivity. Healthy human subjects participated in an oral glucose tolerance test to determine whole-body insulin sensitivity and underwent lumbar puncture. Blood and CSF concentrations of insulin were significantly correlated. The CSF/serum ratio for insulin was significantly associated with whole body insulin sensitivity with reduced insulin transported into the CSF in insulin-resistant subjects. Together, our data suggest that transport of insulin into the CSF relates to peripheral insulin sensitivity and impairs insulin action in the brain. This underlines the need for sensitizing measures in insulin-resistant subjects.

  3. Insulin Promotes the Proliferation of Human Umbilical Cord Matrix-Derived Mesenchymal Stem Cells by Activating the Akt-Cyclin D1 Axis

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    Peng Li

    2017-01-01

    Full Text Available Background. The functions of insulin in mesenchymal stem cells (MSC remain poorly understood. Methods. MSC from human umbilical cord matrix (UCM cultured in serum-free media (SFM with or without insulin were subjected to various molecular biological analyses to determine their proliferation and growth states, expression levels of Akt-cyclin D1 signaling molecules, and in vitro differentiation capacities. Results. Insulin accelerated the G1-S cell cycle progression of UCM-MSC and significantly stimulated their proliferation and growth in SFM. The pro-proliferative action of insulin was associated with augmented cyclin D1 and phosphorylated Akt expression levels. Akt inactivation remarkably abrogated insulin-induced increases in cyclin D1 expression and cell proliferation, indicating that insulin enhances the proliferation of UCM-MSC via acceleration of the G1-S transition mediated by the Akt-cyclin D1 pathway. Additionally, the UCM-MSC propagated in SFM supplemented with insulin exhibited similar specific surface antigen profiles and differentiation capacities as those generated in conventional media containing fetal bovine serum. Conclusions. These findings suggest that insulin acts solely to promote UCM-MSC proliferation without affecting their immunophenotype and differentiation potentials and thus have important implications for utilizing insulin to expand clinical-grade MSC in vitro.

  4. Bovine Lactoferrampin, Human Lactoferricin, and Lactoferrin 1-11 Inhibit Nuclear Translocation of HIV Integrase.

    Science.gov (United States)

    Wang, Winston Yan; Wong, Jack Ho; Ip, Denis Tsz Ming; Wan, David Chi Cheong; Cheung, Randy Chifai; Ng, Tzi Bun

    2016-08-01

    This study aimed to investigate fragments derived from human and bovine lactoferrins for ability to inhibit nuclear translocation of HIV-1 integrase. It was shown that human lactoferricin, human lactoferrin 1-11, and bovine lactoferrampin reduced nuclear distribution of HIV-1 integrase. Bovine lactoferrampin could inhibit both the activity and nuclear translocation of HIV-1 integrase. Human lactoferrampin, bovine lactoferricin, and bovine lactoferrin 1-11 had no effect on HIV-1 integrase nuclear translocation. Human lactoferrampin which inhibited the activity of integrase did not prevent its nuclear translocation. Human lactoferricin and lactoferrin 1-11 did not inhibit HIV-1 integrase nuclear translocation despite their ability to attenuate the enzyme activity. The discrepancy between the findings on reduction of HIV-1 activity and inhibition of nuclear translocation of HIV-1 integrase was due to the different mechanisms involved. A similar reasoning can also be applied to the different inhibitory potencies of the milk peptides on different HIV enzymes, i.e., nuclear translocation.

  5. Bovine and human lactoferricin peptides: chimeras and new cyclic analogs.

    Science.gov (United States)

    Arias, Mauricio; McDonald, Lindsey J; Haney, Evan F; Nazmi, Kamran; Bolscher, Jan G M; Vogel, Hans J

    2014-10-01

    Lactoferrin (LF) is an important antimicrobial and immune regulatory protein present in neutrophils and most exocrine secretions of mammals. The antimicrobial activity of LF has been related to the presence of an antimicrobial peptide sequence, called lactoferricin (LFcin), located in the N-terminal region of the protein. The antimicrobial activity of bovine LFcin is considerably stronger than the human version. In this work, chimera peptides combining segments of bovine and human LFcin were generated in order to study their antimicrobial activity and mechanism of action. In addition, the relevance of the conserved disulfide bridge and the resulting cyclic structure of both LFcins were analyzed by using "click chemistry" and sortase A-catalyzed cyclization of the peptides. The N-terminal region of bovine LFcin (residues 17-25 of bovine LF) proved to be very important for the antimicrobial activity of the chimera peptides against E. coli, when combined with the C-terminal region of human LFcin. Similarly the cyclic bovine LFcin analogs generated by "click chemistry" and sortase A preserved the antimicrobial activity of the original peptide, showing the significance of these two techniques in the design of cyclic antimicrobial peptides. The mechanism of action of bovine LFcin and its active derived peptides was strongly correlated with membrane leakage in E. coli and up to some extent with the ability to induce vesicle aggregation. This mechanism was also preserved under conditions of high ionic strength (150 mM NaCl) illustrating the importance of these peptides in a more physiologically relevant system.

  6. Methicillin resistant S. aureus in human and bovine mastitis.

    Science.gov (United States)

    Holmes, Mark A; Zadoks, Ruth N

    2011-12-01

    Staphylococcus aureus is a ubiquitous organism that causes a variety of diseases including mastitis in cattle and humans. High-level resistance of S. aureus to β-lactams conferred by a mecA gene encoding a modified penicillin binding protein (PBP2a) was first observed in the early 1960's. These methicillin resistant S. aureus (MRSA) have been responsible for both hospital acquired infections (HA-MRSA) and, more recently, community acquired MRSA (CA-MRSA). A small number of human MRSA mastitis cases and outbreaks in maternity or neonatal units have been reported which are generally the result of CA-MRSA. The establishment of the sequence type 398 (ST398) in farm animals, primarily pigs, in the early 2000's has provided a reservoir of infection for humans and dairy cattle, particularly in continental Europe, described as livestock-associated MRSA (LA-MRSA). Prior to the emergence of ST398 there were sporadic reports of MRSA in bovine milk and cases of mastitis, often caused by strains from human associated lineages. Subsequently, there have been several reports describing bovine udder infections caused by ST-398 MRSA. Recently, another group of LA-MRSA strains was discovered in humans and dairy cattle in Europe. This group carries a divergent mecA gene and includes a number of S. aureus lineages (CC130, ST425, and CC1943) that were hitherto thought to be bovine-specific but are now also found as carriage or clinical isolates in humans. The emergence of MRSA in dairy cattle may be associated with contact with other host species, as in the case of ST398, or with the exchange of genetic material between S. aureus and coagulase negative Staphylococcus species, which are the most common species associated with bovine intramammary infections and commonly carry antimicrobial resistance determinants.

  7. Counter-regulatory hormone responses to spontaneous hypoglycaemia during treatment with insulin Aspart or human soluble insulin

    DEFF Research Database (Denmark)

    Brock Jacobsen, I; Vind, B F; Korsholm, Lars

    2011-01-01

    examined in a randomized, double-blinded cross-over study for two periods of 8 weeks. Sixteen patients with type 1 diabetes were subjected to three daily injections of human soluble insulin or Aspart in addition to Neutral Protamine Hagedorn (NPH) insulin twice daily. Each intervention period was followed......-regulatory responses regarding growth hormone, glucagon and ghrelin whereas no differences were found in relation to free fatty acid, cortisol, insulin-like growth factor (IGF)-I, IGF-II and IGF-binding proteins 1 and 2. Treatment with insulin Aspart resulted in well-defined peaks in serum insulin concentrations...... elicited a slightly different physiological response to spontaneous hypoglycaemia compared with human insulin. Keywords hypoglycaemia counter-regulation, insulin Aspart, type 1 diabetes....

  8. Recombinant DNA derived monomeric insulin analogue: comparison with soluble human insulin in normal subjects.

    Science.gov (United States)

    Vora, J P; Owens, D R; Dolben, J; Atiea, J A; Dean, J D; Kang, S; Burch, A; Brange, J

    1988-11-12

    To compare the rate of absorption from subcutaneous tissue and the resulting hypoglycaemic effect of iodine-125 labelled soluble human insulin and a monomeric insulin analogue derived by recombinant DNA technology. Single blind randomised comparison of equimolar doses of 125I labelled soluble human insulin and insulin analogue. Study in normal people at a diabetes research unit and a university department of medical physics. Seven healthy male volunteers aged 20-39 not receiving any other drugs. After an overnight fast and a basal period of one hour two doses (0.05 and 0.1 U/kg) of 125I labelled soluble human insulin and insulin analogue were injected subcutaneously into the anterior abdominal wall on four separate days. To find a fast acting insulin for meal related requirements in insulin dependent diabetics. MEASUREMENTS and main results--Residual radioactivity at the injection site was measured continuously for the first two hours after injection of the 125I labelled preparations and thereafter for five minutes simultaneously with blood sampling. Frequent venous blood samples were obtained over six hours for determination of plasma immunoreactive insulin, insulin analogue, glucose, and glucagon values. Time to 50% of initial radioactivity at the injection site for the insulin analogue compared with soluble insulin was 61 v 135 minutes (p less than 0.05) with 0.05 U/kg and 67 v 145 minutes (p less than 0.001) with 0.1 U/kg. Concentrations in plasma increased faster after the insulin analogue compared with soluble insulin, resulting in higher plasma concentrations between 10 and 150 minutes (0.001 less than p less than 0.05) after 0.05 U/kg and between 40 and 360 minutes (0.001 less than p less than 0.05) after 0.1 U/kg. The hypoglycaemic response to insulin analogue was a plasma glucose nadir at 60 minutes with both doses compared with 90 and 120 minutes with soluble insulin at 0.5 and 0.1 U/kg respectively. The response of glucagon substantiated the earlier and

  9. Human and Bovine Dentin Composition and Its Hybridization Mechanism Assessed by FT-Raman Spectroscopy

    Directory of Open Access Journals (Sweden)

    L. E. S. Soares

    2013-01-01

    Full Text Available FT-Raman spectroscopy was used to study the human and bovine dentin and their interactions with adhesive systems. Ten human (H molars and ten bovine (B teeth were prepared exposing the dentin and then each specimen was divided into two parts. The resulted forty dentin segments were treated either with the total-etch one bottle adhesive (Prime & Bond 2.1, PB or with the single-step self-etching adhesive (Xeno III, X and divided into four groups: HPB (control, HX, BPB, and BX. Each group was analyzed by FT-Raman spectroscopy before and after the adhesive treatment. Six regions of the Raman spectrum were analyzed and the integrated areas of organic and inorganic peaks were calculated. Bovine untreated specimens showed higher peak area of PO4 3−ν2  content than in human specimens. Human untreated specimens had higher peak areas of PO4 3−ν4 and CO3 2−ν1  contents than in bovine specimens. The peak areas of amide III, CH2, and amide I contents were higher in human than in bovine specimens (before treatments. Treated dentin showed no significant statistical differences between the adhesives for both inorganic and organic contents considering the same substrate. However, the differences found between human and bovine specimens after adhesives application show a reduced accuracy of these substrates as a substitute to the human specimens.

  10. Characterization of Staphylococcus aureus strains involved in human and bovine mastitis.

    Science.gov (United States)

    Delgado, Susana; García, Pilar; Fernández, Leonides; Jiménez, Esther; Rodríguez-Baños, Mercedes; del Campo, Rosa; Rodríguez, Juan M

    2011-07-01

    Staphylococcus aureus is one of the main etiological agents of mastitis in different mammalian species. At present, it is unknown whether strains isolated from human mastitis cases share phenotypic properties and genetic background with those obtained from animal mastitis cases. Therefore, the objective of this study was to characterize S. aureus strains isolated from women with lactational mastitis and to compare them with the strains responsible for bovine mastitis and noninfectious strains. All the strains were genotyped by both pulsed field gel electrophoresis and multilocus sequence typing and submitted to a characterization scheme that included diverse assays related to pathogenic potential and antibiotic resistance. Apart from siderophore production, no significant association was observed between the strains from bovine and human mastitis. Statistical differences between human- and bovine-mastitis-associated strains were detected for some traits and virulence determinants, such as the presence of prophages and cna and hlb genes, which were more frequently found within the bovine group. On the contrary, resistance to penicillin was significantly higher among strains isolated from human lactational mastitis, probably related to the common presence of the blaZ gene. A high genetic diversity was found among the strains involved in mastitis in breastfeeding women. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  11. The Activation Pathway of Human Rhodopsin in Comparison to Bovine Rhodopsin*

    Science.gov (United States)

    Kazmin, Roman; Rose, Alexander; Szczepek, Michal; Elgeti, Matthias; Ritter, Eglof; Piechnick, Ronny; Hofmann, Klaus Peter; Scheerer, Patrick; Hildebrand, Peter W.; Bartl, Franz J.

    2015-01-01

    Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (Gt). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences. PMID:26105054

  12. In vivo response of Mesocestoides vogae to human insulin.

    Science.gov (United States)

    Canclini, L; Esteves, A

    2009-02-01

    Successful host invasion by parasitic helminths involves detection and appropriate response to a range of host-derived signals. Insulin signal response pathways are ancient and highly-conserved throughout the metazoans. However, very little is known about helminth insulin signalling and the potential role it may play in host-parasite interactions. The response of Mesocestoides vogae (Cestoda: Cyclophyllidea) larvae to human insulin was investigated, focusing on tyrosine-phosphorylation status, glucose content, survival and asexual reproduction rate. Parasite larvae were challenged with different levels of insulin for variable periods. The parameters tested were influenced by human insulin, and suggested a host-parasite molecular dialogue.

  13. Dietary Sodium Restriction Decreases Insulin Secretion Without Affecting Insulin Sensitivity in Humans

    Science.gov (United States)

    Byrne, Loretta M.; Yu, Chang; Wang, Thomas J.; Brown, Nancy J.

    2014-01-01

    Context: Interruption of the renin-angiotensin-aldosterone system prevents incident diabetes in high-risk individuals, although the mechanism remains unclear. Objective: To test the hypothesis that activation of the endogenous renin-angiotensin-aldosterone system or exogenous aldosterone impairs insulin secretion in humans. Design: We conducted a randomized, blinded crossover study of aldosterone vs vehicle and compared the effects of a low-sodium versus a high-sodium diet. Setting: Academic clinical research center. Participants: Healthy, nondiabetic, normotensive volunteers. Interventions: Infusion of exogenous aldosterone (0.7 μg/kg/h for 12.5 h) or vehicle during low or high sodium intake. Low sodium (20 mmol/d; n = 12) vs high sodium (160 mmol/d; n = 17) intake for 5–7 days. Main Outcome Measures: Change in acute insulin secretory response assessed during hyperglycemic clamps while in sodium balance during a low-sodium vs high-sodium diet during aldosterone vs vehicle. Results: A low-sodium diet increased endogenous aldosterone and plasma renin activity, and acute glucose-stimulated insulin (−16.0 ± 5.6%; P = .007) and C-peptide responses (−21.8 ± 8.4%; P = .014) were decreased, whereas the insulin sensitivity index was unchanged (−1.0 ± 10.7%; P = .98). Aldosterone infusion did not affect the acute insulin response (+1.8 ± 4.8%; P = .72) or insulin sensitivity index (+2.0 ± 8.8%; P = .78). Systolic blood pressure and serum potassium were similar during low and high sodium intake and during aldosterone infusion. Conclusions: Low dietary sodium intake reduces insulin secretion in humans, independent of insulin sensitivity. PMID:25029426

  14. Human and bovine viruses in the Milwaukee River Watershed: hydrologically relevant representation and relations with environmental variables

    Science.gov (United States)

    Corsi, Steven R.; Borchardt, M. A.; Spencer, S. K.; Hughes, Peter E.; Baldwin, Austin K.

    2014-01-01

    To examine the occurrence, hydrologic variability, and seasonal variability of human and bovine viruses in surface water, three stream locations were monitored in the Milwaukee River watershed in Wisconsin, USA, from February 2007 through June 2008. Monitoring sites included an urban subwatershed, a rural subwatershed, and the Milwaukee River at the mouth. To collect samples that characterize variability throughout changing hydrologic periods, a process control system was developed for unattended, large-volume (56–2800 L) filtration over extended durations. This system provided flow-weighted mean concentrations during runoff and extended (24-h) low-flow periods. Human viruses and bovine viruses were detected by real-time qPCR in 49% and 41% of samples (n = 63), respectively. All human viruses analyzed were detected at least once including adenovirus (40% of samples), GI norovirus (10%), enterovirus (8%), rotavirus (6%), GII norovirus (1.6%) and hepatitis A virus (1.6%). Three of seven bovine viruses analyzed were detected including bovine polyomavirus (32%), bovine rotavirus (19%), and bovine viral diarrhea virus type 1 (5%). Human viruses were present in 63% of runoff samples resulting from precipitation and snowmelt, and 20% of low-flow samples. Maximum human virus concentrations exceeded 300 genomic copies/L. Bovine viruses were present in 46% of runoff samples resulting from precipitation and snowmelt and 14% of low-flow samples. The maximum bovine virus concentration was 11 genomic copies/L. Statistical modeling indicated that stream flow, precipitation, and season explained the variability of human viruses in the watershed, and hydrologic condition (runoff event or low-flow) and season explained the variability of the sum of human and bovine viruses; however, no model was identified that could explain the variability of bovine viruses alone. Understanding the factors that affect virus fate and transport in rivers will aid watershed management for minimizing

  15. Towards accurate structural characterization of metal centres in protein crystals: the structures of Ni and Cu T6 bovine insulin derivatives

    DEFF Research Database (Denmark)

    Frankær, Christian Grundahl; Mossin, Susanne; Ståhl, Kenny

    2014-01-01

    Using synchrotron radiation (SR), the crystal structures of T6 bovine insulin complexed with Ni2+ and Cu2+ were solved to 1.50 and 1.45 Å resolution, respectively. The level of detail around the metal centres in these structures was highly limited, and the coordination of water in Cu site II...... of the copper insulin derivative was deteriorated as a consequence of radiation damage. To provide more detail, X-ray absorption spectroscopy (XAS) was used to improve the information level about metal coordination in each derivative. The nickel derivative contains hexacoordinated Ni2+ with trigonal symmetry...... by electron paramagnetic resonance (EPR). The coordination distances were refined from EXAFS with standard deviations within 0.01 Å. The insulin derivative containing Cu2+ is sensitive towards photoreduction when exposed to SR. During the reduction of Cu2+ to Cu+, the coordination geometry of copper changes...

  16. MCF-7 human mammary adenocarcinoma cells exhibit augmented responses to human insulin on a collagen IV surface

    DEFF Research Database (Denmark)

    Listov-Saabye, Nicolai; Jensen, Marianne Blirup; Kiehr, Benedicte

    2009-01-01

    Human mammary cell lines are extensively used for preclinical safety assessment of insulin analogs. However, it is essentially unknown how mitogenic responses can be optimized in mammary cell-based systems. We developed an insulin mitogenicity assay in MCF-7 human mammary adenocarcinoma cells......, under low serum (0.1% FCS) and phenol red-free conditions, with 3H thymidine incorporation as endpoint. Based on EC50 values determined from 10-fold dilution series, beta-estradiol was the most potent mitogen, followed by human IGF-1, human AspB10 insulin and native human insulin. AspB10 insulin...... was significantly more mitogenic than native insulin, validating the ability of the assay to identify hypermitogenic human insulin analogs. With MCF-7 cells on a collagen IV surface, the ranking of mitogens was maintained, but fold mitogenic responses and dynamic range and steepness of dose-response curves were...

  17. Calcineurin inhibitors acutely improve insulin sensitivity without affecting insulin secretion in healthy human volunteers

    DEFF Research Database (Denmark)

    Øzbay, Aygen; Møller, Niels; Juhl, Claus

    2012-01-01

    and tacrolimus has been attributed to both beta cell dysfunction and impaired insulin sensitivity. WHAT THIS STUDY ADDS: This is the first trial to investigate beta cell function and insulin sensitivity using gold standard methodology in healthy human volunteers treated with clinically relevant doses...... of ciclosporin and tacrolimus. We document that both drugs acutely increase insulin sensitivity, while first phase and pulsatile insulin secretion remain unaffected. This study demonstrates that ciclosporin and tacrolimus have similar acute effects on glucose metabolism in healthy humans. AIM The introduction...... of calcineurin inhibitors (CNIs) ciclosporin (CsA) and tacrolimus (Tac) has improved the outcome of organ transplants, but complications such as new onset diabetes mellitus after transplantation (NODAT) cause impairment of survival rates. The relative contribution of each CNI to the pathogenesis and development...

  18. Insulin and the early bovine embryo

    OpenAIRE

    Laskowski, Denise

    2017-01-01

    Metabolic imbalance is a problem in the dairy industry because the metabolic demands of increased milk production can lead to decreased fertility, and more knowledge about improving the management and physical conditions of the cow (the links between fertility, nutrition, milking, and dry period) is needed. Insulin is an important hormone regulating the energy balance in the body, and insulin concentrations change in situations of energy deficiency or excess, both of which a...

  19. Insulin-like growth factor-I and insulin-like growth factor binding proteins in the bovine mammary gland: Receptors, endogenous secretion, and appearance in milk

    International Nuclear Information System (INIS)

    Campbell, P.G.

    1988-01-01

    This is the first study to characterize both insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) in bovine milk, to characterize the IGF-I receptor in the dry and lactating mammary gland, and to report de novo synthesis and secretion of IGF-I and IGFBP from normal mammary tissue. Immunoreactive IGF-I was principally associated with 45 kDa IGFBP in milk. Multiparous cows had a higher IGF-I concentration of 307 ng/ml than primiparous cows at 147 ng/ml. IGF-I concentration on day 56 of lactation was 34 ng/ml for combined parity groups. At parturition, IGF-I mass in blood and milk pools was 1.4 and 1.2 mg, respectively. Binding of 125 I-IGF-I was specific for IGF-I with anIC 50 of 2.2 ng which was a 10- and 1273-fold greater affinity than IGF-II and insulin, respectively. Association constants, as determined by Scatchard analysis, were similar for both pregnant and lactating cows at 3.5 and 4.0 L/nM, respectively. In addition, estimated mean receptor concentration was 0.25 and 0.23 pM/mg protein for pregnant and lactating cows, respectively. In a survey of mammary microscomes prepared from 48 cows, 125 I-IGF-I binding declined with progressing lactation and a similar trend was observed during pregnancy

  20. Comparison of virulence factors and capsular types of Streptococcus agalactiae isolated from human and bovine infections.

    Science.gov (United States)

    Emaneini, Mohammad; Khoramian, Babak; Jabalameli, Fereshteh; Abani, Samira; Dabiri, Hossein; Beigverdi, Reza

    2016-02-01

    Streptococcus agalactiae is a leading cause of human and bovine infections. A total of 194 S. agalactiae isolates, 55 isolates from bovines and 139 from humans, were analyzed for capsular types, virulence genes (scpB, hly, rib, bca and bac) and mobile genetic elements (IS1548 and GBSi1) using polymerase chain reaction (PCR) and multiplex PCR. Capsular type III was predominant (61%), followed by types V, II, Ib, and IV. The scpB, hly, bca and bac virulence genes were only found among human isolates. Twelve and 2 distinct virulence gene profiles were identified among human and bovine isolates respectively. The virulence gene profiles scpB- hly- IS1548- rib-bca (51%) and scpB- hly- IS1548- bca (19%) were only predominant among human isolates. The rib gene was the most common virulence gene in both human and bovine isolates. The study showed a high prevalence of virulence genes in S. agalactiae strains isolated from human infections, these result can support the idea that S. agalactiae isolated from humans and bovines are generally unrelated and probably belonged to separate populations. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Human and bovine viruses in the Milwaukee River watershed: Hydrologically relevant representation and relations with environmental variables

    Energy Technology Data Exchange (ETDEWEB)

    Corsi, S.R., E-mail: srcorsi@usgs.gov [U.S. Geological Survey, Wisconsin Water Science Center, Middleton, WI 53562 (United States); Borchardt, M.A.; Spencer, S.K. [U.S. Department of Agriculture, Agricultural Research Service, 2615 Yellowstone Dr., Marshfield, WI 54449 (United States); Hughes, P.E.; Baldwin, A.K. [U.S. Geological Survey, Wisconsin Water Science Center, Middleton, WI 53562 (United States)

    2014-08-15

    To examine the occurrence, hydrologic variability, and seasonal variability of human and bovine viruses in surface water, three stream locations were monitored in the Milwaukee River watershed in Wisconsin, USA, from February 2007 through June 2008. Monitoring sites included an urban subwatershed, a rural subwatershed, and the Milwaukee River at the mouth. To collect samples that characterize variability throughout changing hydrologic periods, a process control system was developed for unattended, large-volume (56–2800 L) filtration over extended durations. This system provided flow-weighted mean concentrations during runoff and extended (24-h) low-flow periods. Human viruses and bovine viruses were detected by real-time qPCR in 49% and 41% of samples (n = 63), respectively. All human viruses analyzed were detected at least once including adenovirus (40% of samples), GI norovirus (10%), enterovirus (8%), rotavirus (6%), GII norovirus (1.6%) and hepatitis A virus (1.6%). Three of seven bovine viruses analyzed were detected including bovine polyomavirus (32%), bovine rotavirus (19%), and bovine viral diarrhea virus type 1 (5%). Human viruses were present in 63% of runoff samples resulting from precipitation and snowmelt, and 20% of low-flow samples. Maximum human virus concentrations exceeded 300 genomic copies/L. Bovine viruses were present in 46% of runoff samples resulting from precipitation and snowmelt and 14% of low-flow samples. The maximum bovine virus concentration was 11 genomic copies/L. Statistical modeling indicated that stream flow, precipitation, and season explained the variability of human viruses in the watershed, and hydrologic condition (runoff event or low-flow) and season explained the variability of the sum of human and bovine viruses; however, no model was identified that could explain the variability of bovine viruses alone. Understanding the factors that affect virus fate and transport in rivers will aid watershed management for minimizing

  2. Human and bovine viruses in the Milwaukee River watershed: Hydrologically relevant representation and relations with environmental variables

    International Nuclear Information System (INIS)

    Corsi, S.R.; Borchardt, M.A.; Spencer, S.K.; Hughes, P.E.; Baldwin, A.K.

    2014-01-01

    To examine the occurrence, hydrologic variability, and seasonal variability of human and bovine viruses in surface water, three stream locations were monitored in the Milwaukee River watershed in Wisconsin, USA, from February 2007 through June 2008. Monitoring sites included an urban subwatershed, a rural subwatershed, and the Milwaukee River at the mouth. To collect samples that characterize variability throughout changing hydrologic periods, a process control system was developed for unattended, large-volume (56–2800 L) filtration over extended durations. This system provided flow-weighted mean concentrations during runoff and extended (24-h) low-flow periods. Human viruses and bovine viruses were detected by real-time qPCR in 49% and 41% of samples (n = 63), respectively. All human viruses analyzed were detected at least once including adenovirus (40% of samples), GI norovirus (10%), enterovirus (8%), rotavirus (6%), GII norovirus (1.6%) and hepatitis A virus (1.6%). Three of seven bovine viruses analyzed were detected including bovine polyomavirus (32%), bovine rotavirus (19%), and bovine viral diarrhea virus type 1 (5%). Human viruses were present in 63% of runoff samples resulting from precipitation and snowmelt, and 20% of low-flow samples. Maximum human virus concentrations exceeded 300 genomic copies/L. Bovine viruses were present in 46% of runoff samples resulting from precipitation and snowmelt and 14% of low-flow samples. The maximum bovine virus concentration was 11 genomic copies/L. Statistical modeling indicated that stream flow, precipitation, and season explained the variability of human viruses in the watershed, and hydrologic condition (runoff event or low-flow) and season explained the variability of the sum of human and bovine viruses; however, no model was identified that could explain the variability of bovine viruses alone. Understanding the factors that affect virus fate and transport in rivers will aid watershed management for minimizing

  3. Insulin action in the human brain: evidence from neuroimaging studies.

    Science.gov (United States)

    Kullmann, S; Heni, M; Fritsche, A; Preissl, H

    2015-06-01

    Thus far, little is known about the action of insulin in the human brain. Nonetheless, recent advances in modern neuroimaging techniques, such as functional magnetic resonance imaging (fMRI) or magnetoencephalography (MEG), have made it possible to investigate the action of insulin in the brain in humans, providing new insights into the pathogenesis of brain insulin resistance and obesity. Using MEG, the clinical relevance of the action of insulin in the brain was first identified, linking cerebral insulin resistance with peripheral insulin resistance, genetic predisposition and weight loss success in obese adults. Although MEG is a suitable tool for measuring brain activity mainly in cortical areas, fMRI provides high spatial resolution for cortical as well as subcortical regions. Thus, the action of insulin can be detected within all eating behaviour relevant regions, which include regions deeply located within the brain, such as the hypothalamus, midbrain and brainstem, as well as regions within the striatum. In this review, we outline recent advances in the field of neuroimaging aiming to investigate the action of insulin in the human brain using different routes of insulin administration. fMRI studies have shown a significant insulin-induced attenuation predominantly in the occipital and prefrontal cortical regions and the hypothalamus, successfully localising insulin-sensitive brain regions in healthy, mostly normal-weight individuals. However, further studies are needed to localise brain areas affected by insulin resistance in obese individuals, which is an important prerequisite for selectively targeting brain insulin resistance in obesity. © 2015 British Society for Neuroendocrinology.

  4. The structures of T6, T3R3 and R6 bovine insulin: combining X-ray diffraction and absorption spectroscopy

    DEFF Research Database (Denmark)

    Frankær, Christian Grundahl; Knudsen, Marianne Vad; Noren, Katarina

    2012-01-01

    The crystal structures of three conformations, T6, T3R3 and R6, of bovine insulin were solved at 1.40, 1.30 and 1.80 Å resolution, respectively. All conformations crystallized in space group R3. In contrast to the T6 and T3R3 structures, different conformations of the N-terminal B-chain residue Phe......B1 were observed in the R6 insulin structure, resulting in an eightfold doubling of the unit-cell volume upon cooling. The zinc coordination in each conformation was studied by X-ray absorption spectroscopy (XAS), including both EXAFS and XANES. Zinc adopts a tetrahedral coordination in all R3 sites...... molecules, as well as in other high-resolution insulin structures. As the radiation dose for XRD experiments is two orders of magnitude higher compared with that of XAS experiments, the single crystals were exposed to a higher degree of radiation damage that affected the zinc coordination in the T3 sites...

  5. Insulin and C-peptide in human brain neurons (insulin/C-peptide/brain peptides/immunohistochemistry/radioimmunoassay)

    International Nuclear Information System (INIS)

    Dorn, A.; Bernstein, H.G.; Rinne, A.; Hahn, H.J.; Ziegler, M.

    1983-01-01

    The regional distribution and cellular localization of insulin and C-peptide immunoreactivities were studied in human cadaver brains using the indirect immunofluorescence method, the peroxidase-antiperoxidase technique, and radioimmunoassay. Products of the immune reactions to both polypeptides were observed in most nerve cells in all areas of the brain examined. Immunostaining was mainly restricted to the cell soma and proximal dendrites. Radioimmunoassay revealed that human brain contains insulin and C-peptide in concentrations much higher than the blood, the highest being in the hypothalamus. These findings support the hypothesis that the 'brain insulin' is - at least in part - produced in the CNS. (author)

  6. Impaired insulin action in the human brain: causes and metabolic consequences.

    Science.gov (United States)

    Heni, Martin; Kullmann, Stephanie; Preissl, Hubert; Fritsche, Andreas; Häring, Hans-Ulrich

    2015-12-01

    Over the past few years, evidence has accumulated that the human brain is an insulin-sensitive organ. Insulin regulates activity in a limited number of specific brain areas that are important for memory, reward, eating behaviour and the regulation of whole-body metabolism. Accordingly, insulin in the brain modulates cognition, food intake and body weight as well as whole-body glucose, energy and lipid metabolism. However, brain imaging studies have revealed that not everybody responds equally to insulin and that a substantial number of people are brain insulin resistant. In this Review, we provide an overview of the effects of insulin in the brain in humans and the relevance of the effects for physiology. We present emerging evidence for insulin resistance of the human brain. Factors associated with brain insulin resistance such as obesity and increasing age, as well as possible pathogenic factors such as visceral fat, saturated fatty acids, alterations at the blood-brain barrier and certain genetic polymorphisms, are reviewed. In particular, the metabolic consequences of brain insulin resistance are discussed and possible future approaches to overcome brain insulin resistance and thereby prevent or treat obesity and type 2 diabetes mellitus are outlined.

  7. Effects of exercise on insulin binding to human muscle

    International Nuclear Information System (INIS)

    Bonen, A.; Tan, M.H.; Clune, P.; Kirby, R.L.

    1985-01-01

    A procedure was developed to measure insulin binding to human skeletal muscle obtained via the percutaneous muscle biopsy technique. With this method the effects of exercise on insulin binding were investigated. Subjects (n = 9) exercised for 60 min on a bicycle ergometer at intensities ranging from 20-86% maximum O 2 consumption (VO 2 max). Blood samples were obtained before, during, and after exercise and analyzed for glucose and insulin. Muscle samples (250 mg) for the vastus lateralis were obtained 30 min before exercise, at the end of exercise, and 60 min after exercise. Two subjects rested during the experimental period. There was no linear relationship between exercise intensities and the changes in insulin binding to human muscle. At rest (n = 2) and at exercise intensities below 60% VO 2 max (n = 5) no change in insulin binding occurred (P greater than 0.05). However, when exercise occurred at greater than or equal to 69% VO 2 max (n = 4), a pronounced decrement in insulin binding (30-50%) was observed (P less than 0.05). This persisted for 60 min after exercise. These results indicate that insulin binding in human muscle is not altered by 60 min of exercise at less than or equal to 60% VO 2 max but that a marked decrement occurs when exercise is greater than or equal to 69% VO 2 max

  8. Comparing effects of insulin analogues and human insulin on nocturnal glycaemia in hypoglycaemia-prone people with Type 1 diabetes

    DEFF Research Database (Denmark)

    Kristensen, P. L.; Tarnow, L.; Bay, C.

    2017-01-01

    . Conclusions: Treatment with insulin analogue reduces the occurrence of nocturnal hypoglycaemia assessed by nocturnal glucose profiles in people with Type 1 diabetes prone to severe hypoglycaemia. Nocturnal glucose profiles provide a more comprehensive assessment of clinical benefit of insulin regimens......Aims: To assess the difference between analogue and human insulin with regard to nocturnal glucose profiles and risk of hypoglycaemia in people with recurrent severe hypoglycaemia. Methods: A total of 72 people [46 men, mean ± sd age 54 ± 12 years, mean ± sd HbA1c 65 ± 12 mmol/mol (8.1 ± 1.1......%), mean ± sd duration of diabetes 30 ± 14 years], who participated in a 2-year randomized, crossover trial of basal-bolus therapy with insulin detemir/insulin aspart or human NPH insulin/human regular insulin (the HypoAna trial) were studied for 2 nights during each treatment. Venous blood was drawn...

  9. Human and bovine spinal disc mechanics subsequent to trypsin injection.

    Science.gov (United States)

    Alsup, Jeremy; Bishop, Timothy; Eggett, Dennis; Bowden, Anton E

    2017-10-01

    To investigate the biomechanical effects of injections of a protease on the characteristics of bovine coccygeal and human lumbar disc motion segments. Mechanics of treated tissues were measured immediately after injection and 3 h after injection. Motion segments underwent axial rotation and flexion-extension loading. Stiffness and neutral zone parameters experienced significant changes over time, with bovine tissues more strongly affected than human cadaver tissues. This was true in both axial rotation and flexion-extension. The treatment type significantly affected the neutral zone measurements in axial rotation. Hysteresis parameters were impacted by control injections. The extrapolation of bovine coccygeal motion testing results to human lumbar disc mechanics is not yet practical. The injected treatment may have a smaller impact on disc mechanics than time in testing. Viscoelasticity of human lumbar discs may be impacted by any damage to the annulus fibrosis induced by needlestick. Preclinical testing of novel spinal devices is essential to the design validation and regulatory processes, but current testing techniques rely on cadaveric testing of primarily older spines with essentially random amounts of disc degeneration. The present work investigates the viability of using trypsin injections to create a more uniform preclinical model of disc degeneration from a mechanics perspective, for the purpose of testing spinal devices. Such a model would facilitate translation of new spinal technologies to clinical practice.

  10. Histochemical evidence for the differential surface labeling, uptake, and intracellular transport of a colloidal gold-labeled insulin complex by normal human blood cells.

    Science.gov (United States)

    Ackerman, G A; Wolken, K W

    1981-10-01

    A colloidal gold-labeled insulin-bovine serum albumin (GIA) reagent has been developed for the ultrastructural visualization of insulin binding sites on the cell surface and for tracing the pathway of intracellular insulin translocation. When applied to normal human blood cells, it was demonstrated by both visual inspection and quantitative analysis that the extent of surface labeling, as well as the rate and degree of internalization of the insulin complex, was directly related to cell type. Further, the pathway of insulin (GIA) transport via round vesicles and by tubulo-vesicles and saccules and its subsequent fate in the hemic cells was also related to cell variety. Monocytes followed by neutrophils bound the greatest amount of labeled insulin. The majority of lymphocytes bound and internalized little GIA, however, between 5-10% of the lymphocytes were found to bind considerable quantities of GIA. Erythrocytes rarely bound the labeled insulin complex, while platelets were noted to sequester large quantities of the GIA within their extracellular canalicular system. GIA uptake by the various types of leukocytic cells appeared to occur primarily by micropinocytosis and by the direct opening of cytoplasmic tubulo-vesicles and saccules onto the cell surface in regions directly underlying surface-bound GIA. Control procedures, viz., competitive inhibition of GIA labeling using an excess of unlabeled insulin in the incubation medium, preincubation of the GIA reagent with an antibody directed toward porcine insulin, and the incorporation of 125I-insulin into the GIA reagent, indicated the specificity and selectivity of the GIA histochemical procedure for the localization of insulin binding sites.

  11. Histochemical evidence for the differential surface labeling, uptake, and intracellular transport of a colloidal gold-labeled insulin complex by normal human blood cells

    International Nuclear Information System (INIS)

    Ackerman, G.A.; Wolken, K.W.

    1981-01-01

    A colloidal gold-labeled insulin-bovine serum albumin (GIA) reagent has been developed for the ultrastructural visualization of insulin binding sites on the cell surface and for tracing the pathway of intracellular insulin translocation. When applied to normal human blood cells, it was demonstrated by both visual inspection and quantitative analysis that the extent of surface labeling, as well as the rate and degree of internalization of the insulin complex, was directly related to cell type. Further, the pathway of insulin (GIA) transport via round vesicles and by tubulo-vesicles and saccules and its subsequent fate in the hemic cells was also related to cell variety. Monocytes followed by neutrophils bound the greatest amount of labeled insulin. The majority of lymphocytes bound and internalized little GIA, however, between 5-10% of the lymphocytes were found to bind considerable quantities of GIA. Erythrocytes rarely bound the labeled insulin complex, while platelets were noted to sequester large quantities of the GIA within their extracellular canalicular system. GIA uptake by the various types of leukocytic cells appeared to occur primarily by micropinocytosis and by the direct opening of cytoplasmic tubulo-vesicles and saccules onto the cell surface in regions directly underlying surface-bound GIA. Control procedures, viz., competitive inhibition of GIA labeling using an excess of unlabeled insulin in the incubation medium, preincubation of the GIA reagent with an antibody directed toward porcine insulin, and the incorporation of 125I-insulin into the GIA reagent, indicated the specificity and selectivity of the GIA histochemical procedure for the localization of insulin binding sites

  12. Comparative evaluation of various solid phases for the development of coated tube assays for the estimation of progesterone in human serum, bovine serum and bovine milk

    International Nuclear Information System (INIS)

    Karir, Tarveen; Samuel, Grace; Sivaprasad, N.; Venkatesh, Meera

    2009-01-01

    Immobilization of progesterone antibody using three polystyrene surfaces and two progesterone radiotracers for use in the development of a coated tube assay for the evaluation of progesterone levels in human serum, bovine serum and bovine milk was studied. The selection of the solid phase and the tracers were based on the maximum binding, non-specific binding, sensitivity and percentage recovery. Amongst the polystyrene tubes studied, streptavidin coated tubes showed the acceptable assay features such as low non-specific binding (0.5-1.0%), adequate sensitivity (0.13-0.16 ng/ml) and recovery (85-115%) for all the three sample matrices, human serum, bovine serum and bovine milk.

  13. Type 1 Ig-E mediated allergy to human insulin, insulin analogues and beta-lactam antibiotics Hipersensibilidade imediata a insulina humana, análogos de insulina e a antibióticos beta-lactâmicos

    Directory of Open Access Journals (Sweden)

    Pedro Andrade

    2012-12-01

    Full Text Available Insulin, a crucial therapeutic agent for diabetes mellitus, has been rarely associated with hypersensitivity events. We present a 69-year-old type-2 diabetic patient with urticariform lesions on the sites of subcutaneous injection of insulin. The patient denied any known allergies, except for an unspecific cutaneous reaction after intramuscular penicillin administration in childhood. Prick tests revealed positive reactions to all tested human insulins and insulin analogues. Serum IgE levels were above normal range and RAST tests were positive for human, bovine and porcine insulins, as well as beta-lactams. Type 1 IgEmediated allergy to insulin analogues demands a prompt diagnosis and represents a significant therapeutic challenge in diabetic patients.A insulina é um agente indispensável para o controlo da diabetes mellitus. Os efeitos adversos da sua administração, em particular fenómenos de hipersensibilidade, são raros. Apresentamos um doente de 69 anos, diabético do tipo 2, com episódios recorrentes de lesões urticariformes nos locais de administração subcutânea de insulina. Negava alergias medicamentosas, à excepção de reacção não especificada na infância após penicilina intramuscular. Foram realizados testes cutâneos por puntura (prick tests com diversos tipos de insulina humana e análogos, todos com reacções positivas, associando elevação dos níveis de IgE sérica e provas RAST positivas para as insulinas humana, bovina e porcina e para os antibióticos beta-lactâmicos. A alergia a análogos de insulina exige um diagnóstico precoce, originando um desafio terapêutico importante no doente diabético.

  14. Human gut microbes impact host serum metabolome and insulin sensitivity

    DEFF Research Database (Denmark)

    Pedersen, Helle Krogh; Gudmundsdottir, Valborg; Nielsen, Henrik Bjørn

    2016-01-01

    Insulin resistance is a forerunner state of ischaemic cardiovascular disease and type 2 diabetes. Here we show how the human gut microbiome impacts the serum metabolome and associates with insulin resistance in 277 non-diabetic Danish individuals. The serum metabolome of insulin-resistant individ......Insulin resistance is a forerunner state of ischaemic cardiovascular disease and type 2 diabetes. Here we show how the human gut microbiome impacts the serum metabolome and associates with insulin resistance in 277 non-diabetic Danish individuals. The serum metabolome of insulin......-resistant individuals is characterized by increased levels of branched-chain amino acids (BCAAs), which correlate with a gut microbiome that has an enriched biosynthetic potential for BCAAs and is deprived of genes encoding bacterial inward transporters for these amino acids. Prevotella copri and Bacteroides vulgatus...

  15. Comparative innate immune interactions of human and bovine secretory IgA with pathogenic and non-pathogenic bacteria.

    Science.gov (United States)

    Hodgkinson, Alison J; Cakebread, Julie; Callaghan, Megan; Harris, Paul; Brunt, Rachel; Anderson, Rachel C; Armstrong, Kelly M; Haigh, Brendan

    2017-03-01

    Secretory IgA (SIgA) from milk contributes to early colonization and maintenance of commensal/symbiotic bacteria in the gut, as well as providing defence against pathogens. SIgA binds bacteria using specific antigenic sites or non-specifically via its glycans attached to α-heavy-chain and secretory component. In our study, we tested the hypothesis that human and bovine SIgA have similar innate-binding activity for bacteria. SIgAs, isolated from human and bovine milk, were incubated with a selection of commensal, pathogenic and probiotic bacteria. Using flow cytometry, we measured numbers of bacteria binding SIgA and their level of SIgA binding. The percentage of bacteria bound by human and bovine SIgA varied from 30 to 90% depending on bacterial species and strains, but was remarkably consistent between human and bovine SIgA. The level of SIgA binding per bacterial cell was lower for those bacteria that had a higher percentage of SIgA-bound bacteria, and higher for those bacteria that had lower percentage of SIgA-bound bacteria. Overall, human and bovine SIgA interacted with bacteria in a comparable way. This contributes to longer term research about the potential benefits of bovine SIgA for human consumers. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Bovine insulin-phosphatidylcholine mixed Langmuir monolayers: behavior at the air-water interface.

    Science.gov (United States)

    Pérez-López, S; Blanco-Vila, N M; Vila-Romeu, N

    2011-08-04

    The behavior of the binary mixed Langmuir monolayers of bovine insulin (INS) and phosphatidylcholine (PC) spread at the air-water interface was investigated under various subphase conditions. Pure and mixed monolayers were spread on water, on NaOH and phosphate-buffered solutions of pH 7.4, and on Zn(2+)-containing solutions. Miscibility and interactions between the components were studied on the basis of the analysis of the surface pressure (π)-mean molecular area (A) isotherms, surface compression modulus (C(s)(-1))-π curves, and plots of A versus mole fraction of INS (X(INS)). Our results indicate that intermolecular interactions between INS and PC depend on both the monolayer state and the structural characteristics of INS at the interface, which are strongly influenced by the subphase pH and salt content. Brewster angle microscopy (BAM) was applied to investigate the peptide aggregation pattern at the air-water interface in the presence of the studied lipid under any experimental condition investigated. The influence of the lipid on the INS behavior at the interface strongly depends on the subphase conditions.

  17. Influence of human and bovine substrate on the microleakage of two adhesive systems

    Directory of Open Access Journals (Sweden)

    Karoline Guará Brusaca Almeida

    2009-04-01

    Full Text Available The aim of this study was to evaluate in vitro the marginal sealing of two adhesive systems and to analyze the influence of human and bovine substrates on marginal microleakage in enamel. Rectangular-shaped class V cavities (4 mm wide x 2 mm high x 2 mm deep were made as follows: 8 cavities were prepared on the buccal and lingual surfaces of the human teeth with margins located on enamel and 16 cavities were prepared on the buccal surfaces of the bovine teeth. The cavities were randomly assigned to 4 groups of 8 cavities according to the adhesive system and substrate: G1 - Prime & Bond 2.1 (Dentsply/human substrate; G2 - Adhese (Ivoclar/Vivadent/human substrate; G3 - Prime & Bond 2.1 (Dentsply/bovine substrate; G4 - Adhese (Ivoclar/Vivadent/bovine substrate. The cavities were filled with microhybrid composite resin (Fillmagic and after polishing/finishing procedures, the teeth were subjected to a thermocycling regimen of 500 cycles with 1-min immersions in water at 55° ±2°C and 5° ± 2°C. Next, the teeth were coated with two layers of nail polish to within 1 mm of the margin, submerged in a 50% silver nitrate solution for 2 h, rinsed thoroughly in running tap and immersed in developing solution for 8 h. The restorations were bisected resulting in 16 specimens. Microleakage was observed under a stereomicroscope at x25 and recorded using four-point (0-3 scoring system. The data were analyzed statistically by the Mann Whitney U-test at 5% significance level. Leakage was present in all specimens and there was statistically significant difference between the adhesive systems. Adhese self-etching system showed significantly more leakage in both substrates (human - p= 0.0001 and bovine - p= 0.0031. There was no statistically significant difference between human and bovine substrates for either of the adhesive systems based on different bonding mechanisms (Prime & Bond 2.1 - p= 0.6923 and Adhese - p= 0.6109. Neither of the adhesive systems was

  18. Cancer risk among insulin users: comparing analogues with human insulin in the CARING five-country cohort study.

    Science.gov (United States)

    But, Anna; De Bruin, Marie L; Bazelier, Marloes T; Hjellvik, Vidar; Andersen, Morten; Auvinen, Anssi; Starup-Linde, Jakob; Schmidt, Marjanka K; Furu, Kari; de Vries, Frank; Karlstad, Øystein; Ekström, Nils; Haukka, Jari

    2017-09-01

    The aim of this work was to investigate the relationship between use of certain insulins and risk for cancer, when addressing the limitations and biases involved in previous studies. National Health Registries from Denmark (1996-2010), Finland (1996-2011), Norway (2005-2010) and Sweden (2007-2012) and the UK Clinical Practice Research Datalink database (1987-2013) were used to conduct a cohort study on new insulin users (N = 327,112). By using a common data model and semi-aggregate approach, we pooled individual-level records from five cohorts and applied Poisson regression models. For each of ten cancer sites studied, we estimated the rate ratios (RRs) by duration (≤0.5, 0.5-1, 1-2, 2-3, 3-4, 4-5, 5-6 and >6 years) of cumulative exposure to insulin glargine or insulin detemir relative to that of human insulin. A total of 21,390 cancer cases occurred during a mean follow-up of 4.6 years. No trend with cumulative treatment time for insulin glargine relative to human insulin was observed in risk for any of the ten studied cancer types. Of the 136 associations tested in the main analysis, only a few increased and decreased risks were found: among women, a higher risk was observed for colorectal (RR 1.54, 95% CI 1.06, 2.25) and endometrial cancer (RR 1.78, 95% CI 1.07, 2.94) for ≤0.5 years of treatment and for malignant melanoma for 2-3 years (RR 1.92, 95% CI 1.02, 3.61) and 4-5 years (RR 3.55, 95% CI 1.68, 7.47]); among men, a lower risk was observed for pancreatic cancer for 2-3 years (RR 0.34, 95% CI 0.17, 0.66) and for liver cancer for 3-4 years (RR 0.36, 95% CI 0.14, 0.94) and >6 years (RR 0.22, 95% CI 0.05, 0.92). Comparisons of insulin detemir with human insulin also showed no consistent differences. The present multi-country study found no evidence of consistent differences in risk for ten cancers for insulin glargine or insulin detemir use compared with human insulin, at follow-up exceeding 5 years.

  19. Comparison of insulin analogue B9AspB27Glu and soluble human insulin in insulin-treated diabetes.

    Science.gov (United States)

    Kang, S; Owens, D R; Vora, J P; Brange, J

    1990-02-10

    Postprandial plasma glucose excursions and plasma levels of free insulin after subcutaneous bolus injection of a rapidly absorbed monomeric insulin analogue (B9AspB27Glu) or soluble human insulin ('Actrapid HM' U100) were studied in six insulin-treated diabetic subjects. 10 U actrapid or an equimolar amount of the analogue were injected, in random order with an interval of 1 week, immediately before a 500 kcal test meal. Basal insulin levels were similar on the 2 study days (mean 74.1 [SE 5.1] pmol/l, actrapid; 79.7 [13.0] pmol/l, analogue). After injection of actrapid plasma free insulin levels rose slowly, reaching a plateau by 105 min at 222 (19) pmol/l. Injection of the analogue resulted in a rapid early peak at 30 min (798 [112] pmol/l), and levels were significantly higher than those after actrapid between 15 and 210 min. The more physiological plasma insulin levels achieved with the analogue were accompanied by a substantial reduction in postprandial plasma glucose excursions; the integrated area under the incremental plasma glucose curve was 45% lower after the analogue than after actrapid.

  20. Protein Hydrolysates from Non-bovine and Plant Sources Replaces Tryptone in Microbiological Media

    Science.gov (United States)

    Ranganathan, Yamini; Patel, Shifa; Pasupuleti, Vijai K.; Meganathan, R.

    Tryptone (pancreatic digest of casein) is a common ingredient in laboratory and fermentation media for growing wild-type and genetically modified microorganisms. Many of the commercially manufactured products such as human growth hormone, antibiotics, insulin, etc. are produced by recombinant strains grown on materials derived from bovine sources. With the emergence of Bovine Spongiform Encephalopathy (BSE) and the consequent increase in Food and Drug Administration (FDA) regulations, elimination of materials of bovine origin from fermentation media is of paramount importance. To achieve this objective, a number of protein hydrolysates derived from non-bovine animal and plant sources were evaluated. Tryptone in Luria-Bertani (LB) broth was replaced with an equal quantity of alternate protein hydrolysates. Four of the six hydrolysates (one animal and three from plants) were found to efficiently replace the tryptone present in LB-medium as measured by growth rate and growth yield of a recombinant Escherichia coli strain. In addition, we have determined plasmid stability, inducibility and activity of the plasmid encoded β-galactosidase in the recombinant strain grown in the presence of various protein hydrolysates.

  1. Skeletal muscle phosphatidylcholine fatty acids and insulin sensitivity in normal humans.

    Science.gov (United States)

    Clore, J N; Li, J; Gill, R; Gupta, S; Spencer, R; Azzam, A; Zuelzer, W; Rizzo, W B; Blackard, W G

    1998-10-01

    The fatty acid composition of skeletal muscle membrane phospholipids (PL) is known to influence insulin responsiveness in humans. However, the contribution of the major PL of the outer (phosphatidylcholine, PC) and inner (phosphatidylethanolamine, PE) layers of the sarcolemma to insulin sensitivity is not known. Fatty acid composition of PC and PE from biopsies of vastus lateralis from 27 normal men and women were correlated with insulin sensitivity determined by the hyperinsulinemic euglycemic clamp technique at insulin infusion rates of 0.4, 1.0, and 10.0 mU . kg-1 . min-1. Significant variation in the half-maximal insulin concentration (ED50) was observed in the normal volunteers (range 24.0-146.0 microU/ml), which correlated directly with fasting plasma insulin (r = 0.75, P insulin sensitivity was observed in PE (NS). These studies suggest that the fatty acid composition of PC may be of particular importance in the relationship between fatty acids and insulin sensitivity in normal humans.

  2. Fetal and perinatal outcomes in type 1 diabetes pregnancy: a randomized study comparing insulin aspart with human insulin in 322 subjects

    DEFF Research Database (Denmark)

    Hod, Moshe; Damm, Peter; Kaaja, Risto

    2008-01-01

    The objective of the study was a comparison of insulin aspart (IAsp) with human insulin (HI) in basal-bolus therapy with neutral protamine Hagedorn for fetal and perinatal outcomes of type 1 diabetes in pregnancy.......The objective of the study was a comparison of insulin aspart (IAsp) with human insulin (HI) in basal-bolus therapy with neutral protamine Hagedorn for fetal and perinatal outcomes of type 1 diabetes in pregnancy....

  3. Effect of lipopolysaccharide on inflammation and insulin action in human muscle.

    Science.gov (United States)

    Liang, Hanyu; Hussey, Sophie E; Sanchez-Avila, Alicia; Tantiwong, Puntip; Musi, Nicolas

    2013-01-01

    Accumulating evidence from animal studies suggest that chronic elevation of circulating intestinal-generated lipopolysaccharide (LPS) (i.e., metabolic endotoxemia) could play a role in the pathogenesis of insulin resistance. However, the effect of LPS in human muscle is unclear. Moreover, it is unknown whether blockade/down regulation of toll-like receptor (TLR)4 can prevent the effect of LPS on insulin action and glucose metabolism in human muscle cells. In the present study we compared plasma LPS concentration in insulin resistant [obese non-diabetic and obese type 2 diabetic (T2DM)] subjects versus lean individuals. In addition, we employed a primary human skeletal muscle cell culture system to investigate the effect of LPS on glucose metabolism and whether these effects are mediated via TLR4. Obese non-diabetic and T2DM subjects had significantly elevated plasma LPS and LPS binding protein (LBP) concentrations. Plasma LPS (r = -0.46, P = 0.005) and LBP (r = -0.49, P = 0.005) concentrations negatively correlated with muscle insulin sensitivity (M). In human myotubes, LPS increased JNK phosphorylation and MCP-1 and IL-6 gene expression. This inflammatory response led to reduced insulin-stimulated IRS-1, Akt and AS160 phosphorylation and impaired glucose transport. Both pharmacologic blockade of TLR4 with TAK-242, and TLR4 gene silencing, suppressed the inflammatory response and insulin resistance caused by LPS in human muscle cells. Taken together, these findings suggest that elevations in plasma LPS concentration found in obese and T2DM subjects could play a role in the pathogenesis of insulin resistance and that antagonists of TLR4 may improve insulin action in these individuals.

  4. Characterisation of insulin analogues therapeutically available to patients

    KAUST Repository

    Adams, Gary G.

    2018-03-29

    The structure and function of clinical dosage insulin and its analogues were assessed. This included \\'native insulins\\' (human recombinant, bovine, porcine), \\'fast-acting analogues\\' (aspart, glulisine, lispro) and \\'slow-acting analogues\\' (glargine, detemir, degludec). Analytical ultracentrifugation, both sedimentation velocity and equilibrium experiments, were employed to yield distributions of both molar mass and sedimentation coefficient of all nine insulins. Size exclusion chromatography, coupled to multi-angle light scattering, was also used to explore the function of these analogues. On ultracentrifugation analysis, the insulins under investigation were found to be in numerous conformational states, however the majority of insulins were present in a primarily hexameric conformation. This was true for all native insulins and two fast-acting analogues. However, glargine was present as a dimer, detemir was a multi-hexameric system, degludec was a dodecamer (di-hexamer) and glulisine was present as a dimer-hexamer-dihexamer system. However, size-exclusion chromatography showed that the two hexameric fast-acting analogues (aspart and lispro) dissociated into monomers and dimers due to the lack of zinc in the mobile phase. This comprehensive study is the first time all nine insulins have been characterised in this way, the first time that insulin detemir have been studied using analytical ultracentrifugation and the first time that insulins aspart and glulisine have been studied using sedimentation equilibrium. The structure and function of these clinically administered insulins is of critical importance and this research adds novel data to an otherwise complex functional physiological protein.

  5. Sequence-specific 1H-NMR assignments for the aromatic region of several biologically active, monomeric insulins including native human insulin.

    Science.gov (United States)

    Roy, M; Lee, R W; Kaarsholm, N C; Thøgersen, H; Brange, J; Dunn, M F

    1990-06-12

    The aromatic region of the 1H-FT-NMR spectrum of the biologically fully-potent, monomeric human insulin mutant, B9 Ser----Asp, B27 Thr----Glu has been investigated in D2O. At 1 to 5 mM concentrations, this mutant insulin is monomeric above pH 7.5. Coupling and amino acid classification of all aromatic signals is established via a combination of homonuclear one- and two-dimensional methods, including COSY, multiple quantum filters, selective spin decoupling and pH titrations. By comparisons with other insulin mutants and with chemically modified native insulins, all resonances in the aromatic region are given sequence-specific assignments without any reliance on the various crystal structures reported for insulin. These comparisons also give the sequence-specific assignments of most of the aromatic resonances of the mutant insulins B16 Tyr----Glu, B27 Thr----Glu and B25 Phe----Asp and the chemically modified species des-(B23-B30) insulin and monoiodo-Tyr A14 insulin. Chemical dispersion of the assigned resonances, ring current perturbations and comparisons at high pH have made possible the assignment of the aromatic resonances of human insulin, and these studies indicate that the major structural features of the human insulin monomer (including those critical to biological function) are also present in the monomeric mutant.

  6. Insulin binding properties of normal and transformed human epidermal cultured keratinocytes

    International Nuclear Information System (INIS)

    Verrando, P.; Ortonne, J.P.

    1985-01-01

    Insulin binding to its receptors was studied in cultured normal and transformed (A431 line) human epidermal keratinocytes. The specific binding was a temperature-dependent, saturable process. Normal keratinocytes possess a mean value of about 80,000 receptors per cell. Fifteen hours exposure of the cells to insulin lowered their receptor number (about 65% loss in available sites); these reappeared when the hormone was removed from the culture medium. In the A431 epidermoid carcinoma cell line, there is a net decrease in insulin binding (84% of the initial bound/free hormone ratio in comparison with normal cells) essentially related to a loss in receptor affinity for insulin. Thus, cultured human keratinocytes which express insulin receptors may be a useful tool in understanding skin pathology related to insulin disorders

  7. Caenorhabditis elegans DAF-2 as a Model for Human Insulin Receptoropathies

    Directory of Open Access Journals (Sweden)

    David A. Bulger

    2017-01-01

    Full Text Available Human exome sequencing has dramatically increased the rate of identification of disease-associated polymorphisms. However, examining the functional consequences of those variants has created an analytic bottleneck. Insulin-like signaling in Caenorhabditis elegans has long provided a model to assess consequences of human insulin signaling mutations, but this has not been evaluated in the context of current genetic tools. We have exploited strains derived from the Million Mutation Project (MMP and gene editing to explore further the evolutionary relationships and conservation between the human and C. elegans insulin receptors. Of 40 MMP alleles analyzed in the C. elegans insulin-like receptor gene DAF-2, 35 exhibited insulin-like signaling indistinguishable from wild-type animals, indicating tolerated mutations. Five MMP alleles proved to be novel dauer-enhancing mutations, including one new allele in the previously uncharacterized C-terminus of DAF-2. CRISPR-Cas9 genome editing was used to confirm the phenotypic consequence of six of these DAF-2 mutations and to replicate an allelic series of known human disease mutations in a highly conserved tyrosine kinase active site residue, demonstrating the utility of C. elegans for directly modeling human disease. Our results illustrate the challenges associated with prediction of the phenotypic consequences of amino acid substitutions, the value of assaying mutant isoform function in vivo, and how recently developed tools and resources afford the opportunity to expand our understanding even of highly conserved regulatory modules such as insulin signaling. This approach may prove generally useful for modeling phenotypic consequences of candidate human pathogenic mutations in conserved signaling and developmental pathways.

  8. Specific uptake, dissociation, and degradation of 125I-labeled insulin in isolated turtle (Chrysemys dorbigni) thyroid glands

    International Nuclear Information System (INIS)

    Marques, M.; da Silva, R.S.; Turyn, D.; Dellacha, J.M.

    1985-01-01

    Thyroid glands from turtles (Chrysemys dorbigni) pretreated with potassium iodide were incubated with 125 I-insulin in the presence or absence of unlabeled insulin, in order to study its specific uptake. At 24 degrees, the specific uptake reached a plateau at 180 min of incubation. The dose of bovine insulin that inhibited 50% of the 125 I-insulin uptake was 2 micrograms/ml of incubation medium. Most of the radioactive material (71%) extracted from the gland, after 30 min incubation with 125 I-insulin, eluted in the same position as labeled insulin on Sephadex G-50. Only 24% eluted in the salt position. After 240 min incubation, increased amount of radioactivity appeared in the Na 125 I position. When bovine insulin was added together with the labeled hormone, a substantial reduction of radioactivity was observed in the insulin and Na 125 I elution positions. Dissociation studies were performed at 6 degrees in glands preincubated with 125 I-insulin either at 24 or 6 degrees. The percentage of trichloroacetic acid (TCA)-soluble radioactive material in the dissociation medium increased with incubation time at both temperatures. However, the degradation activity was lower at 6 than at 24 degrees. The addition of bovine insulin to the incubation buffer containing 125 I-insulin reduced the radioactive degradation products in the dissociated medium. Chloroquine or bacitracin inhibited the degradation activity. Incubation of thyroid glands with 125 I-hGH or 125 I-BSA showed values of uptake, dissociation, and degradation similar to those experiments in which an excess of bovine insulin was added together with the labeled hormone. Thus, by multiple criteria, such as specific uptake, dissociation, and degradation, the presence of insulin-binding sites in the turtle thyroid gland may be suggested

  9. Identification and characterization of insulin receptors on foetal-mouse brain-cortical cells.

    OpenAIRE

    Van Schravendijk, C F; Hooghe-Peters, E L; De Meyts, P; Pipeleers, D G

    1984-01-01

    The occurrence of insulin receptors was investigated in freshly dissociated brain-cortical cells from mouse embryos. By analogy with classical insulin-binding cell types, binding of 125I-insulin to foetal brain-cortical cells was time- and pH-dependent, only partially reversible, and competed for by unlabelled insulin and closely related peptides. Desalanine-desasparagine-insulin, pig proinsulin, hagfish insulin and turkey insulin were respectively 2%, 4%, 2% and 200% as potent as bovine insu...

  10. Effect of gamma radiation and endodontic treatment on mechanical properties of human and bovine root dentin

    Energy Technology Data Exchange (ETDEWEB)

    Novais, Veridiana Resende; Soares, Priscilla Barbosa Ferreira; Guimaraes, Carlla Martins; Schliebe, Lais Rani Sales Oliveira; Braga, Stella Sueli Lourenco; Soares, Carlos Jose, E-mail: carlosjsoares@ufu.br [Universidade Federal de Uberlandia (UFU), MG (Brazil)

    2016-11-15

    This study evaluated the effect of gamma radiation and endodontic treatment on the microhardness and flexural strength of human and bovine root dentin. Forty single rooted human teeth and forty bovine incisor teeth were collected, cleaned and stored in distilled water at 4 °C. The human and bovine teeth were divided into 4 groups (n=10) resulting from the combination of two study factors: first, regarding the endodontic treatment in 2 levels: with or without endodontic treatment; and second, radiotherapy in two levels: with or without radiotherapy by 60 Gy of Co-60 gamma radiation fractioned into 2 Gy daily doses five days per week. Each tooth was longitudinally sectioned in two parts; one-half was used for the three-point bending test and the other for the Knoop hardness test (KHN). Data were analyzed by 3-way ANOVA and Tukey HSD test (α=0.05). No significant difference was found for flexural strength values. The human dentin had significantly higher KHN than the bovine. The endodontic treatment and radiotherapy resulted in significantly lower KHN irrespective of tooth origin. The results indicated that the radiotherapy had deleterious effects on the microhardness of human and bovine dentin and this effect is increased by the interaction with endodontic therapy. The endodontic treatment adds additional negative effect on the mechanical properties of radiated tooth dentin; the restorative protocols should be designed taking into account this effect. (author)

  11. Effect of gamma radiation and endodontic treatment on mechanical properties of human and bovine root dentin

    International Nuclear Information System (INIS)

    Novais, Veridiana Resende; Soares, Priscilla Barbosa Ferreira; Guimaraes, Carlla Martins; Schliebe, Lais Rani Sales Oliveira; Braga, Stella Sueli Lourenco; Soares, Carlos Jose

    2016-01-01

    This study evaluated the effect of gamma radiation and endodontic treatment on the microhardness and flexural strength of human and bovine root dentin. Forty single rooted human teeth and forty bovine incisor teeth were collected, cleaned and stored in distilled water at 4 °C. The human and bovine teeth were divided into 4 groups (n=10) resulting from the combination of two study factors: first, regarding the endodontic treatment in 2 levels: with or without endodontic treatment; and second, radiotherapy in two levels: with or without radiotherapy by 60 Gy of Co-60 gamma radiation fractioned into 2 Gy daily doses five days per week. Each tooth was longitudinally sectioned in two parts; one-half was used for the three-point bending test and the other for the Knoop hardness test (KHN). Data were analyzed by 3-way ANOVA and Tukey HSD test (α=0.05). No significant difference was found for flexural strength values. The human dentin had significantly higher KHN than the bovine. The endodontic treatment and radiotherapy resulted in significantly lower KHN irrespective of tooth origin. The results indicated that the radiotherapy had deleterious effects on the microhardness of human and bovine dentin and this effect is increased by the interaction with endodontic therapy. The endodontic treatment adds additional negative effect on the mechanical properties of radiated tooth dentin; the restorative protocols should be designed taking into account this effect. (author)

  12. Caenorhabditis elegans DAF-2 as a Model for Human Insulin Receptoropathies.

    Science.gov (United States)

    Bulger, David A; Fukushige, Tetsunari; Yun, Sijung; Semple, Robert K; Hanover, John A; Krause, Michael W

    2017-01-05

    Human exome sequencing has dramatically increased the rate of identification of disease-associated polymorphisms. However, examining the functional consequences of those variants has created an analytic bottleneck. Insulin-like signaling in Caenorhabditis elegans has long provided a model to assess consequences of human insulin signaling mutations, but this has not been evaluated in the context of current genetic tools. We have exploited strains derived from the Million Mutation Project (MMP) and gene editing to explore further the evolutionary relationships and conservation between the human and C. elegans insulin receptors. Of 40 MMP alleles analyzed in the C. elegans insulin-like receptor gene DAF-2, 35 exhibited insulin-like signaling indistinguishable from wild-type animals, indicating tolerated mutations. Five MMP alleles proved to be novel dauer-enhancing mutations, including one new allele in the previously uncharacterized C-terminus of DAF-2 CRISPR-Cas9 genome editing was used to confirm the phenotypic consequence of six of these DAF-2 mutations and to replicate an allelic series of known human disease mutations in a highly conserved tyrosine kinase active site residue, demonstrating the utility of C. elegans for directly modeling human disease. Our results illustrate the challenges associated with prediction of the phenotypic consequences of amino acid substitutions, the value of assaying mutant isoform function in vivo, and how recently developed tools and resources afford the opportunity to expand our understanding even of highly conserved regulatory modules such as insulin signaling. This approach may prove generally useful for modeling phenotypic consequences of candidate human pathogenic mutations in conserved signaling and developmental pathways. Copyright © 2017 Bulger et al.

  13. Bimodal effect on pancreatic β-cells of secretory products from normal or insulin-resistant human skeletal muscle

    DEFF Research Database (Denmark)

    Bouzakri, Karim; Plomgaard, Peter; Berney, Thierry

    2011-01-01

    Type 2 diabetes is characterized by insulin resistance with a relative deficiency in insulin secretion. This study explored the potential communication between insulin-resistant human skeletal muscle and primary (human and rat) β-cells.......Type 2 diabetes is characterized by insulin resistance with a relative deficiency in insulin secretion. This study explored the potential communication between insulin-resistant human skeletal muscle and primary (human and rat) β-cells....

  14. Differential interaction of Apolipoprotein-E isoforms with insulin receptors modulates brain insulin signaling in mutant human amyloid precursor protein transgenic mice.

    Science.gov (United States)

    Chan, Elizabeth S; Chen, Christopher; Cole, Gregory M; Wong, Boon-Seng

    2015-09-08

    It is unclear how human apolipoprotein E4 (ApoE4) increases the risk for Alzheimer's disease (AD). Although Aβ levels can lead to insulin signaling impairment, these experiments were done in the absence of human ApoE. To examine ApoE role, we crossed the human ApoE-targeted replacement mice with mutant human amyloid precursor protein (APP) mice. In 26 week old mice with lower Aβ levels, the expression and phosphorylation of insulin signaling proteins remained comparable among APP, ApoE3xAPP and ApoE4xAPP mouse brains. When the mice aged to 78 weeks, these proteins were markedly reduced in APP and ApoE4xAPP mouse brains. While Aβ can bind to insulin receptor, how ApoE isoforms modulate this interaction remains unknown. Here, we showed that ApoE3 had greater association with insulin receptor as compared to ApoE4, regardless of Aβ42 concentration. In contrast, ApoE4 bound more Aβ42 with increasing peptide levels. Using primary hippocampal neurons, we showed that ApoE3 and ApoE4 neurons are equally sensitive to physiological levels of insulin. However, in the presence of Aβ42, insulin failed to elicit a downstream response only in ApoE4 hippocampal neurons. Taken together, our data show that ApoE genotypes can modulate this Aβ-mediated insulin signaling impairment.

  15. Fibroblast growth factor 21 improves insulin sensitivity and synergizes with insulin in human adipose stem cell-derived (hASC adipocytes.

    Directory of Open Access Journals (Sweden)

    Darwin V Lee

    Full Text Available Fibroblast growth factor 21 (FGF21 has evolved as a major metabolic regulator, the pharmacological administration of which causes weight loss, insulin sensitivity and glucose control in rodents and humans. To understand the molecular mechanisms by which FGF21 exerts its metabolic effects, we developed a human in vitro model of adipocytes to examine crosstalk between FGF21 and insulin signaling. Human adipose stem cell-derived (hASC adipocytes were acutely treated with FGF21 alone, insulin alone, or in combination. Insulin signaling under these conditions was assessed by measuring tyrosine phosphorylation of insulin receptor (InsR, insulin receptor substrate-1 (IRS-1, and serine 473 phosphorylation of Akt, followed by a functional assay using 14C-2-deoxyglucose [14C]-2DG to measure glucose uptake in these cells. FGF21 alone caused a modest increase of glucose uptake, but treatment with FGF21 in combination with insulin had a synergistic effect on glucose uptake in these cells. The presence of FGF21 also effectively lowered the insulin concentration required to achieve the same level of glucose uptake compared to the absence of FGF21 by 10-fold. This acute effect of FGF21 on insulin signaling was not due to IR, IGF-1R, or IRS-1 activation. Moreover, we observed a substantial increase in basal S473-Akt phosphorylation by FGF21 alone, in contrast to the minimal shift in basal glucose uptake. Taken together, our data demonstrate that acute co-treatment of hASC-adipocytes with FGF21 and insulin can result in a synergistic improvement in glucose uptake. These effects were shown to occur at or downstream of Akt, or separate from the canonical insulin signaling pathway.

  16. CHEMERIN (RARRES2) decreases in vitro granulosa cell steroidogenesis and blocks oocyte meiotic progression in bovine species.

    Science.gov (United States)

    Reverchon, Maxime; Bertoldo, Michael J; Ramé, Christelle; Froment, Pascal; Dupont, Joëlle

    2014-05-01

    CHEMERIN, or RARRES2, is a new adipokine that is involved in the regulation of adipogenesis, energy metabolism, and inflammation. Recent data suggest that it also plays a role in reproductive function in rats and humans. Here we studied the expression of CHEMERIN and its three receptors (CMKLR1, GPR1, and CCRL2) in the bovine ovary and investigated the in vitro effects of this hormone on granulosa cell steroidogenesis and oocyte maturation. By RT-PCR, immunoblotting, and immunohistochemistry, we found CHEMERIN, CMKLR1, GPR1, and CCRL2 in various ovarian cells, including granulosa and theca cells, corpus luteum, and oocytes. In cultured bovine granulosa cells, INSULIN, IGF1, and two insulin sensitizers-metformin and rosiglitazone-increased rarres2 mRNA expression whereas they decreased cmklr1, gpr1, and cclr2 mRNA expression. Furthermore, TNF alpha and ADIPONECTIN significantly increased rarres2 and cmklr1 expression, respectively. In cultured bovine granulosa cells, human recombinant CHEMERIN (hRec, 200 ng/ml) reduced production of both progesterone and estradiol, cholesterol content, STAR abundance, CYP19A1 and HMGCR proteins, and the phosphorylation levels of MAPK3/MAPK1 in the presence or absence of FSH (10(-8) M) and IGF1 (10(-8) M). All of these effects were abolished by using an anti-CMKLR1 antibody. In bovine cumulus-oocyte complexes, the addition of hRec (200 ng/ml) in the maturation medium arrested most oocytes at the germinal vesicle stage, and this was associated with a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. Thus, in cultured bovine granulosa cells, hRec decreases steroidogenesis, cholesterol synthesis, and MAPK3/1 phosphorylation, probably through CMKLR1. Moreover, in cumulus-oocyte complexes, it blocked meiotic progression at the germinal vesicle stage and inhibited MAPK3/1 phosphorylation in both the oocytes and cumulus cells during in vitro maturation. © 2014 by the Society for the Study of Reproduction, Inc.

  17. A cluster-randomised intervention trial against Schistosoma japonicum in the Peoples' Republic of China: bovine and human transmission.

    Directory of Open Access Journals (Sweden)

    Darren J Gray

    2009-06-01

    Full Text Available Zoonotic schistosomiasis japonica is a major public health problem in China. Bovines, particularly water buffaloes, are thought to play a major role in the transmission of schistosomiasis to humans in China. Preliminary results (1998-2003 of a praziquantel (PZQ-based pilot intervention study we undertook provided proof of principle that water buffaloes are major reservoir hosts for S. japonicum in the Poyang Lake region, Jiangxi Province.Here we present the results of a cluster-randomised intervention trial (2004-2007 undertaken in Hunan and Jiangxi Provinces, with increased power and more general applicability to the lake and marshlands regions of southern China. The trial involved four matched pairs of villages with one village within each pair randomly selected as a control (human PZQ treatment only, leaving the other as the intervention (human and bovine PZQ treatment. A sentinel cohort of people to be monitored for new infections for the duration of the study was selected from each village. Results showed that combined human and bovine chemotherapy with PZQ had a greater effect on human incidence than human PZQ treatment alone.The results from this study, supported by previous experimental evidence, confirms that bovines are the major reservoir host of human schistosomiasis in the lake and marshland regions of southern China, and reinforce the rationale for the development and deployment of a transmission blocking anti-S. japonicum vaccine targeting bovines.Australian New Zealand Clinical Trials Registry ACTRN12609000263291.

  18. Validation of a radioimmunoassay for rat insulin

    International Nuclear Information System (INIS)

    Delattre, E.; Boschero, A.C.

    1984-01-01

    A methodological approach that permits, in a radioimmunoassay, the evaluation of rat insulin by using bovine insulin as reference is presented. As in general the technics for radioimmunoassay of different substances follow the same principles (competitive inhibition), we believe that the methodology presented here could be used for evaluation of other hormones when the adequated referential, of know biological activity, is not available. (Author) [pt

  19. The functional role of insulin in fertility and embryonic development-What can we learn from the bovine model?

    Science.gov (United States)

    Laskowski, D; Sjunnesson, Y; Humblot, P; Andersson, G; Gustafsson, H; Båge, R

    2016-07-01

    Insulin is a key metabolic hormone that plays a crucial role in regulating energy homeostasis in the body. In addition, insulin-dependent signaling has important functions in reproduction and early embryo development. As metabolism and reproduction are closely linked, metabolic challenges may be the source of reproductive disorders and decreased fertility. This is known for the dairy cow and for other species including the human. Although metabolic disorders in the dairy cow often derive from a failure to adapt to a high milk production, the situation in the human is often linked to emerging conditions and associated diseases in our modern society such as obesity and diabetes, where an excess energy intake causes decreased fertility in women. Both energy excess and energy deficit are associated with a deviation of insulin concentrations in serum and follicular fluid from normal levels. Although many studies have shown that extreme variation in energy supply can negatively influence early embryo development by inducing changes in circulating concentrations of several metabolites or hormones like insulin, several in vitro culture media are still supplemented with insulin in high concentrations. In this review, direct and indirect effects of insulin on fertility will be described. Differences between the in vivo and in vitro situations will also be discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Treating Type 1 Diabetes Mellitus with a Rapid-Acting Analog Insulin Regimen vs. Regular Human Insulin in Germany: A Long-Term Cost-Effectiveness Evaluation.

    Science.gov (United States)

    Valentine, William J; Van Brunt, Kate; Boye, Kristina S; Pollock, Richard F

    2018-06-01

    The aim of the present study was to evaluate the cost effectiveness of rapid-acting analog insulin relative to regular human insulin in adults with type 1 diabetes mellitus in Germany. The PRIME Diabetes Model, a patient-level, discrete event simulation model, was used to project long-term clinical and cost outcomes for patients with type 1 diabetes from the perspective of a German healthcare payer. Simulated patients had a mean age of 21.5 years, duration of diabetes of 8.6 years, and baseline glycosylated hemoglobin of 7.39%. Regular human insulin and rapid-acting analog insulin regimens reduced glycosylated hemoglobin by 0.312 and 0.402%, respectively. Compared with human insulin, hypoglycemia rate ratios with rapid-acting analog insulin were 0.51 (non-severe nocturnal) and 0.80 (severe). No differences in non-severe diurnal hypoglycemia were modeled. Discount rates of 3% were applied to future costs and clinical benefits accrued over the 50-year time horizon. In the base-case analysis, rapid-acting analog insulin was associated with an improvement in quality-adjusted life expectancy of 1.01 quality-adjusted life-years per patient (12.54 vs. 11.53 quality-adjusted life-years). Rapid-acting analog insulin was also associated with an increase in direct costs of €4490, resulting in an incremental cost-effectiveness ratio of €4427 per quality-adjusted life-year gained vs. human insulin. Sensitivity analyses showed that the base case was driven predominantly by differences in hypoglycemia; abolishing these differences reduced incremental quality-adjusted life expectancy to 0.07 quality-adjusted life-years, yielding an incremental cost-effectiveness ratio of €74,622 per quality-adjusted life-year gained. Rapid-acting analog insulin is associated with beneficial outcomes in patients with type 1 diabetes and is likely to be considered cost effective in the German setting vs. regular human insulin.

  1. Effects of pasteurization on adiponectin and insulin concentrations in donor human milk.

    Science.gov (United States)

    Ley, Sylvia H; Hanley, Anthony J; Stone, Debbie; O'Connor, Deborah L

    2011-09-01

    Although pasteurization is recommended before distributing donor human milk in North America, limited data are available on its impact on metabolic hormones in milk. We aimed to investigate the effects of pasteurization on adiponectin and insulin concentrations in donor human milk. The study investigates concentrations of components in donor human milk before and after Holder pasteurization. After the guidelines of the Human Milk Bank Association of North America, human milk samples were pooled to produce 17 distinct batches (4 individuals per batch) and pasteurized at 62.5°C for 30 min. Adiponectin, insulin, energy, fat, total protein, and glucose concentrations were measured pre- and postpasteurization. Pasteurization reduced milk adiponectin and insulin by 32.8 and 46.1%, respectively (both p Pasteurization effects on milk hormone concentrations remained significant after adjusting for fat and energy (beta ± SEE: -4.11 ± 1.27, p = 0.003 for adiponectin; -70.0 ± 15.0, p pasteurization reduced adiponectin and insulin concentrations in donor human milk. In view of emerging knowledge on the importance of milk components, continued work to find the optimal pasteurization process that mitigates risks but promotes retention of bioactive components is needed.

  2. Insulin in human milk and the use of hormones in infant formulas.

    Science.gov (United States)

    Shamir, Raanan; Shehadeh, Naim

    2013-01-01

    Human milk contains a substantial number of hormones and growth factors. Studies in animal models show that some of these peptides (e.g. insulin, insulin-like growth factor 1, IGF-1, epidermal growth factors) have an effect on the small intestine after orogastric administration. Recently, two efforts were made to incorporate growth factors into infant formulas. One of these efforts included the incorporation of IGF-1, and the second is an ongoing effort to evaluate the safety and efficacy of incorporating insulin into infant formulas. The rational and current evidence for adding insulin to infant formulas (presence in human milk, effects of orally administrated insulin on gut maturation, intestinal permeability, systemic effects and preliminary encouraging results of supplementing insulin to a preterm infant formula) is detailed in this review. If the addition of insulin to preterm infant formulas indeed results in better growth and accelerated intestinal maturation, future studies will need to address the supplementation of insulin in term infants and assess the efficacy of such supplementation in enhancing gut maturation and prevention of later noncommunicable diseases such as allergy, autoimmune diseases and obesity. Copyright © 2013 Nestec Ltd., Vevey/S. Karger AG, Basel.

  3. Nasal insulin changes peripheral insulin sensitivity simultaneously with altered activity in homeostatic and reward-related human brain regions.

    Science.gov (United States)

    Heni, M; Kullmann, S; Ketterer, C; Guthoff, M; Linder, K; Wagner, R; Stingl, K T; Veit, R; Staiger, H; Häring, H-U; Preissl, H; Fritsche, A

    2012-06-01

    Impaired insulin sensitivity is a major factor leading to type 2 diabetes. Animal studies suggest that the brain is involved in the regulation of insulin sensitivity. We investigated whether insulin action in the human brain regulates peripheral insulin sensitivity and examined which brain areas are involved. Insulin and placebo were given intranasally. Plasma glucose, insulin and C-peptide were measured in 103 participants at 0, 30 and 60 min. A subgroup (n = 12) was also studied with functional MRI, and blood sampling at 0, 30 and 120 min. For each time-point, the HOMA of insulin resistance (HOMA-IR) was calculated as an inverse estimate of peripheral insulin sensitivity. Plasma insulin increased and subsequently decreased. This excursion was accompanied by slightly decreased plasma glucose, resulting in an initially increased HOMA-IR. At 1 h after insulin spray, the HOMA-IR subsequently decreased and remained lower up to 120 min. An increase in hypothalamic activity was observed, which correlated with the increased HOMA-IR at 30 min post-spray. Activity in the putamen, right insula and orbitofrontal cortex correlated with the decreased HOMA-IR at 120 min post-spray. Central insulin action in specific brain areas, including the hypothalamus, may time-dependently regulate peripheral insulin sensitivity. This introduces a potential novel mechanism for the regulation of peripheral insulin sensitivity and underlines the importance of cerebral insulin action for the whole organism.

  4. Methods for quantifying adipose tissue insulin resistance in overweight/obese humans.

    Science.gov (United States)

    Ter Horst, K W; van Galen, K A; Gilijamse, P W; Hartstra, A V; de Groot, P F; van der Valk, F M; Ackermans, M T; Nieuwdorp, M; Romijn, J A; Serlie, M J

    2017-08-01

    Insulin resistance of adipose tissue is an important feature of obesity-related metabolic disease. However, assessment of lipolysis in humans requires labor-intensive and expensive methods, and there is limited validation of simplified measurement methods. We aimed to validate simplified methods for the quantification of adipose tissue insulin resistance against the assessment of insulin sensitivity of lipolysis suppression during hyperinsulinemic-euglycemic clamp studies. We assessed the insulin-mediated suppression of lipolysis by tracer-dilution of [1,1,2,3,3- 2 H 5 ]glycerol during hyperinsulinemic-euglycemic clamp studies in 125 overweight or obese adults (85 men, 40 women; age 50±11 years; body mass index 38±7 kg m -2 ). Seven indices of adipose tissue insulin resistance were validated against the reference measurement method. Low-dose insulin infusion resulted in suppression of the glycerol rate of appearance ranging from 4% (most resistant) to 85% (most sensitive), indicating a good range of adipose tissue insulin sensitivity in the study population. The reference method correlated with (1) insulin-mediated suppression of plasma glycerol concentrations (r=0.960, PInsulin Resistance (Adipo-IR) index (fasting plasma insulin-NEFA product; r=-0.526, Pinsulin-glycerol product (r=-0.467, PInsulin Resistance Index (fasting plasma insulin-basal lipolysis product; r=0.460, PInsulin Sensitivity Check Index (QUICKI)-NEFA index (r=0.621, Pinsulin resistance (area under the curve ⩾0.801, Pinsulin sensitivity (that is, the antilipolytic action of insulin) can be reliably quantified in overweight and obese humans by simplified index methods. The sensitivity and specificity of the Adipo-IR index and the fasting plasma insulin-glycerol product, combined with their simplicity and acceptable agreement, suggest that these may be most useful in clinical practice.

  5. The anti-papillomavirus activity of human and bovine lactoferricin.

    Science.gov (United States)

    Mistry, Nitesh; Drobni, Peter; Näslund, Jonas; Sunkari, Vivekananda Gupta; Jenssen, Håvard; Evander, Magnus

    2007-09-01

    Human papillomavirus (HPV) cause common warts, laryngeal papilloma and genital condylomata and is necessary for the development of cervical cancer. We have previously found that lactoferrin has antiviral activity against HPV-16 and others have demonstrated that lactoferricin, an N-terminal fragment of lactoferrin, has inhibitory activities against several viruses. Two cell lines and two virus types, HPV-5 and HPV-16, were used to study if lactoferrin and lactoferricin could inhibit HPV pseudovirus (PsV) infection. We demonstrated that bovine lactoferrin (bLf) and human lactoferrin (hLf) were both potent inhibitors of HPV-5 and -16 PsV infections. Among the four lactoferricin derivatives we analyzed, a 15 amino acid peptide from bovine lactoferricin (bLfcin) 17-31 was the most potent inhibitor of both HPV-5 and HPV-16 PsV infection. Among the other derivatives, the human lactoferricin (hLfcin) 1-49 showed some antiviral activity against HPV PsV infection while bLfcin 17-42 inhibited only HPV-5 PsV infection in one of the cell lines. When we studied initial attachment of HPV-16, only bLfcin 17-42 and hLfcin 1-49 had an antiviral effect. This is the first time that lactoferricin was demonstrated to have an inhibitory effect on HPV infection and the antiviral activity differed depending on size, charge and structures of the lactoferricin.

  6. Effect of exercise on insulin action in human skeletal muscle

    DEFF Research Database (Denmark)

    Richter, Erik; Mikines, K J; Galbo, Henrik

    1989-01-01

    The effect of 1 h of dynamic one-legged exercise on insulin action in human muscle was studied in 6 healthy young men. Four hours after one-legged knee extensions, a three-step sequential euglycemic hyperinsulinemic clamp combined with arterial and bilateral femoral vein catheterization...... was performed. Increased insulin action on glucose uptake was found in the exercised compared with the rested thigh at mean plasma insulin concentrations of 23, 40, and 410 microU/ml. Furthermore, prior contractions directed glucose uptake toward glycogen synthesis and increased insulin effects on thigh O2...... consumption and at some insulin concentrations on potassium exchange. In contrast, no change in insulin effects on limb exchange of free fatty acids, glycerol, alanine or tyrosine were found after exercise. Glycogen concentration in rested vastus lateralis muscle did not increase measurably during the clamp...

  7. Celastrol Protects against Antimycin A-Induced Insulin Resistance in Human Skeletal Muscle Cells

    Directory of Open Access Journals (Sweden)

    Mohamad Hafizi Abu Bakar

    2015-05-01

    Full Text Available Mitochondrial dysfunction and inflammation are widely accepted as key hallmarks of obesity-induced skeletal muscle insulin resistance. The aim of the present study was to evaluate the functional roles of an anti-inflammatory compound, celastrol, in mitochondrial dysfunction and insulin resistance induced by antimycin A (AMA in human skeletal muscle cells. We found that celastrol treatment improved insulin-stimulated glucose uptake activity of AMA-treated cells, apparently via PI3K/Akt pathways, with significant enhancement of mitochondrial activities. Furthermore, celastrol prevented increased levels of cellular oxidative damage where the production of several pro-inflammatory cytokines in cultures cells was greatly reduced. Celastrol significantly increased protein phosphorylation of insulin signaling cascades with amplified expression of AMPK protein and attenuated NF-κB and PKC θ activation in human skeletal muscle treated with AMA. The improvement of insulin signaling pathways by celastrol was also accompanied by augmented GLUT4 protein expression. Taken together, these results suggest that celastrol may be advocated for use as a potential therapeutic molecule to protect against mitochondrial dysfunction-induced insulin resistance in human skeletal muscle cells.

  8. Generation of insulin-producing human mesenchymal stem cells using recombinant adeno-associated virus.

    Science.gov (United States)

    Kim, Jeong Hwan; Park, Si-Nae; Suh, Hwal

    2007-02-28

    The purpose of current experiment is the generation of insulin-producing human mesenchymal stem cells as therapeutic source for the cure of type 1 diabetes. Type 1 diabetes is generally caused by insulin deficiency accompanied by the destruction of islet beta-cells. In various trials for the treatment of type 1 diabetes, cell-based gene therapy using stem cells is considered as one of the most useful candidate for the treatment. In this experiment, human mesenchymal stem cells were transduced with AAV which is containing furin-cleavable human preproinsulin gene to generate insulin-producing cells as surrogate beta-cells for the type 1 diabetes therapy. In the rAAV production procedure, rAAV was generated by transfection of AD293 cells. Human mesenchymal stems cells were transduced using rAAV with a various multiplicity of infection. Transduction of recombinant AAV was also tested using beta-galactosidse expression. Cell viability was determined by using MTT assay to evaluate the toxicity of the transduction procedure. Expression and production of Insulin were tested using reverse transcriptase-polymerase chain reaction and immunocytochemistry. Secretion of human insulin and C-peptide from the cells was assayed using enzyme-linked immunosorbent assay. Production of insulin and C-peptide from the test group represented a higher increase compared to the control group. In this study, we examined generation of insulin-producing cells from mesenchymal stem cells by genetic engineering for diabetes therapy. This work might be valuable to the field of tissue engineering for diabetes treatment.

  9. Human exposure to bovine polyomavirus: a zoonosis

    Energy Technology Data Exchange (ETDEWEB)

    Parry, J V; Gardner, S D

    1986-01-01

    A competitive-type solid phase radioimmunoassay (RIA) was developed for the detection of antibody to bovine polyomavirus. Comparison of RIA and counter-immunoelectrophoresis (CIE) results on 273 cattle sera indicated that both techniques were detecting antibody of like specificity. Human sera from 256 blood donors, 219 people recently vaccinated against polio, rubella or rabies, 50 immunosuppressed patients and 472 people with various occupational exposure to cattle were tested for antibody to bovine polyomavirus, the foetal rhesus monkey kidney strain, (anti-FRKV) by RIA. Apart from one blood donor and one of 108 rabies vaccinees only those in close contact with cattle possessed anti-FRKV. Compared with 62 per cent seropositive in the natural hosts, cattle, 71 per cent of veterinary surgeons, 50 per cent of cattle farmers, 40 per cent of abattoir workers, 16 per cent of veterinary institute technical staff and 10 per cent of veterinary students were anti-FRKV positive. Our findings indicate that the theoretical hazard of FRKV infection from undetected contamination of current tissue culture derived vaccines may, in practice, be remote. Proposed wider use of primate kidney cells as substrates for new vaccines may increase this risk.

  10. Diversity and antimicrobial susceptibility profiling of staphylococci isolated from bovine mastitis cases and close human contacts.

    Science.gov (United States)

    Schmidt, T; Kock, M M; Ehlers, M M

    2015-09-01

    The objectives of this study were to examine the diversity of Staphylococcus spp. recovered from bovine intramammary infections and humans working in close contact with the animals and to evaluate the susceptibility of the staphylococcal isolates to different antimicrobials. A total of 3,387 milk samples and 79 human nasal swabs were collected from 13 sampling sites in the KwaZulu-Natal province of South Africa. In total, 146 Staph. aureus isolates and 102 coagulase-negative staphylococci (CNS) were recovered from clinical and subclinical milk samples. Staphylococcusaureus was isolated from 12 (15.2%) of the human nasal swabs and 95 representative CNS were recovered for further characterization. The CNS were identified using multiplex-PCR assays, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and tuf gene sequencing. Seven Staphylococcus spp. were identified among the CNS of bovine origin, with Staph.chromogenes (78.4%) predominating. The predominant CNS species recovered from the human nasal swabs was Staph.epidermidis (80%) followed by Staph.chromogenes (6.3%). The antimicrobial susceptibility of all staphylococcal isolates was evaluated using disk diffusion and was supplemented by screening for specific antimicrobial resistance genes. Ninety-eight (67.1%) Staph.aureus isolates of bovine origin were pansusceptible; 39 (26.7%) isolates were resistant to a single class, and 7 (4.8%) isolates were resistant to 2 classes of antimicrobials. Two Staph. aureus (1.4%) isolates were multidrug-resistant. Resistance to penicillin was common, with 28.8% of the bovine and 75% of the human Staph. aureus isolates exhibiting resistance. A similar observation was made with the CNS, where 37.3% of the bovine and 89.5% of the human isolates were resistant to penicillin. Multidrug-resistance was common among the human CNS, with 39% of the isolates exhibiting resistance to 3 or more classes of antimicrobials. The antimicrobial

  11. Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene

    International Nuclear Information System (INIS)

    Levy, J.R.; Olefsky, J.M.

    1988-01-01

    The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblasts insulin receptors. These cells bind and internalize insulin normally. Biochemically assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4 0 C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37 0 C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation

  12. The stability of human, bovine and avian tuberculin purified protein derivative (PPD).

    Science.gov (United States)

    Maes, Mailis; Giménez, José Francisco; D'Alessandro, Adriana; De Waard, Jacobus H

    2011-11-15

    Guidelines recommend storing tuberculin purified protein derivative (PPD) refrigerated. However, especially in developing countries, maintaining the product refrigerated under field conditions can be difficult, limiting its use. Here we determine the effect of prolonged exposure to high temperatures on the potency of human, bovine and avian tuberculin PPD. Human, bovine and avian tuberculin PPD were stored for several weeks exposed to temperatures ranging from 37º to 100ºC. The potency was evaluated in vivo, in sensitized or naturally infected animals. Most test situations didn't affect the biological activity of the tuberculin PPDs and only very long and extreme incubations (several days at 100 °C) compromised the potency. Tuberculin PPD is very stable and can be stored or transported for long periods without refrigeration. 

  13. A bovine aortic arch in humans

    Directory of Open Access Journals (Sweden)

    María Elena Arnáiz-García

    2014-05-01

    Full Text Available We describe a curious congenital variation of human aortic arch (AA branching pattern termed the “bovine aortic arch”. Rather than arising directly from the AA as a separate branch as occurs in the most common AA branching pattern, the left common carotid artery moves to the right and merges from the brachiocephalic trunk. It is the normal AA branching pattern presented in a number of animals (canines, felines or Macaque monkeys but it has nothing to do with anatomy of AA in ruminant animals, including cattle and buffalo. That is why it is one of the most widely misnomers used in medical literature whose origin is nowadays unknown.

  14. Brain Insulin Resistance at the Crossroads of Metabolic and Cognitive Disorders in Humans.

    Science.gov (United States)

    Kullmann, Stephanie; Heni, Martin; Hallschmid, Manfred; Fritsche, Andreas; Preissl, Hubert; Häring, Hans-Ulrich

    2016-10-01

    Ever since the brain was identified as an insulin-sensitive organ, evidence has rapidly accumulated that insulin action in the brain produces multiple behavioral and metabolic effects, influencing eating behavior, peripheral metabolism, and cognition. Disturbances in brain insulin action can be observed in obesity and type 2 diabetes (T2D), as well as in aging and dementia. Decreases in insulin sensitivity of central nervous pathways, i.e., brain insulin resistance, may therefore constitute a joint pathological feature of metabolic and cognitive dysfunctions. Modern neuroimaging methods have provided new means of probing brain insulin action, revealing the influence of insulin on both global and regional brain function. In this review, we highlight recent findings on brain insulin action in humans and its impact on metabolism and cognition. Furthermore, we elaborate on the most prominent factors associated with brain insulin resistance, i.e., obesity, T2D, genes, maternal metabolism, normal aging, inflammation, and dementia, and on their roles regarding causes and consequences of brain insulin resistance. We also describe the beneficial effects of enhanced brain insulin signaling on human eating behavior and cognition and discuss potential applications in the treatment of metabolic and cognitive disorders. Copyright © 2016 the American Physiological Society.

  15. Specificity and Effector Functions of Human RSV-Specific IgG from Bovine Milk.

    Directory of Open Access Journals (Sweden)

    Gerco den Hartog

    Full Text Available Respiratory syncytial virus (RSV infection is the second most important cause of death in the first year of life, and early RSV infections are associated with the development of asthma. Breastfeeding and serum IgG have been shown to protect against RSV infection. Yet, many infants depend on bovine milk-based nutrition, which at present lacks intact immunoglobulins.To investigate whether IgG purified from bovine milk (bIgG can modulate immune responses against human RSV.ELISAs were performed to analyse binding of bIgG to human respiratory pathogens. bIgG or hRSV was coated to plates to assess dose-dependent binding of bIgG to human Fcγ receptors (FcγR or bIgG-mediated binding of myeloid cells to hRSV respectively. S. Epidermidis and RSV were used to test bIgG-mediated binding and internalisation of pathogens by myeloid cells. Finally, the ability of bIgG to neutralise infection of HEp2 cells by hRSV was evaluated.bIgG recognised human RSV, influenza haemagglutinin and Haemophilus influenza. bIgG bound to FcγRII on neutrophils, monocytes and macrophages, but not to FcγRI and FcγRIII, and could bind simultaneously to hRSV and human FcγRII on neutrophils. In addition, human neutrophils and dendritic cells internalised pathogens that were opsonised with bIgG. Finally, bIgG could prevent infection of HEp2 cells by hRSV.The data presented here show that bIgG binds to hRSV and other human respiratory pathogens and induces effector functions through binding to human FcγRII on phagocytes. Thus bovine IgG may contribute to immune protection against RSV.

  16. Specificity and Effector Functions of Human RSV-Specific IgG from Bovine Milk.

    Science.gov (United States)

    den Hartog, Gerco; Jacobino, Shamir; Bont, Louis; Cox, Linda; Ulfman, Laurien H; Leusen, Jeanette H W; van Neerven, R J Joost

    2014-01-01

    Respiratory syncytial virus (RSV) infection is the second most important cause of death in the first year of life, and early RSV infections are associated with the development of asthma. Breastfeeding and serum IgG have been shown to protect against RSV infection. Yet, many infants depend on bovine milk-based nutrition, which at present lacks intact immunoglobulins. To investigate whether IgG purified from bovine milk (bIgG) can modulate immune responses against human RSV. ELISAs were performed to analyse binding of bIgG to human respiratory pathogens. bIgG or hRSV was coated to plates to assess dose-dependent binding of bIgG to human Fcγ receptors (FcγR) or bIgG-mediated binding of myeloid cells to hRSV respectively. S. Epidermidis and RSV were used to test bIgG-mediated binding and internalisation of pathogens by myeloid cells. Finally, the ability of bIgG to neutralise infection of HEp2 cells by hRSV was evaluated. bIgG recognised human RSV, influenza haemagglutinin and Haemophilus influenza. bIgG bound to FcγRII on neutrophils, monocytes and macrophages, but not to FcγRI and FcγRIII, and could bind simultaneously to hRSV and human FcγRII on neutrophils. In addition, human neutrophils and dendritic cells internalised pathogens that were opsonised with bIgG. Finally, bIgG could prevent infection of HEp2 cells by hRSV. The data presented here show that bIgG binds to hRSV and other human respiratory pathogens and induces effector functions through binding to human FcγRII on phagocytes. Thus bovine IgG may contribute to immune protection against RSV.

  17. Human bovine tuberculosis - remains in the differential.

    LENUS (Irish Health Repository)

    Bilal, Shaukat

    2010-11-01

    Mycobacterium bovis is a pathogen of cattle. The unpasteurized milk of affected cattle is a source of infection in humans. Despite the screening of cattle and the pasteurization of milk, M bovis has not been eradicated. A high index of clinical suspicion is needed in symptomatic patients with a history of possible exposure. At risk groups include animal workers, farmers, meat packers, vets and zoo keepers. Humans are usually infected by the aerosol route. We present two cases of human bovine tuberculosis. One was a presumptive case and the second was a confirmed case. Both responded well to antituberculous therapy. In the confirmed case, there was evidence of transmission to the partner living in the same house. Rifampicin prophylaxis was given to the exposed case. The M. bovis from the confirmed case was isoniazid resistant, in addition to having the well known resistance to pyrazinamide. Isoniazid resistance has been described before in those who are immunocompromised. We describe it in an immunocompetent patient.

  18. Effects of Insulin Detemir and NPH Insulin on Body Weight and Appetite-Regulating Brain Regions in Human Type 1 Diabetes: A Randomized Controlled Trial

    NARCIS (Netherlands)

    van Golen, L.W.; Veltman, D.J.; IJzerman, R.G.; Deijen, J.B.; Heijboer, A.C.; Barkhof, F.; Drent, M.L.; Diamant, M.

    2014-01-01

    Studies in rodents have demonstrated that insulin in the central nervous system induces satiety. In humans, these effects are less well established. Insulin detemir is a basal insulin analog that causes less weight gain than other basal insulin formulations, including the current standard

  19. Effects of insulin detemir and NPH insulin on body weight and appetite-regulating brain regions in human type 1 diabetes: a randomized controlled trial

    NARCIS (Netherlands)

    van Golen, Larissa W.; Veltman, Dick J.; IJzerman, Richard G.; Deijen, Jan Berend; Heijboer, Annemieke C.; Barkhof, Frederik; Drent, Madeleine L.; Diamant, Michaela

    2014-01-01

    Studies in rodents have demonstrated that insulin in the central nervous system induces satiety. In humans, these effects are less well established. Insulin detemir is a basal insulin analog that causes less weight gain than other basal insulin formulations, including the current standard

  20. Fibrillization kinetics of insulin solution in an interfacial shearing flow

    Science.gov (United States)

    Balaraj, Vignesh; McBride, Samantha; Hirsa, Amir; Lopez, Juan

    2015-11-01

    Although the association of fibril plaques with neurodegenerative diseases like Alzheimer's and Parkinson's is well established, in-depth understanding of the roles played by various physical factors in seeding and growth of fibrils is far from well known. Of the numerous factors affecting this complex phenomenon, the effect of fluid flow and shear at interfaces is paramount as it is ubiquitous and the most varying factor in vivo. Many amyloidogenic proteins have been found to denature upon contact at hydrophobic interfaces due to the self-assembling nature of protein in its monomeric state. Here, fibrillization kinetics of insulin solution is studied in an interfacial shearing flow. The transient surface rheological response of the insulin solution to the flow and its effect on the bulk fibrillization process has been quantified. Minute differences in hydrophobic characteristics between two variants of insulin- Human recombinant and Bovine insulin are found to result in very different responses. Results presented will be in the form of fibrillization assays, images of fibril plaques formed, and changes in surface rheological properties of the insulin solution. The interfacial velocity field, measured from images (via Brewster Angle Microscopy), is compared with computations. Supported by NNX13AQ22G, National Aeronautics and Space Administration.

  1. Insulin resistance in human subjects having impaired glucose regulation

    International Nuclear Information System (INIS)

    Khan, S.H.; Khan, F.A.; Ijaz, A.

    2007-01-01

    To determine insulin resistance in human subjects having impaired glucose regulation (IGR) by Homeostasis Model Assessment for Insulin Resistance (HOMA-IR). A total of 100 subjects with impaired glucose regulation were selected for evaluation of metabolic syndrome as per the criteria of National Cholesterol Education Program, Adult Treatment Panel III (NCEP, ATP III), along with 47 healthy age and gender-matched controls. Physical examination to determine blood pressure and waist circumference was carried out and so was sampling for plasma glucose, serum triglycerides, HDL-cholesterol and insulin. Insulin resistance was calculated by the HOMA-IR. Finally, subjects with and without metabolic syndrome were compared with controls (n=47), using one-way ANOVA for studying insulin resistance between groups, with Tukey's post-hoc comparison. The frequency of finding metabolic syndrome in cases of IGR remained 47%. The insulin resistance demonstrated stepwise worsening from control population (mean=1.54, 95 % CI: 1.77 - 2.37) to subjects suffering from only IGR (mean=2.07, 95 % CI: 1.77- 2.37) to metabolic syndrome (mean=2.67, 95 %, CI: 2.34 - 3.00) (p < 0.001). Patients with impaired glucose regulation may have significant insulin resistance. It is, thus, recommended that a vigorous search be made to measure insulin resistance in all cases diagnosed to have impaired glucose regulation. (author)

  2. One Health approach to identify research needs in bovine and human babesioses: workshop report

    Directory of Open Access Journals (Sweden)

    McElwain Terry F

    2010-04-01

    Full Text Available Abstract Background Babesia are emerging health threats to humans and animals in the United States. A collaborative effort of multiple disciplines to attain optimal health for people, animals and our environment, otherwise known as the One Health concept, was taken during a research workshop held in April 2009 to identify gaps in scientific knowledge regarding babesioses. The impetus for this analysis was the increased risk for outbreaks of bovine babesiosis, also known as Texas cattle fever, associated with the re-infestation of the U.S. by cattle fever ticks. Results The involvement of wildlife in the ecology of cattle fever ticks jeopardizes the ability of state and federal agencies to keep the national herd free of Texas cattle fever. Similarly, there has been a progressive increase in the number of cases of human babesiosis over the past 25 years due to an increase in the white-tailed deer population. Human babesiosis due to cattle-associated Babesia divergens and Babesia divergens-like organisms have begun to appear in residents of the United States. Research needs for human and bovine babesioses were identified and are presented herein. Conclusions The translation of this research is expected to provide veterinary and public health systems with the tools to mitigate the impact of bovine and human babesioses. However, economic, political, and social commitments are urgently required, including increased national funding for animal and human Babesia research, to prevent the re-establishment of cattle fever ticks and the increasing problem of human babesiosis in the United States.

  3. Towards accurate structural characterization of metal centres in protein crystals: the structures of Ni and Cu T6 bovine insulin derivatives

    International Nuclear Information System (INIS)

    Frankaer, Christian Grundahl; Mossin, Susanne; Ståhl, Kenny; Harris, Pernille

    2013-01-01

    The level of structural detail around the metal sites in Ni 2+ and Cu 2+ T 6 insulin derivatives was significantly improved by using a combination of single-crystal X-ray crystallography and X-ray absorption spectroscopy. Photoreduction and subsequent radiation damage of the Cu 2+ sites in Cu insulin was followed by XANES spectroscopy. Using synchrotron radiation (SR), the crystal structures of T 6 bovine insulin complexed with Ni 2+ and Cu 2+ were solved to 1.50 and 1.45 Å resolution, respectively. The level of detail around the metal centres in these structures was highly limited, and the coordination of water in Cu site II of the copper insulin derivative was deteriorated as a consequence of radiation damage. To provide more detail, X-ray absorption spectroscopy (XAS) was used to improve the information level about metal coordination in each derivative. The nickel derivative contains hexacoordinated Ni 2+ with trigonal symmetry, whereas the copper derivative contains tetragonally distorted hexacoordinated Cu 2+ as a result of the Jahn–Teller effect, with a significantly longer coordination distance for one of the three water molecules in the coordination sphere. That the copper centre is of type II was further confirmed by electron paramagnetic resonance (EPR). The coordination distances were refined from EXAFS with standard deviations within 0.01 Å. The insulin derivative containing Cu 2+ is sensitive towards photoreduction when exposed to SR. During the reduction of Cu 2+ to Cu + , the coordination geometry of copper changes towards lower coordination numbers. Primary damage, i.e. photoreduction, was followed directly by XANES as a function of radiation dose, while secondary damage in the form of structural changes around the Cu atoms after exposure to different radiation doses was studied by crystallography using a laboratory diffractometer. Protection against photoreduction and subsequent radiation damage was carried out by solid embedment of Cu insulin

  4. A role for SPARC in the moderation of human insulin secretion.

    Directory of Open Access Journals (Sweden)

    Lorna W Harries

    Full Text Available AIMS/HYPOTHESIS: We have previously shown the implication of the multifunctional protein SPARC (Secreted protein acidic and rich in cysteine/osteonectin in insulin resistance but potential effects on beta-cell function have not been assessed. We therefore aimed to characterise the effect of SPARC on beta-cell function and features of diabetes. METHODS: We measured SPARC expression by qRT-PCR in human primary pancreatic islets, adipose tissue, liver and muscle. We then examined the relation of SPARC with glucose stimulated insulin secretion (GSIS in primary human islets and the effect of SPARC overexpression on GSIS in beta cell lines. RESULTS: SPARC was expressed at measurable levels in human islets, adipose tissue, liver and skeletal muscle, and demonstrated reduced expression in primary islets from subjects with diabetes compared with controls (p< = 0.05. SPARC levels were positively correlated with GSIS in islets from control donors (p< = 0.01. Overexpression of SPARC in cultured beta-cells resulted in a 2.4-fold increase in insulin secretion in high glucose conditions (p< = 0.01. CONCLUSIONS: Our data suggest that levels of SPARC are reduced in islets from donors with diabetes and that it has a role in insulin secretion, an effect which appears independent of SPARC's modulation of obesity-induced insulin resistance in adipose tissue.

  5. Heterogeneity of human plasma insulin: techniques for separating immunoreactive components and their determination by radioimmunoassay

    International Nuclear Information System (INIS)

    Souza, Iracelia Torres de Toledo e

    1977-01-01

    When human plasma is filtered on Sephadex G-SO fine, insulin immunoreactivity is recovered in two peaks: 'big insulin', the higher molecular weight component and 'little insulin', the lower molecular component, having elution volumes that correspond to those of porcine proinsulin 125 I and porcine insulin 125 I respectively. The presence of another form of immunoreactive insulin 'big big insulin' was detected from an insuloma suspect and its elution pattern corresponding to serum albumin. The eluates correspondent to 'big' and 'little' insulin as well as 'big big' component were assayed by radioimmunoassay using crystalline human insulin as a standard, porcine insulin 125 tracer and anti insulin serum. The antibody, raised in guinea-pigs, was sensitive and potent being adequate for the assay. The reactivity of insulin and proinsulin was tested against the antibody. The relative proportions of several components of total immunoreactive insulin in plasma were studied in basal conditions in five normal subjects and in the patient JSC with pancreatic insulin-secreting tumor as well as after glucose stimuli in all tolbutamide in JSC. (author)

  6. Heterogeneity of Bovine Peripheral Blood Monocytes

    Directory of Open Access Journals (Sweden)

    Jamal Hussen

    2017-12-01

    Full Text Available Peripheral blood monocytes of several species can be divided into different subpopulations with distinct phenotypic and functional properties. Herein, we aim at reviewing published work regarding the heterogeneity of the recently characterized bovine monocyte subsets. As the heterogeneity of human blood monocytes was widely studied and reviewed, this work focuses on comparing bovine monocyte subsets with their human counterparts regarding their phenotype, adhesion and migration properties, inflammatory and antimicrobial functions, and their ability to interact with neutrophilic granulocytes. In addition, the differentiation of monocyte subsets into functionally polarized macrophages is discussed. Regarding phenotype and distribution in blood, bovine monocyte subsets share similarities with their human counterparts. However, many functional differences exist between monocyte subsets from the two species. In contrast to their pro-inflammatory functions in human, bovine non-classical monocytes show the lowest phagocytosis and reactive oxygen species generation capacity, an absent ability to produce the pro-inflammatory cytokine IL-1β after inflammasome activation, and do not have a role in the early recruitment of neutrophils into inflamed tissues. Classical and intermediate monocytes of both species also differ in their response toward major monocyte-attracting chemokines (CCL2 and CCL5 and neutrophil degranulation products (DGP in vitro. Such differences between homologous monocyte subsets also extend to the development of monocyte-derived macrophages under the influence of chemokines like CCL5 and neutrophil DGP. Whereas the latter induce the differentiation of M1-polarized macrophages in human, bovine monocyte-derived macrophages develop a mixed M1/M2 macrophage phenotype. Although only a few bovine clinical trials analyzed the correlation between changes in monocyte composition and disease, they suggest that functional differences between

  7. Human and bovine viruses and bacteria at three Great Lakes beaches: Environmental variable associations and health risk

    Science.gov (United States)

    Corsi, Steven R.; Borchardt, Mark A.; Carvin, Rebecca B.; Burch, Tucker R; Spencer, Susan K.; Lutz, Michelle A.; McDermott, Colleen M.; Busse, Kimberly M.; Kleinheinz, Gregory; Feng, Xiaoping; Zhu, Jun

    2016-01-01

    Waterborne pathogens were measured at three beaches in Lake Michigan, environmental factors for predicting pathogen concentrations were identified, and the risk of swimmer infection and illness was estimated. Waterborne pathogens were detected in 96% of samples collected at three Lake Michigan beaches in summer, 2010. Samples were quantified for 22 pathogens in four microbial categories (human viruses, bovine viruses, protozoa, and pathogenic bacteria). All beaches had detections of human and bovine viruses and pathogenic bacteria indicating influence of multiple contamination sources at these beaches. Occurrence ranged from 40 to 87% for human viruses, 65–87% for pathogenic bacteria, and 13–35% for bovine viruses. Enterovirus, adenovirus A, Salmonella spp., Campylobacter jejuni, bovine polyomavirus, and bovine rotavirus A were present most frequently. Variables selected in multiple regression models used to explore environmental factors that influence pathogens included wave direction, cloud cover, currents, and water temperature. Quantitative Microbial Risk Assessment was done for C. jejuni, Salmonella spp., and enteroviruses to estimate risk of infection and illness. Median infection risks for one-time swimming events were approximately 3 × 10–5, 7 × 10–9, and 3 × 10–7 for C. jejuni, Salmonella spp., and enteroviruses, respectively. Results highlight the importance of investigating multiple pathogens within multiple categories to avoid underestimating the prevalence and risk of waterborne pathogens.

  8. Differentiation of insulin-producing cells from human neural progenitor cells.

    Directory of Open Access Journals (Sweden)

    Yuichi Hori

    2005-04-01

    Full Text Available BACKGROUND: Success in islet-transplantation-based therapies for type 1 diabetes, coupled with a worldwide shortage of transplant-ready islets, has motivated efforts to develop renewable sources of islet-replacement tissue. Islets and neurons share features, including common developmental programs, and in some species brain neurons are the principal source of systemic insulin. METHODS AND FINDINGS: Here we show that brain-derived human neural progenitor cells, exposed to a series of signals that regulate in vivo pancreatic islet development, form clusters of glucose-responsive insulin-producing cells (IPCs. During in vitro differentiation of neural progenitor cells with this novel method, genes encoding essential known in vivo regulators of pancreatic islet development were expressed. Following transplantation into immunocompromised mice, IPCs released insulin C-peptide upon glucose challenge, remained differentiated, and did not form detectable tumors. CONCLUSION: Production of IPCs solely through extracellular factor modulation in the absence of genetic manipulations may promote strategies to derive transplantable islet-replacement tissues from human neural progenitor cells and other types of multipotent human stem cells.

  9. Insulin-like growth factor I (IGF-I) and long R(3) IGF-I differently affect development and messenger ribonucleic acid abundance for IGF-binding proteins and type IIGF receptors in in vitro produced bovine embryos

    Czech Academy of Sciences Publication Activity Database

    Motlík, Jan; Prelle, K.; Stojkovic, M.; Ewald, D.; Arnold, G. J.; Welf, E.

    2001-01-01

    Roč. 142, - (2001), s. 1309-1316 ISSN 0013-7227 R&D Projects: GA ČR GV524/96/K162; GA AV ČR KSK5052113 Keywords : Insulin Like Growth Factor * bovine embryos Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.971, year: 2001

  10. Human milk insulin is related to maternal plasma insulin and BMI: but other components of human milk do not differ by BMI.

    Science.gov (United States)

    Young, B E; Patinkin, Z; Palmer, C; de la Houssaye, B; Barbour, L A; Hernandez, T; Friedman, J E; Krebs, N F

    2017-09-01

    The impact of maternal BMI and insulin sensitivity on bioactive components of human milk (HM) is not well understood. As the prevalence of obesity and diabetes rises, it is increasingly critical that we understand how maternal BMI and hormones associated with metabolic disease relate to concentrations of bioactive components in HM. This longitudinal cohort design followed 48 breastfeeding mothers through the first four months of lactation, collecting fasting morning HM samples at 2-weeks and 1, 2, 3 and 4-months, and fasting maternal blood at 2-weeks and 4-months. Insulin, glucose, adipokines leptin and adiponectin, appetite regulating hormone ghrelin, marker of oxidative stress 8OHdG and inflammatory cytokines (IL-6, IL-8, and TNF-a) were measured in HM and maternal plasma. A total of 26 normal weight (NW) (BMI=21.4±2.0 kg/m 2 ) and 22 overweight/obese (OW/Ob) (BMI=30.4±4.2 kg/m 2 ) were followed. Of all HM analytes measured, only insulin and leptin were different between groups - consistently higher in the OW/Ob group (leptin: P<0.001; insulin: P<0.03). HM insulin was 98% higher than maternal plasma insulin at 2-weeks and 32% higher at 4-months (P<0.001). Maternal fasting plasma insulin and HOMA-IR were positively related to HM insulin at 2-weeks (P<0.001, R 2 ⩾0.38, n=31), and 4-months (P⩽0.005, R 2 ⩾0.20, n=38). The concentrations of insulin in HM are higher than in maternal plasma and are related to maternal BMI and insulin sensitivity. With the exception of leptin, there were minimal other differences observed in HM composition across a wide range in maternal BMI.

  11. Expression of insulin signalling components in the sensory epithelium of the human saccule

    DEFF Research Database (Denmark)

    Degerman, Eva; Rauch, Uwe; Lindberg, Sven

    2013-01-01

    signalling components in the inner ear is sparce. Our immunohistochemistry approach has shown that the insulin receptor, insulin receptor substrate 1 (IRS1), protein kinase B (PKB) and insulin-sensitive glucose transporter (GLUT4) are expressed in the sensory epithelium of the human saccule, which also...

  12. Frequency of alpha- and beta-haemolysin in Staphylococcus aureus of bovine and human origin - A comparison between pheno- and genotype and variation in phenotypic expression

    DEFF Research Database (Denmark)

    Aarestrup, Frank Møller; Larsen, H.D.; Eriksen, N.H.R.

    1999-01-01

    The phenotypic expression of haemolysins and the presence of genes encoding alpha and beta-haemolysin were determined in 105 Sraphylococcus aureus isolates from bovine mastitis, 100 isolates from the nostrils of healthy humans, and 60 isolates from septicaemia in humans. Furthermore, the possible...... change in expression of haemolysins after subcultivation in human and bovine blood and milk was studied in selected isolates. alpha-haemolysin was expressed phenotypically in 39 (37%) of the bovine isolates, in 59 (59%) of the human carrier isolates, and in 40 (67%) of the isolates from septicaemia. beta......-haemolysin was expressed in 76 (72%) bovine, 11 (11%) carrier, and 8 (13%) septicaemia isolates. Significantly more bovine than human isolates expressed beta-haemolysin and significantly fewer expressed alpha-haemolysin. Genotypically, the gene encoding alpha-haemolysin was detected in all isolates. A significant...

  13. Purification, characterization, and biological activity of insulins from the spotted dogfish, Scyliorhinus canicula, and the hammerhead shark, Sphyrna lewini.

    Science.gov (United States)

    Anderson, W Gary; Ali, Mohamed F; Einarsdóttir, Ingibjörg E; Schäffer, Lauge; Hazon, Neil; Conlon, J Michael

    2002-03-01

    Insulin was purified from pancreatic extracts of two elasmobranch species belonging to different families in the order Carcharhiniformes, the European spotted dogfish, Scyliorhinus canicula (Scyliorhinidae), and the hammerhead shark, Sphyrna lewini (Carcharhinidae). The amino acid sequence of dogfish insulin was established as A-chain GIVDHCCRNT(10)CSLYDLEGYC(20)NQ and B-chain LPSQHLCGSH(10)LVETLYFVCG(20)QKGFYYVPKV(30). The primary structure of hammerhead shark insulin was similar to that of dogfish insulin with only 2 amino acid substitutions at A8 (R --> H) and B30 (V --> I). The elasmobranch insulins were markedly different from human insulin (17 amino acid substitutions) but all the residues in human insulin that are believed to be important in determining the receptor binding conformation (B6, B8, B11, B13, B23, B24, B25, A2, A3, and A19) have been conserved in the elasmobranch insulins with the exception of the conservative substitution Phe --> Tyr at B25. Consistent with this, dogfish and human insulin showed almost identical binding affinity to the recombinant solubilized human insulin receptor (K(D) values of 14.0 and 18.6 pM, respectively; relative potency 133%). Previous studies have shown that bovine insulin produces severe and sustained hypoglycemia in elasmobranchs but the effect is of slow onset. Bolus arterial injections of dogfish insulin (10 nmol x kg(-1)) into unanesthetized, fasting dogfish (n = 9) produced no changes in blood glucose, 3-hydroxybutyrate, and acetoacetate concentrations over a 4-h period. In a second series of experiments (n = 7), dogfish insulin (10 nmol x kg(-1)) produced a significant (P < 0.05) fall in blood glucose after 12 h that persisted for at least 48 h, but no change in ketone body concentrations. The data indicate that the metabolic actions of an endogenous elasmobranch insulin in an elasmobranch are similar to those previously described for mammalian insulin.

  14. Regulation of lipogenesis by glucocorticoids and insulin in human adipose tissue.

    Directory of Open Access Journals (Sweden)

    Laura L Gathercole

    Full Text Available Patients with glucocorticoid (GC excess, Cushing's syndrome, develop a classic phenotype characterized by central obesity and insulin resistance. GCs are known to increase the release of fatty acids from adipose, by stimulating lipolysis, however, the impact of GCs on the processes that regulate lipid accumulation has not been explored. Intracellular levels of active GC are dependent upon the activity of 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1 and we have hypothesized that 11β-HSD1 activity can regulate lipid homeostasis in human adipose tissue (Chub-S7 cell line and primary cultures of human subcutaneous (sc and omental (om adipocytes. Across adipocyte differentiation, lipogenesis increased whilst β-oxidation decreased. GC treatment decreased lipogenesis but did not alter rates of β-oxidation in Chub-S7 cells, whilst insulin increased lipogenesis in all adipocyte cell models. Low dose Dexamethasone pre-treatment (5 nM of Chub-S7 cells augmented the ability of insulin to stimulate lipogenesis and there was no evidence of adipose tissue insulin resistance in primary sc cells. Both cortisol and cortisone decreased lipogenesis; selective 11β-HSD1 inhibition completely abolished cortisone-mediated repression of lipogenesis. GCs have potent actions upon lipid homeostasis and these effects are dependent upon interactions with insulin. These in vitro data suggest that manipulation of GC availability through selective 11β-HSD1 inhibition modifies lipid homeostasis in human adipocytes.

  15. Leptin responses to bovine interferon- α and insulin in cattle

    African Journals Online (AJOL)

    Egyptian Journal of Biochemistry and Molecular Biology ... IFN- α injection produced a rapid increase in glucose and insulin levels but leptin levels did not show any alteration after the injection. ... Insulin levels rapidly increased in the blood and consequently a significant decrease in blood glucose level was recorded.

  16. A method for culturing human hair follicle cells.

    Science.gov (United States)

    Weterings, P J; Vermorken, A J; Bloemendal, H

    1981-01-01

    For the first time a method for culturing human hair follicle cells is described. The bovine eye lens capsule, a basement membrane-like structure, is used as the substrate for the cultures. In a culture medium supplemented with hydrocortisone and insulin about 70% of the original follicles will form growing colonies of diploid keratinocytes.

  17. Human blood-brain barrier insulin-like growth factor receptor

    International Nuclear Information System (INIS)

    Duffy, K.R.; Pardridge, W.M.; Rosenfeld, R.G.

    1988-01-01

    Insulin-like growth factor (IGF)-1 and IGF-2, may be important regulatory molecules in the CNS. Possible origins of IGFs in brain include either de novo synthesis or transport of circulating IGFs from blood into brain via receptor mediated transcytosis mechanisms at the brain capillary endothelial wall, ie, the blood-brain barrier (BBB). In the present studies, isolated human brain capillaries are used as an in vitro model system of the human BBB and the characteristics of IGF-1 or IGF-2 binding to this preparation were assessed. The total binding of IGF-2 at 37 degrees C exceeded 130% per mg protein and was threefold greater than the total binding for IGF-1. However, at 37 degrees C nonsaturable binding equaled total binding, suggesting that endocytosis is rate limiting at physiologic temperatures. Binding studies performed at 4 degrees C slowed endocytosis to a greater extent than membrane binding, and specific binding of either IGF-1 or IGF-2 was detectable. Scatchard plots for either peptide were linear and the molar dissociation constant of IGF-1 and IGF-2 binding was 2.1 +/- 0.4 and 1.1 +/- 0.1 nmol/L, respectively. Superphysiologic concentrations of porcine insulin inhibited the binding of both IGF-1 (ED50 = 2 micrograms/mL) and IGF-2 (ED50 = 0.5 microgram/mL). Affinity cross linking of 125 I-IGF-1, 125 I-IGF-2, and 125 I-insulin to isolated human brain capillaries was performed using disuccinimidylsuberate (DSS). These studies revealed a 141 kd binding site for both IGF-1 and IGF-2, and a 133 kd binding site for insulin

  18. Serological analysis of human IgG and IgE anti-insulin antibodies by solid-phase radioimmunoassays

    International Nuclear Information System (INIS)

    Hamilton, R.G.; Rendell, M.; Adkinson, N.F. Jr.

    1980-01-01

    A single solid-phase assay system which is useful for quantitative measurement of both IgG and IgE anti-insulin antibodies in human serum has been developed. Insulin-specific immunoglobulins are absorbed from human serum by excess quantities of insulin-agarose. After washes to remove unbound immunoglobulins, radioiodinated Staph A or rabbit anti-human IgE is added to detect bound IgG or IgE anbitodies, respectively

  19. Effect of adiponectin on bovine granulosa cell steroidogenesis, oocyte maturation and embryo development

    Directory of Open Access Journals (Sweden)

    Coyral-Castel Stéphanie

    2010-03-01

    Full Text Available Abstract Background Adiponectin is an adipokine, mainly produced by adipose tissue. It regulates several reproductive processes. The protein expression of the adiponectin system (adiponectin, its receptors, AdipoR1 and AdipoR2 and the APPL1 adaptor in bovine ovary and its role on ovarian cells and embryo, remain however to be determined. Methods Here, we identified the adiponectin system in bovine ovarian cells and embryo using RT-PCR, immunoblotting and immunohistochemistry. Furthermore, we investigated in vitro the effects of recombinant human adiponectin (10 micro g/mL on proliferation of granulosa cells (GC measured by [3H] thymidine incorporation, progesterone and estradiol secretions measured by radioimmunoassay in the culture medium of GC, nuclear oocyte maturation and early embryo development. Results We show that the mRNAs and proteins for the adiponectin system are present in bovine ovary (small and large follicles and corpus luteum and embryo. Adiponectin, AdipoR1 and AdipoR2 were more precisely localized in oocyte, GC and theca cells. Adiponectin increased IGF-1 10(-8 M-induced GC proliferation (P Conclusions In bovine species, adiponectin decreased insulin-induced steroidogenesis and increased IGF-1-induced proliferation of cultured GC through a potential involvement of ERK1/2 MAPK pathway, whereas it did not modify oocyte maturation and embryo development in vitro.

  20. Expression profiling of insulin action in human myotubes

    DEFF Research Database (Denmark)

    Hansen, L.; Gaster, Michael; Oakeley, E.J.

    2004-01-01

    Myotube cultures from patients with type 2 diabetes mellitus (T2DM) represent an experimental in vitro model of T2DM that offers a possibility to perform gene expression studies under standardized conditions. During a time-course of insulin stimulation (1 microM) at 5.5 mM glucose for 0 (no insulin......, metabolic enzymes, and finally cell cycle regulating genes. One-hundred-forty-four genes were differentially expressed in myotubes from donors with type 2 diabetes compared with control subjects, including HSP70, apolipoprotein D/E, tropomyosin, myosin, and actin previously reported from in vivo studies...... of diabetic skeletal muscle. We conclude, (i) that insulin induces a time-dependent inflammatory and pro-angiogenic transcriptional response in cultured human myotubes, (ii) that myotubes in vitro retain a gene expression pattern specific for type 2 diabetes and sharing five genes with that of type 2 diabetic...

  1. Tissue-specific expression of transfected human insulin genes in pluripotent clonal rat insulinoma lines induced during passage in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Madsen, O.D.; Andersen, L.C.; Michelsen, B.; Owerbach, D.; Larsson, L.I.; Lernmark, A.; Steiner, D.F. (Hagedorn Research Laboratory, Gentofte (Denmark))

    1988-09-01

    The pluripotent rat islet tumor cell line MSL-G2 expresses primarily glucagon or cholecystokinin and not insulin in vitro but changes phenotype completely after prolonged in vivo cultivation to yield small-sized hypoglycemic tumors composed almost entirely of insulin-producing beta cells. When a genomic DNA fragment containing the coding and upstream regulatory regions of the human insulin gene was stably transfected into MSL-G2 cells no measurable amounts of insulin or insulin mRNA were detected in vitro. However, successive transplantation of two transfected clones resulted in hypoglycemic tumors that efficiently coexpressed human and rat insulin as determined by human C-peptide-specific immunoreagents. These results demonstrate that cis-acting tissue-specific insulin gene enhancer elements are conserved between rat and human insulin genes. The authors propose that the in vivo differentiation of MSL-G2 cells and transfected subclones into insulin-producing cells reflects processes of natural beta-cell ontogeny leading to insulin gene expression.

  2. Tissue-specific expression of transfected human insulin genes in pluripotent clonal rat insulinoma lines induced during passage in vivo

    International Nuclear Information System (INIS)

    Madsen, O.D.; Andersen, L.C.; Michelsen, B.; Owerbach, D.; Larsson, L.I.; Lernmark, A.; Steiner, D.F.

    1988-01-01

    The pluripotent rat islet tumor cell line MSL-G2 expresses primarily glucagon or cholecystokinin and not insulin in vitro but changes phenotype completely after prolonged in vivo cultivation to yield small-sized hypoglycemic tumors composed almost entirely of insulin-producing beta cells. When a genomic DNA fragment containing the coding and upstream regulatory regions of the human insulin gene was stably transfected into MSL-G2 cells no measurable amounts of insulin or insulin mRNA were detected in vitro. However, successive transplantation of two transfected clones resulted in hypoglycemic tumors that efficiently coexpressed human and rat insulin as determined by human C-peptide-specific immunoreagents. These results demonstrate that cis-acting tissue-specific insulin gene enhancer elements are conserved between rat and human insulin genes. The authors propose that the in vivo differentiation of MSL-G2 cells and transfected subclones into insulin-producing cells reflects processes of natural beta-cell ontogeny leading to insulin gene expression

  3. Interleukin-1β inhibits insulin signaling and prevents insulin-stimulated system A amino acid transport in primary human trophoblasts.

    Science.gov (United States)

    Aye, Irving L M H; Jansson, Thomas; Powell, Theresa L

    2013-12-05

    Interleukin-1β (IL-1β) promotes insulin resistance in tissues such as liver and skeletal muscle; however the influence of IL-1β on placental insulin signaling is unknown. We recently reported increased IL-1β protein expression in placentas of obese mothers, which could contribute to insulin resistance. In this study, we tested the hypothesis that IL-1β inhibits insulin signaling and prevents insulin-stimulated amino acid transport in cultured primary human trophoblast (PHT) cells. Cultured trophoblasts isolated from term placentas were treated with physiological concentrations of IL-1β (10pg/ml) for 24h. IL-1β increased the phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser307 (inhibitory) and decreased total IRS-1 protein abundance but did not affect insulin receptor β expression. Furthermore, IL-1β inhibited insulin-stimulated phosphorylation of IRS-1 (Tyr612, activation site) and Akt (Thr308) and prevented insulin-stimulated increase in PI3K/p85 and Grb2 protein expression. IL-1β alone stimulated cRaf (Ser338), MEK (Ser221) and Erk1/2 (Thr202/Tyr204) phosphorylation. The inflammatory pathways nuclear factor kappa B and c-Jun N-terminal kinase, which are involved in insulin resistance, were also activated by IL-1β treatment. Moreover, IL-1β inhibited insulin-stimulated System A, but not System L amino acid uptake, indicating functional impairment of insulin signaling. In conclusion, IL-1β inhibited the insulin signaling pathway by inhibiting IRS-1 signaling and prevented insulin-stimulated System A transport, thereby promoting insulin resistance in cultured PHT cells. These findings indicate that conditions which lead to increased systemic maternal or placental IL-1β levels may attenuate the effects of maternal insulin on placental function and consequently fetal growth. Published by Elsevier Ireland Ltd.

  4. Quality control of insulin radioreceptor assay for human erythrocytes. Effect of ageing of mono-125I-Tyr-A14-insulin preparation

    International Nuclear Information System (INIS)

    Marttinen, A.; Pasternack, A.; Koivula, T.; Jokela, H.; Lehtinen, M.

    1984-01-01

    The quality control of insulin radioreceptor assay for human erythrocytes is based on the storage of erythrocyte preparations in Hepes buffer of pH 8.0, containing 10 g/l of albumin and 20 mmol/l of glucose. The change of erythrocytes into spherocytes and crenated cells reduces the apparent number of insulin receptors in a relatively constant way by less than 8% a week after 10 days of storage. At the same time the dissociation constants of the insulin-receptor complex increase rapidly. Thus the use of a preparation must be limited to controlling the determination of the insulin binding sites of erythrocytes, and not to the measurement of the affinities of the receptors. When mono- 125 I-Tyr-A14-insulin gets old, a slow decrease in the insulin binding sites can be measured, but the dissociation constants of the insulin receptor complex are not affected. (author)

  5. Regulation of autophagy in human skeletal muscle: effects of exercise, exercise training and insulin stimulation

    Science.gov (United States)

    Fritzen, Andreas M.; Madsen, Agnete B.; Kleinert, Maximilian; Treebak, Jonas T.; Lundsgaard, Anne‐Marie; Jensen, Thomas E.; Richter, Erik A.; Wojtaszewski, Jørgen; Kiens, Bente

    2016-01-01

    Key points Regulation of autophagy in human muscle in many aspects differs from the majority of previous reports based on studies in cell systems and rodent muscle.An acute bout of exercise and insulin stimulation reduce human muscle autophagosome content.An acute bout of exercise regulates autophagy by a local contraction‐induced mechanism.Exercise training increases the capacity for formation of autophagosomes in human muscle.AMPK activation during exercise seems insufficient to regulate autophagosome content in muscle, while mTORC1 signalling via ULK1 probably mediates the autophagy‐inhibiting effect of insulin. Abstract Studies in rodent muscle suggest that autophagy is regulated by acute exercise, exercise training and insulin stimulation. However, little is known about the regulation of autophagy in human skeletal muscle. Here we investigate the autophagic response to acute one‐legged exercise, one‐legged exercise training and subsequent insulin stimulation in exercised and non‐exercised human muscle. Acute one‐legged exercise decreased (Pexercise in human muscle. The decrease in LC3‐II/LC3‐I ratio did not correlate with activation of 5′AMP activated protein kinase (AMPK) trimer complexes in human muscle. Consistently, pharmacological AMPK activation with 5‐aminoimidazole‐4‐carboxamide riboside (AICAR) in mouse muscle did not affect the LC3‐II/LC3‐I ratio. Four hours after exercise, insulin further reduced (Pexercised and non‐exercised leg in humans. This coincided with increased Ser‐757 phosphorylation of Unc51 like kinase 1 (ULK1), which is suggested as a mammalian target of rapamycin complex 1 (mTORC1) target. Accordingly, inhibition of mTOR signalling in mouse muscle prevented the ability of insulin to reduce the LC3‐II/LC3‐I ratio. In response to 3 weeks of one‐legged exercise training, the LC3‐II/LC3‐I ratio decreased (Pexercise and insulin stimulation reduce muscle autophagosome content, while exercise

  6. Structure, antihyperglycemic activity and cellular actions of a novel diglycated human insulin

    DEFF Research Database (Denmark)

    O'Harte, F P; Boyd, A C; McKillop, A M

    2000-01-01

    Human insulin was glycated under hyperglycemic reducing conditions and a novel diglycated form (M(r) 6135.1 Da) was purified by RP-HPLC. Endoproteinase Glu-C digestion combined with mass spectrometry and automated Edman degradation localized glycation to Gly(1) and Phe(1) of the insulin A- and B-...

  7. Short-acting insulin analogues versus regular human insulin for adults with type 1 diabetes mellitus.

    Science.gov (United States)

    Fullerton, Birgit; Siebenhofer, Andrea; Jeitler, Klaus; Horvath, Karl; Semlitsch, Thomas; Berghold, Andrea; Plank, Johannes; Pieber, Thomas R; Gerlach, Ferdinand M

    2016-06-30

    Short-acting insulin analogue use for people with diabetes is still controversial, as reflected in many scientific debates. To assess the effects of short-acting insulin analogues versus regular human insulin in adults with type 1 diabetes. We carried out the electronic searches through Ovid simultaneously searching the following databases: Ovid MEDLINE(R), Ovid MEDLINE(R) In-Process & Other Non-Indexed Citations, Ovid MEDLINE(R) Daily and Ovid OLDMEDLINE(R) (1946 to 14 April 2015), EMBASE (1988 to 2015, week 15), the Cochrane Central Register of Controlled Trials (CENTRAL; March 2015), ClinicalTrials.gov and the European (EU) Clinical Trials register (both March 2015). We included all randomised controlled trials with an intervention duration of at least 24 weeks that compared short-acting insulin analogues with regular human insulins in the treatment of adults with type 1 diabetes who were not pregnant. Two review authors independently extracted data and assessed trials for risk of bias, and resolved differences by consensus. We graded overall study quality using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) instrument. We used random-effects models for the main analyses and presented the results as odds ratios (OR) with 95% confidence intervals (CI) for dichotomous outcomes. We identified nine trials that fulfilled the inclusion criteria including 2693 participants. The duration of interventions ranged from 24 to 52 weeks with a mean of about 37 weeks. The participants showed some diversity, mainly with regard to diabetes duration and inclusion/exclusion criteria. The majority of the trials were carried out in the 1990s and participants were recruited from Europe, North America, Africa and Asia. None of the trials was carried out in a blinded manner so that the risk of performance bias, especially for subjective outcomes such as hypoglycaemia, was present in all of the trials. Furthermore, several trials showed inconsistencies in

  8. Platelet-rich plasma can replace fetal bovine serum in human meniscus cell cultures

    NARCIS (Netherlands)

    Gonzales, V.K.; Mulder, E.L.W. de; Boer, T. den; Hannink, G.; Tienen, T.G. van; Heerde, W.L. van; Buma, P.

    2013-01-01

    Concerns over fetal bovine serum (FBS) limit the clinical application of cultured tissue-engineered constructs. Therefore, we investigated if platelet-rich plasma (PRP) can fully replace FBS for meniscus tissue engineering purposes. Human PRP and platelet-poor plasma (PPP) were isolated from three

  9. Lipid content and response to insulin are not invariably linked in human muscle cells

    OpenAIRE

    Aguer , Céline; Mercier , Jacques; Kitzmann , Magali

    2009-01-01

    Abstract In type 2 diabetes, a strong correlation between intramyocellular lipid accumulation and insulin resistance exists but whether intramyocellular accumulation is a cause or a consequence of insulin resistance is not clear. Lipid accumulation and response to insulin were evaluated in primary human myotubes derived from non-diabetic subjects and type 2 diabetic patients. Myotubes derived from type 2 diabetic patients had a defective response to insulin without showing a signif...

  10. Anti-Müllerian hormone inhibits activation and growth of bovine ovarian follicles in vitro and is localized to growing follicles.

    Science.gov (United States)

    Yang, M Y; Cushman, R A; Fortune, J E

    2017-05-01

    Does anti-Müllerian hormone (AMH) inhibit activation (initiation of growth) of primordial follicles and attenuate the growth of primary follicles in cattle, an excellent animal model for human ovarian follicular development? AMH inhibited activation of bovine primordial follicles and attenuated the growth of activated follicles in vitro. In mice null mutant for AMH, the pool of primordial follicles is depleted prematurely and AMH inhibits follicle activation in vitro. Results of studies with human ovarian tissue in vitro were inconsistent. Our previous work provided indirect evidence that AMH inhibits follicle activation in bovine ovaries. Pieces of fetal bovine ovarian cortex (2 pieces/culture well), obtained during mid or late pregnancy, were cultured in control medium or with graded doses of AMH for 2, 10 or 12 days. Effects of treatment on follicle activation and growth were determined by histological morphometry; follicles in every 20th histological section were staged (primordial or primary), counted, and measured. In addition, AMH was immunolocalized in bovine ovaries obtained at various times during pregnancy (n = 20 ovaries). Bovine fetal ovaries at mid or late gestation were obtained at a commercial abattoir. Pieces of ovarian cortex were cultured without or with AMH and fixed for histological morphometry on Day 0 and at the end of culture. Treatments were applied to duplicate cultures from each of two or three fetuses. In 12-day cultures, addition of AMH was delayed until the third day. Histological analysis provided information about the types, numbers and sizes of follicles in cortical pieces before and after treatments. Ovaries obtained during the second and third trimesters were assessed for the presence of AMH by immunohistochemistry. AMH (100-500 ng/ml) inhibited follicle activation in response to an activator (insulin) in ovarian cortical pieces from fetal ovaries in late gestation. Dose-dependent inhibitory effects on the diameters of primary

  11. Tissue-specific methylation of human insulin gene and PCR assay for monitoring beta cell death.

    Directory of Open Access Journals (Sweden)

    Mohamed I Husseiny

    Full Text Available The onset of metabolic dysregulation in type 1 diabetes (T1D occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy.

  12. Palmitate and insulin synergistically induce IL-6 expression in human monocytes

    Directory of Open Access Journals (Sweden)

    Lumpkin Charles K

    2010-11-01

    Full Text Available Abstract Background Insulin resistance is associated with a proinflammatory state that promotes the development of complications such as type 2 diabetes mellitus (T2DM and atherosclerosis. The metabolic stimuli that initiate and propagate proinflammatory cytokine production and the cellular origin of proinflammatory cytokines in insulin resistance have not been fully elucidated. Circulating proinflammatory monocytes show signs of enhanced inflammation in obese, insulin resistant subjects and are thus a potential source of proinflammatory cytokine production. The specific, circulating metabolic factors that might stimulate monocyte inflammation in insulin resistant subjects are poorly characterized. We have examined whether saturated nonesterified fatty acids (NEFA and insulin, which increase in concentration with developing insulin resistance, can trigger the production of interleukin (IL-6 and tumor necrosis factor (TNF-α in human monocytes. Methods Messenger RNA and protein levels of the proinflammatory cytokines IL-6 and TNF-α were measured by quantitative real-time PCR (qRT-PCR and Luminex bioassays. Student's t-test was used with a significance level of p Results Esterification of palmitate with coenzyme A (CoA was necessary, while β-oxidation and ceramide biosynthesis were not required, for the induction of IL-6 and TNF-α in THP-1 monocytes. Monocytes incubated with insulin and palmitate together produced more IL-6 mRNA and protein, and more TNF-α protein, compared to monocytes incubated with palmitate alone. Incubation of monocytes with insulin alone did not affect the production of IL-6 or TNF-α. Both PI3K-Akt and MEK/ERK signalling pathways are important for cytokine induction by palmitate. MEK/ERK signalling is necessary for synergistic induction of IL-6 by palmitate and insulin. Conclusions High levels of saturated NEFA, such as palmitate, when combined with hyperinsulinemia, may activate human monocytes to produce

  13. Evidence of the protein content of bovine and human dental pulps by the action of endodontic irrigation solutions through electrophoretic patterns

    Directory of Open Access Journals (Sweden)

    María E López

    2013-01-01

    Full Text Available Background: Sodium dodecyl sulfate polyacrylamide gel electrophoresis let to show the protein content of different tissues. Dental pulp contains connective tissue which is removed during the endodontic treatment. Many studies consider bovine rather than human pulp tissue because of its size. Aim: To evidence the protein content of bovine and human dental pulps and the action of endodontic irrigation solutions through electrophoretic patterns. Materials and Methods: Extracts of human and bovine dental pulps were prepared. Sodium hypochlorite, calcium hydroxide, chlorhexidine and ethylenediamine tetraacetic acid were used as irrigating solutions. Results: Bovine and human pulps have a small difference in two bands of proteins present between 74 kDa and 80 kDa. The denaturizing capacity of sodium hypochlorite and the washing action of calcium hydroxide and chlorhexidine were evidenced. Ethylenediamine tetraacetic acid solution was shown to contain proteins continuously during the endodontic root canal washing. Conclusions: Differences in pulp tissues and the action of irrigating solutions on their protein content would help on the understanding of the biological process of the endodontic treatment.

  14. The basal kinetic parameters of glycogen synthase in human myotube cultures are not affected by chronic high insulin exposure

    DEFF Research Database (Denmark)

    Gaster, M; Schrøder, H D; Handberg, A

    2001-01-01

    results show that chronic exposure of human myotubes to high insulin with or without high glucose did not affect the basal kinetic parameters but abolished the reactivity of GS to acute insulin stimulation. We suggest that insulin induced insulin resistance of GS is caused by a failure of acute insulin......There is no consensus regarding the results from in vivo and in vitro studies on the impact of chronic high insulin and/or high glucose exposure on acute insulin stimulation of glycogen synthase (GS) kinetic parameters in human skeletal muscle. The aim of this study was to evaluate the kinetic...... parameters of glycogen synthase activity in human myotube cultures at conditions of chronic high insulin combined or not with high glucose exposure, before and after a subsequent acute insulin stimulation. Acute insulin stimulation significantly increased the fractional activity (FV(0.1)) of GS, increased...

  15. Trends in the use and cost of human and analogue insulins in a Colombian population, 2011-2015.

    Science.gov (United States)

    Torres, D R; Portilla, A; Machado-Duque, M E; Machado-Alba, J E

    2017-12-01

    Diabetes mellitus is a common disease among the general population and imposes considerable costs on health care systems. Insulin is used to treat type 1 diabetes mellitus and as an adjuvant to oral agents in advanced stages of type 2 diabetes mellitus. The objective was to describe the trends in use and cost of human and analogue insulins for Colombian patients. Descriptive retrospective analysis of prescriptions of human and analogue insulins on a monthly basis for the period from July 1, 2011 to February 2, 2015. Information was collected for the database population of two insurance companies. Frequencies and proportions were calculated; estimated economic impact was expressed as net cost and cost per thousand inhabitants per day. During the observation period, there was continuous growth in use of insulin, mainly in analogue forms (34.0% growth). At the start of the study, 10.4% of subjects were using an analogue insulin; this figure was 62.6% at the end of the study. In 2012, the average cost per 1000 inhabitants/day was US$1.7 for analogue and US$0.8 for human insulins. At the end of the observation period these costs had risen to US$9.2 for analogue (441.1% increase) and fallen to US$0.5 for human insulin (58.3% decrease). There has been an increase in the unit cost and frequency of use of insulin analogues for anti-diabetic therapy in Colombian patients. Moreover, there is controversy over whether insulin analogues are a more cost-effective treatment than human insulins for the general diabetic population. Copyright © 2017 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved.

  16. Effects of green tea polyphenols, insulin-like growth factor I and glucose on developmental competence of bovine oocytes

    Directory of Open Access Journals (Sweden)

    Zhengguang Wang

    2012-12-01

    Full Text Available The present study examined the effects of green tea polyphenols (GTP, insulin-like growth factor-I (IGF-I and glucose on oocyte in vitro maturation, subsequent embryo development and blastocyst quality in bovine. Cumulus-oocyte complexes (COC were aspirated from the ovaries and cultured in synthetic oviduct fluid supplemented with MEM amino acids (SOFaa media supplemented with one of the following supplements: GTP (0, 10, 15 and 20 µM, IGF-I (0, 50, 100 and 150 ng/mL or glucose (0, 1.5, 5.6 and 20 mM for 24 h. The results showed that oocytes cultured in media supplemented with 15 µM GTP, 100 ng/mL IGF-I and 5.6 mM glucose, in separate experiments, have higher cleavage and blastocyst rates compared with oocytes cultured in media without or with other concentration of GTP, IGF-I and glucose. Then these three substances with the concentration above were added together into SOFaa media and constituted a modified medium (Modified SOFaa. The COC were cultured in control SOFaa media and modified SOFaa media, respectively. The results showed that modified SOFaa media increased the intracellular glutathione concentration of matured oocytes, blastocyst rates and total cell numbers and cell numbers of inner cell mass per blastocyst compared with the control. Supplementing of GTP, IGF-I and glucose synchronously to maturation media can increase the intracellular GSH concentration of oocytes after in vitro maturation, and improve the embryo development and blastocyst quality in bovine.

  17. Interaction of Coxiella burnetii Strains of Different Sources and Genotypes with Bovine and Human Monocyte-Derived Macrophages

    Directory of Open Access Journals (Sweden)

    Katharina Sobotta

    2018-01-01

    Full Text Available Most human Q fever infections originate from small ruminants. By contrast, highly prevalent shedding of Coxiella (C. burnetii by bovine milk rarely results in human disease. We hypothesized that primary bovine and human monocyte-derived macrophages (MDM represent a suitable in vitro model for the identification of strain-specific virulence properties at the cellular level. Twelve different C. burnetii strains were selected to represent different host species and multiple loci variable number of tandem repeat analysis (MLVA genotypes. Infection efficiency and replication of C. burnetii were monitored by cell culture re-titration and qPCR. Expression of immunoregulatory factors after MDM infection was measured by qRT-PCR and flow cytometry. Invasion, replication and MDM response differed between C. burnetii strains but not between MDMs of the two hosts. Strains isolated from ruminants were less well internalized than isolates from humans and rodents. Internalization of MLVA group I strains was lower compared to other genogroups. Replication efficacy of C. burnetii in MDM ranged from low (MLVA group III to high (MLVA group IV. Infected human and bovine MDM responded with a principal up-regulation of pro-inflammatory cytokines such as IL-1β, IL-12, and TNF-α. However, MLVA group IV strains induced a pronounced host response whereas infection with group I strains resulted in a milder response. C. burnetii infection marginally affected polarization of MDM. Only one C. burnetii strain of MLVA group IV caused a substantial up-regulation of activation markers (CD40, CD80 on the surface of bovine and human MDM. The study showed that replication of C. burnetii in MDM and the subsequent host cell response is genotype-specific rather than being determined by the host species pointing to a clear distinction in C. burnetii virulence between the genetic groups.

  18. Targeting the human lysozyme gene on bovine αs1- casein gene ...

    African Journals Online (AJOL)

    Targeting an exogenous gene into a favorable gene locus and for expression under endogenous regulators is an ideal method in mammary gland bioreactor research. For this purpose, a gene targeting vector was constructed to targeting the human lysozyme gene on bovine αs1-casein gene locus. In this case, the ...

  19. Geographical Analysis for Detecting High-Risk Areas for Bovine/Human Rabies Transmitted by the Common Hematophagous Bat in the Amazon Region, Brazil.

    Directory of Open Access Journals (Sweden)

    Fernanda A G de Andrade

    Full Text Available The common hematophagous bat, Desmodus rotundus, is one of the main wild reservoirs of rabies virus in several regions in Latin America. New production practices and changed land use have provided environmental features that have been very favorable for D. rotundus bat populations, making this species the main transmitter of rabies in the cycle that involves humans and herbivores. In the Amazon region, these features include a mosaic of environmental, social, and economic components, which together creates areas with different levels of risk for human and bovine infections, as presented in this work in the eastern Brazilian Amazon.We geo-referenced a total of 175 cases of rabies, of which 88% occurred in bovines and 12% in humans, respectively, and related these cases to a number of different geographical and biological variables. The spatial distribution was analyzed using the Kernel function, while the association with independent variables was assessed using a multi-criterion Analytical Hierarchy Process (AHP technique.The spatiotemporal analysis of the occurrence of rabies in bovines and humans found reduction in the number of cases in the eastern state of Pará, where no more cases were recorded in humans, whereas high infection rates were recorded in bovines in the northeastern part of the state, and low rates in the southeast. The areas of highest risk for bovine rabies are found in the proximity of rivers and highways. In the case of human rabies, the highest concentration of high-risk areas was found where the highway network coincides with high densities of rural and indigenous populations.The high-risk areas for human and bovine rabies are patchily distributed, and related to extensive deforested areas, large herds of cattle, and the presence of highways. These findings provide an important database for the generation of epidemiological models that could support the development of effective prevention measures and controls.

  20. Is Insulin Action in the Brain Relevant in Regulating Blood Glucose in Humans?

    Science.gov (United States)

    Dash, Satya; Xiao, Changting; Morgantini, Cecilia; Koulajian, Khajag; Lewis, Gary F

    2015-07-01

    In addition to its direct action on the liver to lower hepatic glucose production, insulin action in the central nervous system (CNS) also lowers hepatic glucose production in rodents after 4 hours. Although CNS insulin action (CNSIA) modulates hepatic glycogen synthesis in dogs, it has no net effect on hepatic glucose output over a 4-hour period. The role of CNSIA in regulating plasma glucose has recently been examined in humans and is the focus of this review. Intransal insulin (INI) administration increases CNS insulin concentration. Hence, INI can address whether CNSIA regulates plasma glucose concentration in humans. We and three other groups have sought to answer this question, with differing conclusions. Here we will review the critical aspects of each study, including its design, which may explain these discordant conclusions. The early glucose-lowering effect of INI is likely due to spillover of insulin into the systemic circulation. In the presence of simultaneous portal and CNS hyperinsulinemia, portal insulin action is dominant. INI administration does lower plasma glucose independent of peripheral insulin concentration (between ∼3 and 6 h after administration), suggesting that CNSIA may play a role in glucose homeostasis in the late postprandial period when its action is likely greatest and portal insulin concentration is at baseline. The potential physiological role and purpose of this pathway are discussed in this review. Because the effects of INI are attenuated in patients with type 2 diabetes and obesity, this is unlikely to be of therapeutic utility.

  1. Regulation of autophagy in human skeletal muscle: effects of exercise, exercise training and insulin stimulation

    DEFF Research Database (Denmark)

    Fritzen, Andreas Mæchel; Madsen, Agnete Louise Bjerregaard; Kleinert, Maximilian

    2016-01-01

    Studies in rodent muscle suggest that autophagy is regulated by acute exercise, exercise training and insulin stimulation. However, little is known about the regulation of autophagy in human skeletal muscle. Here we investigate the autophagic response to acute one-legged exercise, one-legged exer......Studies in rodent muscle suggest that autophagy is regulated by acute exercise, exercise training and insulin stimulation. However, little is known about the regulation of autophagy in human skeletal muscle. Here we investigate the autophagic response to acute one-legged exercise, one......-legged exercise training as well as in response to subsequent insulin stimulation in exercised and non-exercised human muscle. Acute one-legged exercise decreased (phuman muscle....... The decrease in LC3-II/LC3-I ratio did not correlate with activation of AMPK trimer complexes in human muscle. Consistently, pharmacological AMPK activation with AICAR in mouse muscle did not affect the LC3-II/LC3-I ratio. Four hours after exercise, insulin further reduced (p

  2. Differentiation of human-induced pluripotent stem cells into insulin-producing clusters.

    Science.gov (United States)

    Shaer, Anahita; Azarpira, Negar; Vahdati, Akbar; Karimi, Mohammad Hosein; Shariati, Mehrdad

    2015-02-01

    In diabetes mellitus type 1, beta cells are mostly destroyed; while in diabetes mellitus type 2, beta cells are reduced by 40% to 60%. We hope that soon, stem cells can be used in diabetes therapy via pancreatic beta cell replacement. Induced pluripotent stem cells are a kind of stem cell taken from an adult somatic cell by "stimulating" certain genes. These induced pluripotent stem cells may be a promising source of cell therapy. This study sought to produce isletlike clusters of insulin-producing cells taken from induced pluripotent stem cells. A human-induced pluripotent stem cell line was induced into isletlike clusters via a 4-step protocol, by adding insulin, transferrin, and selenium (ITS), N2, B27, fibroblast growth factor, and nicotinamide. During differentiation, expression of pancreatic β-cell genes was evaluated by reverse transcriptase-polymerase chain reaction; the morphologic changes of induced pluripotent stem cells toward isletlike clusters were observed by a light microscope. Dithizone staining was used to stain these isletlike clusters. Insulin produced by these clusters was evaluated by radio immunosorbent assay, and the secretion capacity was analyzed with a glucose challenge test. Differentiation was evaluated by analyzing the morphology, dithizone staining, real-time quantitative polymerase chain reaction, and immunocytochemistry. Gene expression of insulin, glucagon, PDX1, NGN3, PAX4, PAX6, NKX6.1, KIR6.2, and GLUT2 were documented by analyzing real-time quantitative polymerase chain reaction. Dithizone-stained cellular clusters were observed after 23 days. The isletlike clusters significantly produced insulin. The isletlike clusters could increase insulin secretion after a glucose challenge test. This work provides a model for studying the differentiation of human-induced pluripotent stem cells to insulin-producing cells.

  3. Quality control of insulin radioreceptor assay for human erythrocytes. Effect of ageing of mono-/sup 125/I-Tyr-A14-insulin preparation

    Energy Technology Data Exchange (ETDEWEB)

    Marttinen, A; Pasternack, A [Tampere Univ. (Finland). Dept. of Clinical Sciences; Koivula, T; Jokela, H; Lehtinen, M [Tampere Univ. Central Hospital (Finland). Dept. of Clinical Chemistry

    1984-09-01

    The quality control of insulin radioreceptor assay for human erythrocytes is based on the storage of erythrocyte preparations in Hepes buffer of pH 8.0, containing 10 g/l of albumin and 20 mmol/l of glucose. The change of erythrocytes into spherocytes and crenated cells reduces the apparent number of insulin receptors in a relatively constant way by less than 8% a week after 10 days of storage. At the same time the dissociation constants of the insulin-receptor complex increase rapidly. Thus the use of a preparation must be limited to controlling the determination of the insulin binding sites of erythrocytes, and not to the measurement of the affinities of the receptors. When mono-/sup 125/I-Tyr-A14-insulin gets old, a slow decrease in the insulin binding sites can be measured, but the dissociation constants of the insulin receptor complex are not affected.

  4. Glucose metabolism in pigs expressing human genes under an insulin promoter.

    Science.gov (United States)

    Wijkstrom, Martin; Bottino, Rita; Iwase, Hayoto; Hara, Hidetaka; Ekser, Burcin; van der Windt, Dirk; Long, Cassandra; Toledo, Frederico G S; Phelps, Carol J; Trucco, Massimo; Cooper, David K C; Ayares, David

    2015-01-01

    Xenotransplantation of porcine islets can reverse diabetes in non-human primates. The remaining hurdles for clinical application include safe and effective T-cell-directed immunosuppression, but protection against the innate immune system and coagulation dysfunction may be more difficult to achieve. Islet-targeted genetic manipulation of islet-source pigs represents a powerful tool to protect against graft loss. However, whether these genetic alterations would impair islet function is unknown. On a background of α1,3-galactosyltransferase gene-knockout (GTKO)/human (h)CD46, additional genes (hCD39, human tissue factor pathway inhibitor, porcine CTLA4-Ig) were inserted in different combinations under an insulin promoter to promote expression in islets (confirmed by immunofluorescence). Seven pigs were tested for baseline and glucose/arginine-challenged levels of glucose, insulin, C-peptide, and glucagon. This preliminary study did not show definite evidence of β-cell deficiencies, even when three transgenes were expressed under the insulin promoter. Of seven animals, all were normoglycemic at fasting, and five of seven had normal glucose disposal rates after challenge. All animals exhibited insulin, C-peptide, and glucagon responses to both glucose and arginine challenge; however, significant interindividual variation was observed. Multiple islet-targeted transgenic expression was not associated with an overtly detrimental effect on islet function, suggesting that complex genetic constructs designed for islet protection warrants further testing in islet xenotransplantation models. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Comparison of subcutaneous soluble human insulin and insulin analogues (AspB9, GluB27; AspB10; AspB28) on meal-related plasma glucose excursions in type I diabetic subjects.

    Science.gov (United States)

    Kang, S; Creagh, F M; Peters, J R; Brange, J; Vølund, A; Owens, D R

    1991-07-01

    To compare postprandial glucose excursions and plasma free insulin-analogue levels after subcutaneous injection of three novel human insulin analogues (AspB10; AspB9, GluB27; and AspB28) with those after injection of soluble human insulin (Actrapid HM U-100). Six male subjects with insulin-dependent diabetes, at least 1 wk apart and after an overnight fast and basal insulin infusion, received 72 nmol (approximately 12 U) s.c. of soluble human insulin 30 min before, or 72 nmol of each of the three analogues immediately before, a standard 500-kcal meal. Mean basal glucoses were similar on the 4 study days. Compared to human insulin (6.3 +/- 0.8 mM), mean +/- SE peak incremental glucose rises were similar after analogues AspB10 (5.4 +/- 0.8 mM) and AspB9, GluB27 (5.4 +/- 0.7 mM) and significantly lower after analogue AspB28 (3.6 +/- 1.2 mM, P less than 0.02). Relative to soluble human insulin (100% +/- SE21), incremental areas under the glucose curve between 0 and 240 min were 79% +/- 34 (AspB10, NS), 70% +/- 29 (AspB9, GluB27, NS), and 43% +/- 23 (AspB28, P less than 0.02). Basal plasma free insulin levels were similar on the 4 study days. Plasma free insulin-analogue levels rose rapidly to peak 30 min after injection at 308 +/- 44 pM (AspB10); 1231 +/- 190 pM (AspB9, GluB27) and 414 +/- 42 pM (AspB28) and were significantly higher than corresponding (i.e., 30 min postmeal) plasma free insulin levels of 157 +/- 15 pM (P less than 0.02 in each case). Plasma profiles of the insulin analogues were more physiological than that of human insulin after subcutaneous injection. All three analogues given immediately before the meal are at least as effective as soluble human insulin given 30 min earlier. These analogues are promising potential candidates for short-acting insulins of the future.

  6. PKCδ regulates hepatic insulin sensitivity and hepatosteatosis in mice and humans

    DEFF Research Database (Denmark)

    Bezy, Olivier; Tran, Thien T; Pihlajamäki, Jussi

    2011-01-01

    C57BL/6J and 129S6/Sv (B6 and 129) mice differ dramatically in their susceptibility to developing diabetes in response to diet- or genetically induced insulin resistance. A major locus contributing to this difference has been mapped to a region on mouse chromosome 14 that contains the gene encoding...... tolerance, and reduced hepatosteatosis with aging. Conversely, mice with liver-specific overexpression of PKCδ developed hepatic insulin resistance characterized by decreased insulin signaling, enhanced lipogenic gene expression, and hepatosteatosis. Therefore, changes in the expression and regulation...... of PKCδ between strains of mice and in obese humans play an important role in the genetic risk of hepatic insulin resistance, glucose intolerance, and hepatosteatosis; and thus PKCδ may be a potential target in the treatment of metabolic syndrome....

  7. Insulin resistance and the mitochondrial link. Lessons from cultured human myotubes

    DEFF Research Database (Denmark)

    Gaster, Michael

    2007-01-01

    In order to better understand the impact of reduced mitochondrial function for the development of insulin resistance and cellular metabolism, human myotubes were established from lean, obese, and T2D subjects and exposed to mitochondrial inhibitors, either affecting the electron transport chain...... lipid uptake. The metabolic phenotype during respiratory uncoupling resembled the above picture, except for an increase in glucose and palmitate oxidation. Antimycin A and oligomycin treatment induced insulin resistance at the level of glucose and palmitate uptake in all three study groups while......, at the level of glycogen synthesis, insulin resistance was only seen in lean myotubes. Primary insulin resistance in diabetic myotubes was significantly worsened at the level of glucose and lipid uptake. The present study is the first convincing data linking functional mitochondrial impairment per se...

  8. Characterization and regulation of the bovine stearoyl-CoA desaturase gene promoter

    International Nuclear Information System (INIS)

    Keating, Aileen F.; Kennelly, John J.; Zhao Fengqi

    2006-01-01

    The bovine stearoyl-CoA desaturase (Scd) gene plays an important role in the bovine mammary gland where substrates such as stearic and vaccenic acids are converted to oleic acid and conjugated linoleic acid (CLA), respectively. Up to 90% of the CLA in bovine milk is formed due to the action of this enzyme in the mammary gland. The areas of the bovine promoter of importance in regulating this key enzyme were examined and an area of 36 bp in length was identified as having a critical role in transcriptional activation and is designated the Scd transcriptional enhancer element (STE). Electrophoretic mobility shift assay detected three binding complexes on this area in Mac-T cell nuclear extracts. Treatment of cells with CLA caused a significant reduction in transcriptional activity, with this effect being mediated through the STE region. The bovine Scd gene promoter was up-regulated by insulin and down-regulated by oleic acid

  9. Electrophoretic demonstration of glycoproteins, lipoproteins, and phosphoproteins in human and bovine enamel

    DEFF Research Database (Denmark)

    Kirkeby, S; Moe, D; Bøg-Hansen, T C

    1990-01-01

    Enamel proteins from fully mineralized human molars and from bovine tooth germs were separated by electrophoresis. The gels were stained for detection of glycoproteins, lipoproteins, and phosphoproteins. Glycoproteins were shown by periodic acid-Schiff staining and lectin blotting. In mature human...... enamel a number of high molecular weight proteins could be demonstrated after ethylenediaminetetra-acetic acid demineralization and subsequent Triton X-100 extraction. These proteins are suggested to be lipoproteins. Phosphoproteins could only be visualized in enamel matrix from the tooth germs....

  10. Antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni

    Science.gov (United States)

    Bovine NK-lysins, which are functionally and structurally similar to human granulysin and porcine NK-lysin, are predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Although antimicrobial activity of bovine NK-lysin has been assessed for several bacterial pathogens, not all t...

  11. Studies on interaction of insulin and insulin receptor in rat liver cell membranes

    Energy Technology Data Exchange (ETDEWEB)

    Sakai, Y; Hara, H; Kawate, R; Kawasaki, T [Hiroshima Univ. (Japan). School of Medicine

    1975-07-01

    Rat liver was homogenized with a Polytron PT 20 ST and fractionated by differential centrifugation. Prepared plasma membranes (100 ..mu..g protein) were incubated with enzymatically iodinated /sup 125/I-insulin (0.3 ng, specific activity 107 ..mu..Ci/..mu..g) in 25 mM Tris-HCl buffer, pH 7.5, containing 0.9% NaCl and 1% bovine serum albumin. The 12,000xg- and 17,000xg-sediments obtained after subfractionation of liver homogenates showed almost equally high specific binding activity with /sup 125/I-insulin and less activity was detected in the 600 g-, 5,000 g- and 40,000 g- sediments and the 40,000 g- supernatant. Specific binding of insulin with the membrane fraction was time-, temperature- and ionic strength-dependent. The highest binding was obtained under conditions in which the membrane fraction was incubated with insulin for 24 hours at 4/sup 0/C in the buffer containing 1 M NaCl. Under these conditions, specific binding of /sup 125/I-insulin was 26.8% of the total radioactivity. The effect of native insulin on the binding of /sup 125/I-insulin with the membrane fraction was studied in the range of 0--6.4 x 10/sup 5/ ..mu..U/ml of unlabeled insulin and a distinct competitive displacement of /sup 125/I-insulin with native insulin was observed between 10 and 10/sup 4/ ..mu..U/ml. Kinetic studies by Scatchard plot analysis of the above results revealed heterogeneity in insulin receptors or receptor sites, one with a high affinity of 10/sup 9/ M/sup -1/ order and the other with a low affinity of 10/sup 8/ M/sup -1/ order. Both affinities were also affected by temperature and ionic strength.

  12. Effect of Human Myotubes-Derived Media on Glucose-Stimulated Insulin Secretion

    Directory of Open Access Journals (Sweden)

    Maria L. Mizgier

    2017-01-01

    Full Text Available Fasting to postprandial transition requires a tight adjustment of insulin secretion to its demand, so tissue (e.g., skeletal muscle glucose supply is assured while hypo-/hyperglycemia are prevented. High muscle glucose disposal after meals is pivotal for adapting to increased glycemia and might drive insulin secretion through muscle-released factors (e.g., myokines. We hypothesized that insulin influences myokine secretion and then increases glucose-stimulated insulin secretion (GSIS. In conditioned media from human myotubes incubated with/without insulin (100 nmol/L for 24 h, myokines were qualitatively and quantitatively characterized using an antibody-based array and ELISA-based technology, respectively. C57BL6/J mice islets and Wistar rat beta cells were incubated for 24 h with control and conditioned media from noninsulin- and insulin-treated myotubes prior to GSIS determination. Conditioned media from insulin-treated versus nontreated myotubes had higher RANTES but lower IL6, IL8, and MCP1 concentration. Qualitative analyses revealed that conditioned media from noninsulin- and insulin-treated myotubes expressed 32 and 23 out of 80 myokines, respectively. Islets incubated with conditioned media from noninsulin-treated myotubes had higher GSIS versus control islets (p<0.05. Meanwhile, conditioned media from insulin-treated myotubes did not influence GSIS. In beta cells, GSIS was similar across conditions. In conclusion, factors being present in noninsulin-stimulated muscle cell-derived media appear to influence GSIS in mice islets.

  13. ADAMTS13 expression in human chondrosarcoma cells induced by insulin

    Directory of Open Access Journals (Sweden)

    Rıdvan Fırat

    2014-06-01

    Full Text Available Objectives: A Disintegrin-like Metalloproteinase with Thrombospondin Motifs (ADAMTS proteins is a proteinase enzyme group that primarily located in the extracellular matrix (ECM. Insulin has been known to stimulate proteoglycan biosynthesis in chondrosarcoma chondrocytes and thereby the levels of ADAMTS proteins. The aim of this study is to evaluate the time-dependent effects of insulin on the ADAMTS13 expression in OUMS-27 human chondrosarcoma cell line to test the hypothesis that insulin diminishes ADAMTS13 expression because of its anabolic effects. Methods: To test this hypothesis OUMS-27 cells were cultured in Dulbecco’s modified Eagle’ medium (DMEM containing 10μg/mL insulin. The medium containing insulin was changed every other day up to 11th day. Cells were harvested at 1, 3, 7, and 11th days and protein and RNA isolations were performed at the proper times. The levels of RNA expression of ADAMTS13 was quantified by qRT-PCR using appropriate primers while protein levels was detected by Western blot technique using anti-ADAMTS13 antibody. Results: Although there was a decrease in both RNA and protein levels in insulin-applied groups compared to the control cells, it was not statistically significant. Conclusion: Under the light of our findings, it is suggested that insulin does not participate in regulation of ADAMTS13 in OUMS-27 chondrosarcoma cells. J Clin Exp Invest 2014; 5 (2: 226-232

  14. Roles of circulating WNT-signaling proteins and WNT-inhibitors in human adiposity, insulin resistance, insulin secretion, and inflammation.

    Science.gov (United States)

    Almario, R U; Karakas, S E

    2015-02-01

    Wingless-type MMTV integration site family member (WNT) signaling and WNT-inhibitors have been implicated in regulation of adipogenesis, insulin resistance, pancreatic function, and inflammation. Our goal was to determine serum proteins involved in WNT signaling (WNT5 and WISP2) and WNT inhibition (SFRP4 and SFRP5) as they relate to obesity, serum adipokines, insulin resistance, insulin secretion, and inflammation in humans. Study population comprised 57 insulin resistant women with polycystic ovary syndrome (PCOS) and 27 reference women. In a cross-sectional study, blood samples were obtained at fasting, during oral, and frequently sampled intravenous glucose tolerance tests. Serum WNT5, WISP2, and SFRP4 concentrations did not differ between PCOS vs. reference women. Serum WNT5 correlated inversely with weight both in PCOS and reference women, and correlated directly with insulin response during oral glucose tolerance test in PCOS women. Serum WISP2 correlated directly with fatty acid binding protein 4. Serum SFRP5 did not differ between obese (n=32) vs. nonobese (n=25) PCOS women, but reference women had lower SFRP5 (pPCOS groups). Serum SFRP5 correlated inversely with IL-1β, TNF-α, cholesterol, and apoprotein B. These findings demonstrated that WNT5 correlated inversely with adiposity and directly with insulin response, and the WNT-inhibitor SFRP5 may be anti-inflammatory. Better understanding of the role of WNT signaling in obesity, insulin resistance, insulin secretion, lipoprotein metabolism, and inflammation is important for prevention and treatment of metabolic syndrome, diabetes and cardiovascular disease. © Georg Thieme Verlag KG Stuttgart · New York.

  15. Bovine Tuberculosis, A Zoonotic Disease

    Directory of Open Access Journals (Sweden)

    Tarmudji

    2008-12-01

    Full Text Available Bovine tuberculosis is caused by the infection of Mycobacterium tuberculosis var. bovis (M. bovis. This species is one of Mycobacterium tuberculosis complex, can infect wide range of hosts: cattle and other domesticated animals, wild mammals and humans (zoonotic. M. bovis bacterium from infected hosts can be transmitted to other susceptible animals and humans through respiratory excretes and secretion materials. Humans can be infected with M. bovis by ingested M. bovis contaminated animal products, unpasteurised milk from tuberculosis cows or through respiratory route of contaminated aerosol. Bovine tuberculosis at the first stage does not show any clinical sign but as the disease progress in the next stage which may take several months or years, clinical signs may arise, suh as: fluctuative body temperature, anorexia, lost body weight, coughing, oedema of lymph nodes, increased respiratory frequencies. Pathological lesion of bovine tuberculosis is characterised by the formation of granulomas (tubercles, in which bacterial cells have been localised, most in lymph nodes and pulmonum, but can occur in other organs. The granulomas usually arise in small nodules or tubercles appear yellowish either caseus, caseo-calcareus or calcified. In Indonesia, bovine tuberculosis occurred in dairy cattle since 1905 through the imported dairy cows from Holland and Australian. It was unfortunate that until recently, there were not many research and surveilances of bovine tuberculosis conducted in this country, so the distribution of bovine tuberculosis is unknown. Early serological diagnosis can be done on live cattle by means of tuberculin tests under field conditions. Confirmation can be done by isolation and identification of excreted and secreted samples from the slaughter house. Antibiotic treatment and vaccination were uneffective, therefore the effective control of bovine tuberculosis is suggested by tuberculin tests and by slaughtering the selected

  16. Insulin increases phosphorylation of mitochondrial proteins in human skeletal muscle in vivo

    DEFF Research Database (Denmark)

    Zhao, Xiaolu; Bak, Steffen; Pedersen, Andreas James Thestrup

    2014-01-01

    , we investigated the effect of insulin on the phosphorylation of mitochondrial proteins in human skeletal muscle in vivo. Using a combination of TiO2 phosphopeptide-enrichment, HILIC fractionation, and LC−MS/MS, we compared the phosphoproteomes of isolated mitochondria from skeletal muscle samples...... obtained from healthy individuals before and after 4 h of insulin infusion. In total, we identified 207 phosphorylation sites in 95 mitochondrial proteins. Of these phosphorylation sites, 45% were identified in both basal and insulin-stimulated samples. Insulin caused a 2-fold increase in the number...... of different mitochondrial phosphopeptides (87 ± 7 vs 40 ± 7, p = 0.015) and phosphoproteins (46 ± 2 vs 26 ± 3, p = 0.005) identified in each mitochondrial preparation. Almost half of the mitochondrial phosphorylation sites (n = 94) were exclusively identified in the insulin-stimulated state and included...

  17. Effect of alcohol on insulin secretion and viability of human pancreatic islets

    Directory of Open Access Journals (Sweden)

    Nikolić Dragan

    2017-01-01

    Full Text Available Introduction/Objective. There are controversial data in the literature on the topic of effects of alcohol on insulin secretion, apoptosis, and necrosis of the endocrine and exocrine pancreas. The goal of this research was to determine how alcohol affects the insulin secretion and viability of human adult pancreatic islets in vitro during a seven-day incubation. Methods. Human pancreatic tissue was digested with Collagenase XI, using a non-automated method. Cultures were incubated in Roswell Park Memorial Institute (RPMI medium containing alcohol (10 μl of alcohol in 100 ml of medium. Insulin stimulation index (SI and viability of the islets were determined on the first, third, and seventh day of cultivation. Results. Analysis of the viability of the islets showed that there wasn’t significant difference between the control and the test group. In the test group, viability of the cultures declined with the time of incubation. SI of the test group was higher compared to the control group, by 50% and 25% on the first and third day of cultivation, respectively. On the seventh day, insulin secretion was reduced by 25%. The difference was not statistically significant (p > 0.05. In the test group, significant decline in insulin secretion was found on the third and seventh day of incubation (p ≤ 0.05. Conclusion. Alcohol can increase or decrease insulin secretion of islets cultures, which may result in an inadequate response of pancreatic β-cells to blood glucose, leading to insulin resistance, and increased risk of developing type 2 diabetes. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 41002

  18. Disruption of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Integrity Contributes to Muscle Insulin Resistance in Mice and Humans.

    Science.gov (United States)

    Tubbs, Emily; Chanon, Stéphanie; Robert, Maud; Bendridi, Nadia; Bidaux, Gabriel; Chauvin, Marie-Agnès; Ji-Cao, Jingwei; Durand, Christine; Gauvrit-Ramette, Daphné; Vidal, Hubert; Lefai, Etienne; Rieusset, Jennifer

    2018-04-01

    Modifications of the interactions between endoplasmic reticulum (ER) and mitochondria, defined as mitochondria-associated membranes (MAMs), were recently shown to be involved in the control of hepatic insulin action and glucose homeostasis, but with conflicting results. Whereas skeletal muscle is the primary site of insulin-mediated glucose uptake and the main target for alterations in insulin-resistant states, the relevance of MAM integrity in muscle insulin resistance is unknown. Deciphering the importance of MAMs on muscle insulin signaling could help to clarify this controversy. Here, we show in skeletal muscle of different mice models of obesity and type 2 diabetes (T2D) a marked disruption of ER-mitochondria interactions as an early event preceding mitochondrial dysfunction and insulin resistance. Furthermore, in human myotubes, palmitate-induced insulin resistance is associated with a reduction of structural and functional ER-mitochondria interactions. Importantly, experimental increase of ER-mitochondria contacts in human myotubes prevents palmitate-induced alterations of insulin signaling and action, whereas disruption of MAM integrity alters the action of the hormone. Lastly, we found an association between altered insulin signaling and ER-mitochondria interactions in human myotubes from obese subjects with or without T2D compared with healthy lean subjects. Collectively, our data reveal a new role of MAM integrity in insulin action of skeletal muscle and highlight MAM disruption as an essential subcellular alteration associated with muscle insulin resistance in mice and humans. Therefore, reduced ER-mitochondria coupling could be a common alteration of several insulin-sensitive tissues playing a key role in altered glucose homeostasis in the context of obesity and T2D. © 2018 by the American Diabetes Association.

  19. [Therapeutic qualities of a lente preparation of swine insulins manufactured by Pharmachem].

    Science.gov (United States)

    Andreev, D; Bozadzhieva, E; Pampulov, L; Penchev, I; Uzunova, B

    1977-01-01

    The therapeutic effect of swine insulin preparation Lente, "Pharmachim" production was studied in 50 diabetics. Its hypoglycemizing activitis compared with that of standardized insulin Lente--"Novo", extract of bovine pancreas. The Bulgarian preparation was found to be very similar to the preparation Lente-Novo in its pharma-codynamic properties and could successfully be used in the diabetes melitus treatment. Insulin Lente Pharmachim is presumed to have lower antigenicity as an extract of swine pancreas, which is actually its great advantage for the therapeutic practice.

  20. RFX6 Regulates Insulin Secretion by Modulating Ca2+ Homeostasis in Human β Cells

    Directory of Open Access Journals (Sweden)

    Vikash Chandra

    2014-12-01

    Full Text Available Development and function of pancreatic β cells involve the regulated activity of specific transcription factors. RFX6 is a transcription factor essential for mouse β cell differentiation that is mutated in monogenic forms of neonatal diabetes. However, the expression and functional roles of RFX6 in human β cells, especially in pathophysiological conditions, are poorly explored. We demonstrate the presence of RFX6 in adult human pancreatic endocrine cells. Using the recently developed human β cell line EndoC-βH2, we show that RFX6 regulates insulin gene transcription, insulin content, and secretion. Knockdown of RFX6 causes downregulation of Ca2+-channel genes resulting in the reduction in L-type Ca2+-channel activity that leads to suppression of depolarization-evoked insulin exocytosis. We also describe a previously unreported homozygous missense RFX6 mutation (p.V506G that is associated with neonatal diabetes, which lacks the capacity to activate the insulin promoter and to increase Ca2+-channel expression. Our data therefore provide insights for understanding certain forms of neonatal diabetes.

  1. Cultured bovine brain capillary endothelial cells (BBCEC) - a blood-brain barrier model for studying the binding and internalization of insulin and insulin-like growth factor 1

    International Nuclear Information System (INIS)

    Keller, B.T.; Borchardt, R.T.

    1987-01-01

    Cultured bovine brain capillary endothelial cells (BBCEC) have previously been reported by their laboratory as a working model for studying nutrient and drug transport and metabolism at the blood-brain barrier. In the present study, they have utilized this culture system to investigate the binding and internalization of [ 125 I]-labelled insulin (INS) and insulin-like growth factor 1(IGF-1) by BBCEC. After 2 hrs at 23 0 C, the specific binding of INS and IGF-1 was 1.6% and 13.6%, respectively. At 37 0 C, the maximum specific binding was 0.9% for INS and 5.8% for IGF-1. Using an acid-wash technique to assess peptide internalization, it was observed that, at 37 0 C, approximately 60% of the bound INS rapidly became resistant to acid treatment, a value which was constant over 2 hr. With IGF-1, a similar proportion of the bound material, 62%, became resistant by 30 min, but subsequently decreased to 45% by 2 hr. Scatchard analysis of competitive binding studies indicated the presence of two binding sites for each protein, having K/sub d/'s of 0.82 nM and 19.2 nM for INS and 0.39 nM and 3.66 nM for IGF-1. Little change in the amount of INS binding was observed over a four-day interval as the cultures became a confluent monolayer. The present report of binding and internalization of these proteins suggests that the BBCEC may utilize a receptor-mediated process to internalize and/or transport (transcytosis) INS and IGF-1 from the circulation

  2. LC-MS/MS analysis of permethylated free oligosaccharides and N-glycans derived from human, bovine, and goat milk samples.

    Science.gov (United States)

    Dong, Xue; Zhou, Shiyue; Mechref, Yehia

    2016-06-01

    Oligosaccharides in milk not only provide nutrition to the infants but also have significant immune biofunctions such as inhibition of pathogen binding to the host cell. The main component in milk oligosaccharides is free oligosaccharides. Since the proteins in milk are highly glycosylated, N-glycans in milk also play an import role. In this study, we investigated the permethylated free oligosaccharides and N-glycans extracted from bovine, goat, and human milks using LC-MS/MS. Quantitation profiles of free oligosaccharides and N-glycans were reported. The number of free oligosaccharides observed in bovine, goat, and human milk samples (without isomeric consideration) were 11, 8, and 11, respectively. Human milk had more complex free oligosaccharides structures than the other two milk samples. Totally 58, 21, and 43 N-glycan structures (without isomeric consideration) were associated with whey proteins extracted from bovine, goat, and human milk samples, respectively. Bovine milk free oligosaccharides and N-glycans from whey proteins were highly sialylated and to a lesser extend fucosylated. Goat and human milk free oligosaccharides and N-glycans from whey proteins were both highly fucosylated. Also, the isomeric glycans in milk samples were determined by porous graphitic carbon LC at elevated temperatures. For example, separation of human milk free oligosaccharide Gal-GlcNAc-(Fuc)-Gal-Glc and Gal-GlcNAc-Gal-Glc-Fuc isomers was achieved using porous graphitic carbon column. Permethylation of the glycan structures facilitated the interpretation of MS/MS. For example, internal cleavage and glycosidic bond cleavage are readily distinguished in the tandem mass spectra of permethylated glycans. This feature resulted in the identification of several isomers. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. 77 FR 29914 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

    Science.gov (United States)

    2012-05-21

    ... Spongiform Encephalopathy; Importation of Bovines and Bovine Products AGENCY: Animal and Plant Health... derived from bovines with regard to bovine spongiform encephalopathy. This action will allow interested... importation of live bovines and products derived from bovines with regard to bovine spongiform encephalopathy...

  4. Alternate Phosphorylation/O-GlcNAc Modification on Human Insulin IRSs: A Road towards Impaired Insulin Signaling in Alzheimer and Diabetes

    Directory of Open Access Journals (Sweden)

    Zainab Jahangir

    2014-01-01

    Full Text Available Impaired insulin signaling has been thought of as important step in both Alzheimer’s disease (AD and type 2 diabetes mellitus (T2DM. Posttranslational modifications (PTMs regulate functions and interaction of insulin with insulin receptors substrates (IRSs and activate insulin signaling downstream pathways via autophosphorylation on several tyrosine (TYR residues on IRSs. Two important insulin receptor substrates 1 and 2 are widely expressed in human, and alternative phosphorylation on their serine (Ser and threonine (Thr residues has been known to block the Tyr phosphorylation of IRSs, thus inhibiting insulin signaling and promoting insulin resistance. Like phosphorylation, O-glycosylation modification is important PTM and inhibits phosphorylation on same or neighboring Ser/Thr residues, often called Yin Yang sites. Both IRS-1 and IRS-2 have been shown to be O-glycosylated; however exact sites are not determined yet. In this study, by using neuronal network based prediction methods, we found more than 50 Ser/Thr residues that have potential to be O-glycosylated and may act as possible sites as well. Moreover, alternative phosphorylation and O-glycosylation on IRS-1 Ser-312, 984, 1037, and 1101 may act as possible therapeutic targets to minimize the risk of AD and T2DM.

  5. Validation of methods for measurement of insulin secretion in humans in vivo

    DEFF Research Database (Denmark)

    Kjems, L L; Christiansen, E; Vølund, A

    2000-01-01

    To detect and understand the changes in beta-cell function in the pathogenesis of type 2 diabetes, an accurate and precise estimation of prehepatic insulin secretion rate (ISR) is essential. There are two common methods to assess ISR, the deconvolution method (by Eaton and Polonsky)-considered th......To detect and understand the changes in beta-cell function in the pathogenesis of type 2 diabetes, an accurate and precise estimation of prehepatic insulin secretion rate (ISR) is essential. There are two common methods to assess ISR, the deconvolution method (by Eaton and Polonsky...... of these mathematical techniques for quantification of insulin secretion have been tested in dogs, but not in humans. In the present studies, we examined the validity of both methods to recover the known infusion rates of insulin and C-peptide mimicking ISR during an oral glucose tolerance test. ISR from both......, and a close agreement was found for the results of an oral glucose tolerance test. We also studied whether C-peptide kinetics are influenced by somatostatin infusion. The decay curves after bolus injection of exogenous biosynthetic human C-peptide, the kinetic parameters, and the metabolic clearance rate were...

  6. Short-and long-term glucocorticoid treatment enhances insulin signalling in human subcutaneous adipose tissue

    OpenAIRE

    Gathercole, LL; Morgan, SA; Bujalska, IJ; Stewart, PM; Tomlinson, JW

    2011-01-01

    Background: Endogenous or exogenous glucocorticoid (GC) excess (Cushing's syndrome) is characterized by increased adiposity and insulin resistance. Although GCs cause global insulin resistance in vivo, we have previously shown that GCs are able to augment insulin action in human adipose tissue, contrasting with their action in skeletal muscle. Cushing's syndrome develops following chronic GC exposure and, in addition, is a state of hyperinsulinemia. Objectives: We have therefore compared the ...

  7. Ancestral genomic duplication of the insulin gene in tilapia: An analysis of possible implications for clinical islet xenotransplantation using donor islets from transgenic tilapia expressing a humanized insulin gene.

    Science.gov (United States)

    Hrytsenko, Olga; Pohajdak, Bill; Wright, James R

    2016-07-03

    Tilapia, a teleost fish, have multiple large anatomically discrete islets which are easy to harvest, and when transplanted into diabetic murine recipients, provide normoglycemia and mammalian-like glucose tolerance profiles. Tilapia insulin differs structurally from human insulin which could preclude their use as islet donors for xenotransplantation. Therefore, we produced transgenic tilapia with islets expressing a humanized insulin gene. It is now known that fish genomes may possess an ancestral duplication and so tilapia may have a second insulin gene. Therefore, we cloned, sequenced, and characterized the tilapia insulin 2 transcript and found that its expression is negligible in islets, is not islet-specific, and would not likely need to be silenced in our transgenic fish.

  8. Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS.

    Science.gov (United States)

    Yang, Mei; Cao, Xueyan; Wu, Rina; Liu, Biao; Ye, Wenhui; Yue, Xiqing; Wu, Junrui

    2017-09-01

    Whey, an essential source of dietary nutrients, is widely used in dairy foods for infants. A total of 584 whey proteins in human and bovine colostrum and mature milk were identified and quantified by the isobaric tag for relative and absolute quantification (iTRAQ) proteomic method. The 424 differentially expressed whey proteins were identified and analyzed according to gene ontology (GO) annotation, Kyoto encyclopedia of genes and genomes (KEGG) pathway, and multivariate statistical analysis. Biological processes principally involved biological regulation and response to stimulus. Major cellular components were extracellular region part and extracellular space. The most prevalent molecular function was protein binding. Twenty immune-related proteins and 13 proteins related to enzyme regulatory activity were differentially expressed in human and bovine milk. Differentially expressed whey proteins participated in many KEGG pathways, including major complement and coagulation cascades and in phagosomes. Whey proteins show obvious differences in expression in human and bovine colostrum and mature milk, with consequences for biological function. The results here increase our understanding of different whey proteomes, which could provide useful information for the development and manufacture of dairy products and nutrient food for infants. The advanced iTRAQ proteomic approach was used to analyze differentially expressed whey proteins in human and bovine colostrum and mature milk.

  9. Bovine tuberculosis at the human-livestock-wildlife interface: Is it a public health problem in Tanzania? A review

    Directory of Open Access Journals (Sweden)

    Bugwesa Z. Katale

    2012-06-01

    Full Text Available Despite the apparent public health concern about Bovine tuberculosis (BTB in Tanzania, little has been done regarding the zoonotic importance of the disease and raising awareness of the community to prevent the disease. Bovine tuberculosis is a potential zoonotic disease that can infect a variety of hosts, including humans. The presence of multiple hosts including wild animals, inefficient diagnostic techniques, absence of defined national controls and eradication programs could impede the control of bovine TB. In Tanzania, the diagnosis of Mycobacterium bovis in animals is mostly carried out by tuberculin skin testing, meat inspection in abattoirs and only rarely using bacteriological techniques. The estimated prevalence of BTB in animals in Tanzania varies and ranges across regions from 0.2% to 13.3%, which is likely to be an underestimate if not confirmed by bacteriology or molecular techniques. Mycobacterium bovis has been detected and isolated from different animal species and has been recovered in 10% of apparently healthy wildebeest that did not show lesions at post-mortem. The transmission of the disease from animals to humans can occur directly through the aerosol route and indirectly by consumption of raw milk. This poses an emerging disease threat in the current era of HIV confection in Tanzania and elsewhere. Mycobacterium bovis is one of the causative agents of human extra pulmonary tuberculosis. In Tanzania there was a significant increase (116.6% of extrapulmonary cases reported between 1995 and 2009, suggesting the possibility of widespread M. bovis and Mycobacterium tuberculosis infection due to general rise of Human Immunodeficiency virus (HIV. This paper aims to review the potential health and economic impact of bovine tuberculosis and challenges to its control in order to safeguard human and animal population in Tanzania.

  10. Generation of high-yield insulin producing cells from human bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Jafarian, Arefeh; Taghikhani, Mohammad; Abroun, Saeid; Pourpak, Zahra; Allahverdi, Amir; Soleimani, Masoud

    2014-07-01

    Allogenic islet transplantation is a most efficient approach for treatment of diabetes mellitus. However, the scarcity of islets and long term need for an immunosuppressant limits its application. Recently, cell replacement therapies that generate of unlimited sources of β cells have been developed to overcome these limitations. In this study we have described a stage specific differentiation protocol for the generation of insulin producing islet-like clusters from human bone marrow mesenchymal stem cells (hBM-MSCs). This specific stepwise protocol induced differentiation of hMSCs into definitive endoderm, pancreatic endoderm and pancreatic endocrine cells that expressed of sox17, foxa2, pdx1, ngn3, nkx2.2, insulin, glucagon, somatostatin, pancreatic polypeptide, and glut2 transcripts respectively. In addition, immunocytochemical analysis confirmed protein expression of the above mentioned genes. Western blot analysis discriminated insulin from proinsulin in the final differentiated cells. In derived insulin producing cells (IPCs), secreted insulin and C-peptide was in a glucose dependent manner. We have developed a protocol that generates effective high-yield human IPCs from hBM-MSCs in vitro. These finding suggest that functional IPCs generated by this procedure can be used as a cell-based approach for insulin dependent diabetes mellitus.

  11. Anticancer activities of bovine and human lactoferricin-derived peptides.

    Science.gov (United States)

    Arias, Mauricio; Hilchie, Ashley L; Haney, Evan F; Bolscher, Jan G M; Hyndman, M Eric; Hancock, Robert E W; Vogel, Hans J

    2017-02-01

    Lactoferrin (LF) is a mammalian host defense glycoprotein with diverse biological activities. Peptides derived from the cationic region of LF possess cytotoxic activity against cancer cells in vitro and in vivo. Bovine lactoferricin (LFcinB), a peptide derived from bovine LF (bLF), exhibits broad-spectrum anticancer activity, while a similar peptide derived from human LF (hLF) is not as active. In this work, several peptides derived from the N-terminal regions of bLF and hLF were studied for their anticancer activities against leukemia and breast-cancer cells, as well as normal peripheral blood mononuclear cells. The cyclized LFcinB-CLICK peptide, which possesses a stable triazole linkage, showed improved anticancer activity, while short peptides hLF11 and bLF10 were not cytotoxic to cancer cells. Interestingly, hLF11 can act as a cell-penetrating peptide; when combined with the antimicrobial core sequence of LFcinB (RRWQWR) through either a Pro or Gly-Gly linker, toxicity to Jurkat cells increased. Together, our work extends the library of LF-derived peptides tested for anticancer activity, and identified new chimeric peptides with high cytotoxicity towards cancerous cells. Additionally, these results support the notion that short cell-penetrating peptides and antimicrobial peptides can be combined to create new adducts with increased potency.

  12. Sexing bovine pre-implantation embryos using the polymerase ...

    African Journals Online (AJOL)

    The paper aims to present a bovine model for human embryo sexing. Cows were super-ovulated, artificially inseminated and embryos were recovered 7 days later. Embryo biopsy was performed; DNA was extracted from blastomeres and amplified using bovine-specific and bovine-Y-chromosomespecific primers, followed ...

  13. Characterization of the growth of murine fibroblasts that express human insulin receptors. II. Interaction of insulin with other growth factors

    International Nuclear Information System (INIS)

    Randazzo, P.A.; Jarett, L.

    1990-01-01

    The effects of insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin on DNA synthesis were studied in murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental NIH 3T3 cells. In NIH 3T3/HIR cells, individual growth factors in serum-free medium stimulated DNA synthesis with the following relative efficacies: insulin greater than or equal to 10% fetal calf serum greater than PDGF greater than IGF-1 much greater than EGF. In comparison, the relative efficacies of these factors in stimulating DNA synthesis by NIH 3T3 cells were 10% fetal calf serum greater than PDGF greater than EGF much greater than IGF-1 = insulin. In NIH 3T3/HIR cells, EGF was synergistic with 1-10 ng/ml insulin but not with 100 ng/ml insulin or more. Synergy of PDGF or IGF-1 with insulin was not detected. In the parental NIH 3T3 cells, insulin and IGF-1 were found to be synergistic with EGF (1 ng/ml), PDGF (100 ng/ml), and PDGF plus EGF. In NIH 3T3/HIR cells, the lack of interaction of insulin with other growth factors was also observed when the percentage of cells synthesizing DNA was examined. Despite insulin's inducing only 60% of NIH 3T3/HIR cells to incorporate thymidine, addition of PDGF, EGF, or PDGF plus EGF had no further effect. In contrast, combinations of growth factors resulted in 95% of the parental NIH 3T3 cells synthesizing DNA. The independence of insulin-stimulated DNA synthesis from other mitogens in the NIH 3T3/HIR cells is atypical for progression factor-stimulated DNA synthesis and is thought to be partly the result of insulin receptor expression in an inappropriate context or quantity

  14. Impairments of mecA gene detection in bovine Staphylococcus spp.

    Directory of Open Access Journals (Sweden)

    Dayanne Araújo de Melo

    2014-09-01

    Full Text Available Staphylococcus aureus antimicrobial resistance, especially to beta-lactams, favors treatment failures and its persistence in herd environment. This work aimed to develop a more specific primer for mecA gene detection based on the comparison of the conserved regions from distinct host origins and also investigated the presence of homologue mecA LGA251 in bovine strains. A total of 43 Staphylococcus spp. were included in this study, comprising 38 bovine S. aureus, two human and three equine coagulase-negative staphylococci (CNS. Phenotypical methicillin-resistance detection was performed through oxacillin agar-screening and cefoxitin disk-diffusion test. None isolate tested positive for mecA LGA251 gene. For mecA gene PCR, new primers were designed based on the sequences of human S. aureus (HE681097 and bovine S. sciuri (AY820253 mecA. The new primers based on the S. aureus mecA sequence amplified fragments of human and equine CNS and the ones based on S. sciuri mecA sequence only yielded fragments for S. aureus bovine strains. Multiples alignments of mecA gene sequences from bovine, human and equine revealed punctual but significant differences in bovine strains that can lead to the mecA gene detection impairment. The observed divergences of mecA gene sequences are not a matter of animal or human origin, it is a specificity of bovine samples.

  15. Destabilization of Human Insulin Fibrils by Peptides of Fruit Bromelain Derived From Ananas comosus (Pineapple).

    Science.gov (United States)

    Das, Sromona; Bhattacharyya, Debasish

    2017-12-01

    Deposition of insulin aggregates in human body leads to dysfunctioning of several organs. Effectiveness of fruit bromelain from pineapple in prevention of insulin aggregate was investigated. Proteolyses of bromelain was done as par human digestive system and the pool of small peptides was separated from larger peptides and proteins. Under conditions of growth of insulin aggregates from its monomers, this pool of peptides restricted the reaction upto formation of oligomers of limited size. These peptides also destabilized preformed insulin aggregates to oligomers. These processes were followed fluorimetrically using Thioflavin T and 1-ANS, size-exclusion HPLC, dynamic light scattering, atomic force microscopy, and transmission electron microscopy. Sequences of insulin (A and B chains) and bromelain were aligned using Clustal W software to predict most probable sites of interactions. Synthetic tripeptides corresponding to the hydrophobic interactive sites of bromelain showed disaggregation of insulin suggesting specificity of interactions. The peptides GG and AAA serving as negative controls showed no potency in destabilization of aggregates. Disaggregation potency of the peptides was also observed when insulin was deposited on HepG2 liver cells where no formation of toxic oligomers occurred. Amyloidogenic des-octapeptide (B23-B30 of insulin) incapable of cell signaling showed cytotoxicity similar to insulin. This toxicity could be neutralized by bromelain derived peptides. FT-IR and far-UV circular dichroism analysis indicated that disaggregated insulin had structure distinctly different from that of its hexameric (native) or monomeric states. Based on the stoichiometry of interaction and irreversibility of disaggregation, the mechanism/s of the peptides and insulin interactions has been proposed. J. Cell. Biochem. 118: 4881-4896, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  16. Insulin aspart in diabetic pregnancy

    DEFF Research Database (Denmark)

    Mathiesen, Elisabeth R

    2008-01-01

    in insulin requirements during pregnancy necessitate short-acting insulins for postprandial control of hyperglycemia. The fast-acting insulin analogue insulin aspart has been tested in a large, randomized trial of pregnant women with Type 1 diabetes and offers benefits in control of postprandial...... hyperglycemia with a tendency towards fewer episodes of severe hypoglycemia compared with human insulin. Treatment with insulin aspart was associated with a tendency toward fewer fetal losses and preterm deliveries than treatment with human insulin. Insulin aspart could not be detected in the fetal circulation...... and no increase in insulin antibodies was found. Thus, the use of insulin aspart in pregnancy is regarded safe....

  17. Anticancer activities of bovine and human lactoferricin-derived peptides

    NARCIS (Netherlands)

    Arias, M.; Hilchie, A.L.; Haney, E.F.; Bolscher, J.G.M.; Hyndman, M.E.; Hancock, R.E.W.; Vogel, H.J.

    2017-01-01

    Lactoferrin (LF) is a mammalian host defense glycoprotein with diverse biological activities. Peptides derived from the cationic region of LF possess cytotoxic activity against cancer cells in vitro and in vivo. Bovine lactoferricin (LFcinB), a peptide derived from bovine LF (bLF), exhibits

  18. Proteomic Analysis of Bovine Nucleolus

    Institute of Scientific and Technical Information of China (English)

    Amrutlal K.Patel; Doug Olson; Suresh K. Tikoo

    2010-01-01

    Nucleolus is the most prominent subnuclear structure, which performs a wide variety of functions in the eu-karyotic cellular processes. In order to understand the structural and functional role of the nucleoli in bovine cells,we analyzed the proteomie composition of the bovine nueleoli. The nucleoli were isolated from Madin Darby bo-vine kidney cells and subjected to proteomie analysis by LC-MS/MS after fractionation by SDS-PAGE and strongcation exchange chromatography. Analysis of the data using the Mascot database search and the GPM databasesearch identified 311 proteins in the bovine nucleoli, which contained 22 proteins previously not identified in theproteomic analysis of human nucleoli. Analysis of the identified proteins using the GoMiner software suggestedthat the bovine nueleoli contained proteins involved in ribosomal biogenesis, cell cycle control, transcriptional,translational and post-translational regulation, transport, and structural organization.

  19. The effect of exercise on the absorption of inhaled human insulin in healthy volunteers

    DEFF Research Database (Denmark)

    Petersen, Astrid Heide; Kohler, Gerd; Korsatko, Stefan

    2008-01-01

    overall absorption. Aims To investigate the effect of moderate exercise on the absorption of inhaled insulin. Methods A single-centre, randomized, open-label, three-period cross-over trial was carried out in 12 nonsmoking healthy subjects. A dose of 3.5 mg inhaled human insulin was administered via...

  20. High prevalence of human anti-bovine IgG antibodies as the major cause of false positive reactions in two-site immunoassays based on monoclonal antibodies

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Koch, Claus; Jensen, Charlotte H

    2004-01-01

    were purified by protein G affinity chromatography from culture supernatant containing 10% (v/v) fetal calf serum (FCS). Human anti-animal IgG (bovine, mouse, horse, and swine) antibodies and human anti-bovine serum albumin antibodies were measured using an ELISA design, with direct bridging...... of the solid phase and biotinylated antigens. The false positive reactions were abolished by addition of 1% (v/v) bovine serum to the dilution buffer (DB). Human anti-bovine IgG antibodies (HABIA) were detected in 99 out of 104 sera from blood donors (50 females; 54 males). HABIA levels in male sera (n = 54......) were positively correlated to the false positive signals in the PP14 ELISA (r = 0.923; p detected in the donor sera, but levels and frequencies were lower compared to that of HABIA. Furthermore, HABIA were...

  1. Antigen-binding radioimmunoassays for human IgG antibodies to bovine ν-lactoglobulin

    International Nuclear Information System (INIS)

    Turner, M.W.; Paganelli, R.; Levinsky, R.J.; Williams, A.

    1983-01-01

    A double antibody antigen-binding assay for the detection of human IgG antibodies to the bovine milk allergen ν-lactoglobulin is described. The levels of such antibodies in patients with established cows' milk protein intolerance were significantly higher than the levels observed in a healthy control group (P<0.01). The assay showed excellent correlation with a solid phase antigen binding assay (rsub(s) = 0.8, P<0.001). (Auth.)

  2. A human model of dietary saturated fatty acid induced insulin resistance.

    Science.gov (United States)

    Koska, Juraj; Ozias, Marlies K; Deer, James; Kurtz, Julie; Salbe, Arline D; Harman, S Mitchell; Reaven, Peter D

    2016-11-01

    Increased consumption of high-fat diets is associated with the development of insulin resistance and type 2 diabetes. Current models to study the mechanisms of high-fat diet-induced IR in humans are limited by their long duration or low efficacy. In the present study we developed and characterized an acute dietary model of saturated fatty acid-enriched diet induced insulin resistance. High caloric diets enriched with saturated fatty acids (SFA) or carbohydrates (CARB) were evaluated in subjects with normal and impaired glucose tolerance (NGT or IGT). Both diets were compared to a standard eucaloric American Heart Association (AHA) control diet in a series of crossover studies. Whole body insulin resistance was estimated as steady state plasma glucose (SSPG) concentrations during the last 30min of a 3-h insulin suppression test. SSPG was increased after a 24-h SFA diet (by 83±74% vs. control, n=38) in the entire cohort, which was comprised of participants with NGT (92±82%, n=22) or IGT (65±55%, n=16) (all pinsulin resistance in both NGT and IGT subjects. Insulin resistance persisted overnight after the last SFA meal and was attenuated by one day of a healthy diet. This model offers opportunities for identifying early mechanisms and potential treatments of dietary saturated fat induced insulin resistance. Published by Elsevier Inc.

  3. The Investigation of ADAMTS16 in Insulin-Induced Human Chondrosarcoma Cells.

    Science.gov (United States)

    Cakmak, Ozlem; Comertoglu, Ismail; Firat, Ridvan; Erdemli, Haci Kemal; Kursunlu, S Fatih; Akyol, Sumeyya; Ugurcu, Veli; Altuntas, Aynur; Adam, Bahattin; Demircan, Kadir

    2015-08-01

    A disintegrin-like metalloproteinase with thrombospondin motifs (ADAMTS) is a group of proteins that have enzymatic activity secreted by cells to the outside extracellular matrix. Insulin induces proteoglycan biosynthesis in chondrosarcoma chondrocytes. The purpose of the present in vitro study is to assess the time course effects of insulin on ADAMTS16 expression in OUMS-27 (human chondrosarcoma) cell line to examine whether insulin regulates ADAMTS16 expression as well as proteoglycan biosynthesis with multifaceted properties or not. Chondrosarcoma cells were cultured in Dulbecco's modified Eagle's medium having either 10 μg/mL insulin or not. While the experiment was going on, the medium containing insulin had been changed every other day. Cells were harvested at 1st, 3rd, 7th, and 11th days; subsequently, RNA and proteins were isolated in every experimental group according to their time interval. RNA expression of ADAMTS was estimated by quantitative real-time polymerase chain reaction (qRT-PCR) by using primers. Immunoreactive protein levels were encountered by the western blot protein detection technique by using proper anti-ADAMTS16 antibodies. ADAMTS16 mRNA expression level of chondrosarcoma cells was found to be insignificantly decreased in chondrosarcoma cells induced by insulin detected by the qRT-PCR instrument. On the other hand, there was a gradual decrease in immune-reactant ADAMTS16 protein amount by the time course in insulin-treated cell groups when compared with control cells. It has been suggested that insulin might possibly regulate ADAMTS16 levels/activities in OUMS-27 chondrosarcoma cells taking a role in extracellular matrix turnover.

  4. Human skeletal muscle perilipin 2 and 3 expression varies with insulin sensitivity

    DEFF Research Database (Denmark)

    Vigelsø Hansen, Andreas; Prats Gavalda, Clara; Ploug, Thorkil

    2013-01-01

    Background: Impaired insulin sensitivity may partly arise from a dysregulated lipid metabolism in human skeletal muscle. This study investigates the expression levels of perilipin 2, 3, and 5, and four key lipases in human skeletal muscle from the subjects that exhibit a range from normal to very...

  5. Differential roles of MAPK-Erk1/2 and MAPK-p38 in insulin or insulin-like growth factor-I (IGF-I) signaling pathways for progesterone production in human ovarian cells.

    Science.gov (United States)

    Seto-Young, D; Avtanski, D; Varadinova, M; Park, A; Suwandhi, P; Leiser, A; Parikh, G; Poretsky, L

    2011-06-01

    Insulin and insulin like-growth factor-I (IGF-I) participate in the regulation of ovarian steroidogenesis. In insulin resistant states ovaries remain sensitive to insulin because insulin can activate alternative signaling pathways, such as phosphatidylinositol-3-kinase (PI-3 kinase) and mitogen-activated protein-kinase (MAPK) pathways, as well as insulin receptors and type 1 IGF receptors. We investigated the roles of MAPK-Erk1/2 and MAPK-p38 in insulin and IGF-I signaling pathways for progesterone production in human ovarian cells. Human ovarian cells were cultured in tissue culture medium in the presence of varying concentrations of insulin or IGF-I, with or without PD98059, a specific MAPK-Erk1/2 inhibitor, with or without SB203580, a specific MAPK-p38 inhibitor or with or without a specific PI-3-kinase inhibitor LY294002. Progesterone concentrations were measured using radioimmunoassay. PD98059 alone stimulated progesterone production in a dose-dependent manner by up to 65% (pprogesterone production by 13-18% (pprogesterone production by 17-20% (pprogesterone production by 20-30% (pprogesterone production by 40-60% (pprogesterone synthesis while SB203580 abolished insulin-induced progesterone production. Either PD98059 or SB203580 abolished IGF-I-induced progesterone production. Both MAPK-Erk1/2 and MAPK-p38 participate in IGF-I-induced signaling pathways for progesterone production, while insulin-induced progesterone production requires MAPK-p38, but not MAPK-Erk1/2. These studies provide further evidence for divergence of insulin and IGF-I signaling pathways for human ovarian cell steroidogenesis. © Georg Thieme Verlag KG Stuttgart · New York.

  6. The effect of feeding frequency on insulin and ghrelin responses in human subjects

    DEFF Research Database (Denmark)

    Solomon, Thomas; Chambers, Edward S; Jeukendrup, Asker E

    2008-01-01

    Recent work shows that increased meal frequency reduces ghrelin responses in sheep. Human research suggests there is an interaction between insulin and ghrelin. The effect of meal frequency on this interaction is unknown. Therefore, we investigated the effect of feeding frequency on insulin...... and ghrelin responses in human subjects. Five healthy male volunteers were recruited from the general population: age 24 (SEM 2)years, body mass 75.7 (SEM 3.2) kg and BMI 23.8 (SEM 0.8) kg/m(2). Volunteers underwent three 8-h feeding regimens: fasting (FAST); low-frequency(two) meal ingestion (LOFREQ......(MEAL)); high-frequency (twelve) meal ingestion (HIFREQ(MEAL)). Meals were equi-energetic within trials,consisting of 64% carbohydrate, 23% fat and 13% protein. Total energy intake was equal between feeding trials. Total area under the curve for serum insulin and plasma ghrelin responses did not differ between...

  7. Enzymatic amplification of a flow-injected thermometric enzyme-linked immunoassay for human insulin.

    Science.gov (United States)

    Mecklenburg, M; Lindbladh, C; Li, H; Mosbach, K; Danielsson, B

    1993-08-01

    A flow-injected thermometric enzyme linked immunoassay for human insulin which employs the lactate dehydrogenase/lactate oxidase (LDH/LOD) substrate recycling system for signal amplification is described. The system is composed of two columns, an immunosorbent column containing immobilized anti-insulin antibodies for sensing and a recycling column containing immobilized LDH/LOD/Catalase for detection. The effect of flow rates, conjugate concentrations, and chromatographic support material upon the sensitivity of the assay are investigated. The assay has a detection limit of 0.025 microgram/ml and a linear range from 0.05 to 2 micrograms/ml. This corresponds to a 10-fold increase in sensitivity over the unamplified system. A recombinant human insulin-proinsulin conjugate was also tested. The results show that enzymatic amplification can be employed to increase the sensitivity and reproducibility of flow injection assay-based biosensors. The implications of these results upon on-line analysis are discussed.

  8. Penetration of 38% hydrogen peroxide into the pulp chamber in bovine and human teeth submitted to office bleach technique.

    Science.gov (United States)

    Camargo, Samira Esteves Afonso; Valera, Marcia Carneiro; Camargo, Carlos Henrique Ribeiro; Gasparoto Mancini, Maria Nadir; Menezes, Marcia Maciel

    2007-09-01

    This study evaluated the pulp chamber penetration of peroxide bleaching agent in human and bovine teeth after office bleach technique. All the teeth were sectioned 3 mm apical of the cement-enamel junction and were divided into 2 groups, A (70 third human molars) and B (70 bovine lateral incisors), that were subdivided into A1 and B1 restored by using composite resin, A2 and B2 by using glass ionomer cement, and A3 and B3 by using resin-modified glass ionomer cement; A4, A5, B4, and B5 were not restored. Acetate buffer was placed in the pulp chamber, and the bleaching agent was applied for 40 minutes as follows: A1-A4 and B1-B4, 38% hydrogen peroxide exposure and A5 and B5, immersion into distilled water. The buffer solution was transferred to a glass tube in which leuco crystal violet and horseradish peroxidase were added, producing a blue solution. The optical density of the blue solution was determined by spectrophotometer and converted into microgram equivalents of hydrogen peroxide. Data were submitted to analysis of variance and Dunnett, Kruskal-Wallis, and Tukey tests (5%). A higher level of hydrogen peroxide penetrated into the pulp chamber in resin-modified glass ionomer cements in bovine (0.79 +/- 0.61 microg) and human (2.27 +/- 0.41 microg) groups. The bleaching agent penetration into the pulp chamber was higher in human teeth for any experimental situation. The penetration of the hydrogen peroxide depends on restorative materials, and under the conditions of this study human teeth are more susceptible to penetration of bleaching agent into the pulp chamber than bovine teeth.

  9. Differences in glucose-stimulated insulin secretion in vitro of islets from human, nonhuman primate, and porcine origin.

    Science.gov (United States)

    Mueller, Kate R; Balamurugan, A N; Cline, Gary W; Pongratz, Rebecca L; Hooper, Rebecca L; Weegman, Bradley P; Kitzmann, Jennifer P; Taylor, Michael J; Graham, Melanie L; Schuurman, Henk-Jan; Papas, Klearchos K

    2013-01-01

    Porcine islet xenotransplantation is considered a potential cell-based therapy for type 1 diabetes. It is currently being evaluated in diabetic nonhuman primates (NHP) to assess safety and efficacy of the islet product. However, due to a variety of distinct differences between the respective species, including the insulin secretory characteristics of islets, the suitability and predictive value of the preclinical model in the extrapolation to the clinical setting remain a critical issue. Islets isolated from human (n = 3), NHP (n = 2), adult pig (AP, n = 3), and juvenile pig (JP, n = 4) pancreata were perifused with medium at basal glucose (2.5 mm) followed by high glucose (16.7 mm) concentrations. The total glucose-stimulated insulin secretion (GSIS) was calculated from generated insulin secretion profiles. Nonhuman primate islets exhibited GSIS 3-fold higher than AP islets, while AP and JP islets exhibited GSIS 1/3 and 1/30 of human islets, respectively. The insulin content of NHP and AP islets was similar to that of human islets, whereas that of JP islets was 1/5 of human islets. Despite the fact that human, NHP, and AP islets contain similar amounts of insulin, the much higher GSIS for NHP islets than for AP and JP islets suggests the need for increased dosing of islets from JP and AP in pig-to-NHP transplantation. Porcine islet xenotransplantation to humans may require significantly higher dosing given the lower GSIS of AP islets compared to human islets. © 2013 John Wiley & Sons A/S.

  10. Effect of Technological Treatments on Human-Like Leptin Level in Bovine Milk for Human Consumption.

    Science.gov (United States)

    Magistrelli, Damiano; Rosi, Fabia

    2014-07-23

    In this experiment, raw milk and commercially available full-cream UHT milk, semi-skimmed UHT milk, skimmed UHT milk, full-cream pasteurized milk, semi-skimmed pasteurized milk and infant formulas for babies between 6 and 12 months of age were analyzed by RIA, with a method using an antibody directed against human leptin and human leptin as reference standard. Raw milk and full-cream UHT milk did not differ for human-like leptin. Leptin content of full-cream pasteurized milk was not different to that of full-cream UHT milk, but it was 14% lower ( p raw milk. Human-like leptin level of semi-skimmed UHT milk was not different to that of semi-skimmed pasteurized milk, but it was 30% lower ( p pasteurized milks. In skimmed UHT milk, leptin was 40% lower ( p milk. Leptin was correlated ( p milks. Results suggest that the heat treatment (pasteurization or UHT) is not a modifier of human-like leptin content of edible commercial bovine milks, whereas the skimming process significantly reduces milk leptin level.

  11. Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.

    Science.gov (United States)

    Blans, Kristine; Hansen, Maria S; Sørensen, Laila V; Hvam, Michael L; Howard, Kenneth A; Möller, Arne; Wiking, Lars; Larsen, Lotte B; Rasmussen, Jan T

    2017-01-01

    Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.

  12. Analyses of insulin-potentiating fragments of human growth hormone by computative simulation; essential unit for insulin-involved biological responses.

    Science.gov (United States)

    Ohkura, K; Hori, H

    2000-07-01

    We analyzed the structural features of insulin-potentiating fragments of human growth hormone by computative simulations. The peptides were designated from the N-terminus sequences of the hormone positions at 1-15 (hGH(1-15); H2N-Phe1-Pro2-Thr3-Ile4-Pro5-Leu6-Ser7-Arg8-L eu9-Phe10-Asp11-Asn12-Ala13-Met14-Leu15 -COOH), 6-13 (hGH(6-13)), 7-13 (hGH(7-13)) and 8-13 (hGH(8-13)), which enhanced insulin-producing hypoglycemia. In these peptide molecules, ionic bonds were predicted to form between 8th-arginyl residue and 11th-aspartic residue, and this intramolecular interaction caused the formation of a macrocyclic structure containing a tetrapeptide Arg8-Leu9-Phe10-Asp11. The peptide positions at 6-10 (hGH(6-10)), 9-13 (hGH(9-13)) and 10-13 (hGH(10-13)) did not lead to a macrocyclic formation in the molecules, and had no effect on the insulin action. Although beta-Ala13hGH(1-15), in which the 13th-alanine was replaced by a beta-alanyl residue, had no effect on insulin-producing hypoglycemia, the macrocyclic region (Arg8-Leu9-Phe10-Asp11) was observed by the computative simulation. An isothermal vibration analysis of both of beta-Ala13hGH(1-15) and hGH(1-15) peptide suggested that beta-Ala13hGH(1-15) is molecule was more flexible than hGH(1-15); C-terminal carboxyl group of Leu15 easily accessed to Arg8 and inhibited the ionic bond formation between Arg8 and Asp11 in beta-Ala13hGH(1-15). The peptide of hGH(8-13) dose-dependently enhanced the insulin-involved fatty acid synthesis in rat white adipocytes, and stabilized the C6-NBD-PC (1-acyl-2-[6-[(7-nitro-2,1,3benzoxadiazol-4-yl)amino]-caproyl]-sn- glycero-3-phosphatidylcholine) model membranes. In contrast, hGH(9-13) had no effect both on the fatty acid synthesis and the membrane stability. In the same culture conditions as the fatty acid synthesis assay, hGH(8-13) had no effect on the transcript levels of glucose transporter isoforms (GLUT 1, 4) and hexokinase isozymes (HK I, II) in rat white adipocytes. Judging from

  13. Acetabular reconstruction with human and bovine freeze-dried bone grafts and a reinforcement device

    Directory of Open Access Journals (Sweden)

    Ricardo Rosito

    2008-01-01

    Full Text Available BACKGROUND: This is a cohort trial (1997-2005 of 49 patients submitted to an acetabular component revision of a total hip arthroplasty, using impacted human and bovine freeze-dried cancellous bone grafts (H&FDBG and a reinforcement device. OBJECTIVE: To compare clinical/radiographic graft incorporation capability between cancellous bone grafts. PATIENTS/METHODS: There were two groups: I (n=26 receiving human grafts and II (n=25 receiving bovine grafts. The average follow-up times were 55 and 49 months, respectively. Clinical analysis was based on the Merle d'Aubigné and Postel score, and the radiographic analysis involved an established score based on Conn's et al. criteria for radiographic bone incorporation. RESULTS: No clinical/radiographic differences were found between the groups and both showed an overall rate of 88.5% and 76% of graft incorporation (p=0.424. CONCLUSION: The results presented here are comparable to those in the literature with the use of deep-FG. Therefore, cancellous bone grafts can be safely and adequately used in acetabular component revision in total hip arthroplasty.

  14. Effect of iodination site on binding of radiolabeled ligand by insulin antibodies and insulin autoantibodies

    International Nuclear Information System (INIS)

    Diaz, J.L.; Wilkin, T.J.

    1988-01-01

    Four human insulins and four porcine insulins, each monoiodinated to the same specific activity at one of the four tyrosine residues (A14, A19, B16, B26) and purified by reversed-phase liquid chromatography, were tested in a radiobinding assay against a panel of insulin-antibody (IA)-positive sera from 10 insulin-treated diabetics and insulin-autoantibody-positive (IAA) sera from 10 nondiabetics. Of the 10 IAA-positive sera, five were fully cross reactive with both insulin species, and five were specific for human insulin. The rank order of binding of sera with the four ligands from each species was random for IA (mean rank values of 1.9 for A14, 2.0 for A19, 2.5 for B16, and 3.6 for B26 from a possible ranking range of 1 to 4), but more consistent for non-human-insulin-specific IAA (mean rank values 1.3 for A14, 3.8 for A19, 1.7 for B16, and 3.2 for B26 for labeled human insulins; 1.2 for A14, 4.0 for A19, 1.8 for B16, and 3.0 for B26 for labeled porcine insulins). The rank order of binding was virtually uniform for human-insulin-specific IAA (mean values 1.2 for A14, 3.0 for A19, 1.8 for B16, and 4.0 for B26). The influence of iodination site on the binding of labeled insulin appears to be dependent on the proximity of the labeled tyrosine to the antibody binding site and the clonal diversity, or restriction, of insulin-binding antibodies in the test serum. When IA and IAA are measured, the implications of this study regarding the choice of assay ligand may be important

  15. Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

    International Nuclear Information System (INIS)

    Bayne, M.L.; Applebaum, J.; Chicchi, G.G.; Hayes, N.S.; Green, B.G.; Cascieri, M.A.

    1988-01-01

    Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe -1 , Val 1 , Asn 2 , Gln 3 , His 4 , Ser 8 , His 9 , Glu 12 , Tyr 15 , Leu 16 ]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has >1000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln 3 , Ala 4 ] IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr 15 , Leu 16 ] IGH-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. The peptide in which these four-point mutations are combined, [Gln 3 , Ala 4 , Tyr 15 ,Leu 16 ]IGF-I, has 600-fold reduced affinity for the serum binding proteins. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, These peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I

  16. Scavenger receptor-mediated recognition of maleyl bovine plasma albumin and the demaleylated protein in human monocyte macrophages

    International Nuclear Information System (INIS)

    Haberland, M.E.; Fogelman, A.M.

    1985-01-01

    Maleyl bovine plasma albumin competed on an equimolar basis with malondialdehyde low density lipoprotein (LDL) in suppressing the lysosomal hydrolysis of 125 I-labeled malondialdehyde LDL mediated by the scavenger receptor of human monocyte macrophages. Maleyl bovine plasma albumin, in which 94% of the amino groups were modified, exhibited an anodic mobility in agarose electrophoresis 1.7 times that of the native protein. Incubation of maleyl bovine plasma albumin at pH 3.5 regenerated the free amino groups and restored the protein to the same electrophoretic mobility as native albumin. Although ligands recognized by the scavenger receptor typically are anionic, the authors propose that addition of new negative charge achieved by maleylation, rather than directly forming the receptor binding site(s), induces conformational changes in albumin as a prerequisite to expression of the recognition domain(s). They conclude that the primary sequence of albumin, rather than addition of new negative charge, provides the recognition determinant(s) essential for interaction of maleyl bovine plasma albumin with the scavenger receptor

  17. Monoclonal antibody to the type I insulin-like growth factor (IGF-I) receptor blocks IGF-I receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-I and insulin in human skin fibroblasts

    International Nuclear Information System (INIS)

    Flier, J.S.; Usher, P.; Moses, A.C.

    1986-01-01

    Insulin and insulin-like growth factor type I (IGF-I) stimulate an overlapping spectrum of biological responses in human skin fibroblasts. Although insulin and IGF-I are known to stimulate the incorporation of [ 3 H]thymidine into DNA in these cells, the identify of the receptor(s) that mediates this effect has not been fully clarified. The mouse anti-human IGF-I receptor antibody αIR-3 binds with specificity to IGF-I but not to insulin receptors in human placental membranes; it also specifically inhibits the binding of 125 I-labeled IGF-I but not 125 I-labeled insulin to suspensions of human skin fibroblasts in a dose-dependent manner. αIR-3 competitively inhibits IGF-I-mediated stimulation of [ 3 H]thymidine incorporation into DNA. This inhibition is dependent on the concentration of αIR-3 and in the presence of a fixed antibody concentration can be partially overcome by high concentrations of IGF-I. In contrast, at concentrations of 3 H]thymidine incorporation is not inhibited by αIR-3. However, the incremental effects of higher concentrations (> 1 μg/ml) of insulin on [ 3 H]thymidine incorporation are inhibited by αIR-3. αIR-3 is a highly specific antagonist of IGF-I receptor-mediated mitogenesis in human skin fibroblasts. By using this antibody, it is shown directly that insulin can act through the IGF-I receptor to stimulate DNA synthesis but can also activate this effect through the insulin receptor itself

  18. Insulin resistance alters islet morphology in nondiabetic humans

    DEFF Research Database (Denmark)

    Mezza, Teresa; Muscogiuri, Giovanna; Sorice, Gian Pio

    2014-01-01

    Type 2 diabetes is characterized by poor glucose uptake in metabolic tissues and manifests when insulin secretion fails to cope with worsening insulin resistance. In addition to its effects on skeletal muscle, liver, and adipose tissue metabolism, it is evident that insulin resistance also affects...... pancreatic β-cells. To directly examine the alterations that occur in islet morphology as part of an adaptive mechanism to insulin resistance, we evaluated pancreas samples obtained during pancreatoduodenectomy from nondiabetic subjects who were insulin-resistant or insulin-sensitive. We also compared...... insulin sensitivity, insulin secretion, and incretin levels between the two groups. We report an increased islet size and an elevated number of β- and α-cells that resulted in an altered β-cell-to-α-cell area in the insulin- resistant group. Our data in this series of studies suggest that neogenesis from...

  19. Iontophoresis of monomeric insulin analogues in vitro: effects of insulin charge and skin pretreatment.

    Science.gov (United States)

    Langkjaer, L; Brange, J; Grodsky, G M; Guy, R H

    1998-01-23

    The aim of this study was to investigate the influence of association state and net charge of human insulin analogues on the rate of iontophoretic transport across hairless mouse skin, and the effect of different skin pretreatments on said transport. No insulin flux was observed with anodal delivery probably because of degradation at the Ag/AgCl anode. The flux during cathodal iontophoresis through intact skin was insignificant for human hexameric insulin, and only low and variable fluxes were observed for monomeric insulins. Using stripped skin on the other hand, the fluxes of monomeric insulins with two extra negative charges were 50-100 times higher than that of hexameric human insulin. Introducing three additional charges led to a further 2-3-fold increase in flux. Wiping the skin gently with absolute alcohol prior to iontophoresis resulted in a 1000-fold increase in transdermal transport of insulin relative to that across untreated skin, i.e. to almost the same level as stripping the skin. The alcohol pretreatment reduced the electrical resistance of the skin, presumably by lipid extraction. In conclusion, monomeric insulin analogues with at least two extra negative charges can be iontophoretically delivered across hairless mouse skin, whereas insignificant flux is observed with human, hexameric insulin. Wiping the skin with absolute alcohol prior to iontophoresis gave substantially improved transdermal transport of monomeric insulins resulting in clinically relevant delivery rates for basal treatment.

  20. Apoptotic death of Listeria monocytogenes-infected human macrophages induced by lactoferricin B, a bovine lactoferrin-derived peptide.

    Science.gov (United States)

    Longhi, C; Conte, M P; Ranaldi, S; Penta, M; Valenti, P; Tinari, A; Superti, F; Seganti, L

    2005-01-01

    Listeria monocytogenes, an intracellular facultative food-borne pathogen, was reported to induce apoptosis in vitro and in vivo in a variety of cell types with the exception of murine macrophages. These cells represent the predominant compartment of bacterial multiplication and die as a result of necrosis. In this study we showed that human non-activated and IFN-gamma-activated macrophagic-like (THP-1) cells infected with L. monocytogenes, mainly die by necrosis rather than by an apoptotic process. Two natural products derived from bovine milk, lactoferrin and its derivative peptide lactoferricin B, are capable of regulating the fate of infected human macrophages. Bovine lactoferrin treatment of macrophages protects them from L. monocytogenes-induced death whereas lactoferricin B, its derivative peptide, determines a shifting of the equilibrium from necrosis to apoptosis.

  1. WNT5A-JNK regulation of vascular insulin resistance in human obesity.

    Science.gov (United States)

    Farb, Melissa G; Karki, Shakun; Park, Song-Young; Saggese, Samantha M; Carmine, Brian; Hess, Donald T; Apovian, Caroline; Fetterman, Jessica L; Bretón-Romero, Rosa; Hamburg, Naomi M; Fuster, José J; Zuriaga, María A; Walsh, Kenneth; Gokce, Noyan

    2016-12-01

    Obesity is associated with the development of vascular insulin resistance; however, pathophysiological mechanisms are poorly understood. We sought to investigate the role of WNT5A-JNK in the regulation of insulin-mediated vasodilator responses in human adipose tissue arterioles prone to endothelial dysfunction. In 43 severely obese (BMI 44±11 kg/m 2 ) and five metabolically normal non-obese (BMI 26±2 kg/m 2 ) subjects, we isolated arterioles from subcutaneous and visceral fat during planned surgeries. Using videomicroscopy, we examined insulin-mediated, endothelium-dependent vasodilator responses and characterized adipose tissue gene and protein expression using real-time polymerase chain reaction and Western blot analyses. Immunofluorescence was used to quantify endothelial nitric oxide synthase (eNOS) phosphorylation. Insulin-mediated vasodilation was markedly impaired in visceral compared to subcutaneous vessels from obese subjects (p<0.001), but preserved in non-obese individuals. Visceral adiposity was associated with increased JNK activation and elevated expression of WNT5A and its non-canonical receptors, which correlated negatively with insulin signaling. Pharmacological JNK antagonism with SP600125 markedly improved insulin-mediated vasodilation by sixfold (p<0.001), while endothelial cells exposed to recombinant WNT5A developed insulin resistance and impaired eNOS phosphorylation (p<0.05). We observed profound vascular insulin resistance in the visceral adipose tissue arterioles of obese subjects that was associated with up-regulated WNT5A-JNK signaling and impaired endothelial eNOS activation. Pharmacological JNK antagonism markedly improved vascular endothelial function, and may represent a potential therapeutic target in obesity-related vascular disease. © The Author(s) 2016.

  2. Biological activity of alligator, avian, and mammalian insulin in juvenile alligators: plasma glucose and amino acids.

    Science.gov (United States)

    Lance, V A; Elsey, R M; Coulson, R A

    1993-02-01

    The biological activity of alligator, turkey, and bovine insulin on plasma glucose and plasma amino acids was tested in fasted juvenile alligators. Preliminary experiments showed that the stress associated with taking the initial blood sample resulted in a hyperglycemic response lasting more than 24 hr. Despite repeated bleedings no additional hyperglycemic events occurred, and blood glucose declined slowly over the next 7 days. Under these conditions the smallest dose of insulin eliciting a hypoglycemic response was 40 micrograms/kg body wt. A dose of 400 micrograms/kg body wt of either alligator or bovine insulin caused a pronounced hypoglycemia by 12 hr postinjection. Maximum decline in plasma glucose occurred at 24 to 36 hr with a slow return to control levels by 120 hr. There were no significant differences in the hypoglycemic responses to any of the three insulins tested. The decline in plasma amino acids was much more rapid than the decline in plasma glucose in response to insulin. Even at the 40 micrograms/kg body wt dose a significant difference from saline-injected control was seen at 2 hr postinjection. Maximum decline in plasma amino acids occurred at 8 to 12 hr with a return to baseline by 36 hr. These results show that the relatively conservative changes in the sequence of alligator insulin (three amino acid substitutions in the B-chain compared with that of chicken) have little effect on biological activity and that alligator insulin receptors do not appear to discriminate among the three insulins.

  3. Chemical stability of insulin. 1. Hydrolytic degradation during storage of pharmaceutical preparations.

    Science.gov (United States)

    Brange, J; Langkjaer, L; Havelund, S; Vølund, A

    1992-06-01

    Hydrolysis of insulin has been studied during storage of various preparations at different temperatures. Insulin deteriorates rapidly in acid solutions due to extensive deamidation at residue AsnA21. In neutral formulations deamidation takes place at residue AsnB3 at a substantially reduced rate under formation of a mixture of isoAsp and Asp derivatives. The rate of hydrolysis at B3 is independent of the strength of the preparation, and in most cases the species of insulin, but varies with storage temperature and formulation. Total transformation at B3 is considerably reduced when insulin is in the crystalline as compared to the amorphous or soluble state, indicating that formation of the rate-limiting cyclic imide decreases when the flexibility of the tertiary structure is reduced. Neutral solutions containing phenol showed reduced deamidation probably because of a stabilizing effect of phenol on the tertiary structure (alpha-helix formation) around the deamidating residue, resulting in a reduced probability for formation of the intermediate imide. The ratio of isoAsp/Asp derivative was independent of time and temperature, suggesting a pathway involving only intermediate imide formation, without any direct side-chain hydrolysis. However, increasing formation of Asp relative to isoAsp derivative was observed with decreasing flexibility of the insulin three-dimensional structure in the formulation. In certain crystalline suspensions a cleavage of the peptide bond A8-A9 was observed. Formation of this split product is species dependent: bovine greater than porcine greater than human insulin. The hydrolytic cleavage of the peptide backbone takes place only in preparations containing rhombohedral crystals in addition to free zinc ions.

  4. mRNA related to insulin family in human placenta

    International Nuclear Information System (INIS)

    Younes, M.A.; D'Agostino, J.B.; Frazier, M.L.; Besch, P.K.

    1986-01-01

    The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A + ) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A + ) RNA templates. Five hundred transformants were initially screened by colony hybridization using a 32 P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II

  5. mRNA related to insulin family in human placenta

    Energy Technology Data Exchange (ETDEWEB)

    Younes, M.A.; D' Agostino, J.B.; Frazier, M.L.; Besch, P.K.

    1986-03-01

    The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A/sup +/) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A/sup +/) RNA templates. Five hundred transformants were initially screened by colony hybridization using a /sup 32/P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II.

  6. Characterization of the growth of murine fibroblasts that express human insulin receptors. I. The effect of insulin in the absence of other growth factors

    International Nuclear Information System (INIS)

    Randazzo, P.A.; Morey, V.A.; Polishook, A.K.; Jarett, L.

    1990-01-01

    The effect of insulin on the growth of murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental cells (NIH/3T3) was characterized. Insulin in the absence of other mitogens increased the rate of incorporation of thymidine into NIH 3T3/HIR cells with a half-maximal response occurring at an insulin concentration of 35 ng/ml and a maximal response that was equivalent to that elicited by 10% fetal calf serum. The thymidine incorporation rate was increased by 12 h, was maximal at approximately 16 h, and returned to basal rates at 24 h after the addition of insulin. Insulin induced a maximum of 65% of cells to incorporate thymidine. The increased DNA synthesis was accompanied by net growth. Addition of insulin to the NIH 3T3/HIR cells resulted in increased DNA content with a half-maximal response occurring at approximately 30 ng/ml insulin and a maximal response equivalent to that elicited by serum. An increase in cell number detected after the addition of insulin to the NIH 3T3/HIR suggests that the cells had progressed through mitosis. Insulin did not increase the rate of thymidine incorporation, DNA content, or number of the parental NIH 3T3 cells. These data show that insulin, in the absence of a second mitogen, is able to induce NIH 3T3/HIR fibroblasts to traverse the cell cycle

  7. Streptococcus dysgalactiae subsp. dysgalactiae isolated from milk of the bovine udder as emerging pathogens: In vitro and in vivo infection of human cells and zebrafish as biological models.

    Science.gov (United States)

    Alves-Barroco, Cinthia; Roma-Rodrigues, Catarina; Raposo, Luís R; Brás, Catarina; Diniz, Mário; Caço, João; Costa, Pedro M; Santos-Sanches, Ilda; Fernandes, Alexandra R

    2018-03-25

    Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is a major cause of bovine mastitis and has been regarded as an animal-restricted pathogen, although rare infections have been described in humans. Previous studies revealed the presence of virulence genes encoded by phages of the human pathogen Group A Streptococcus pyogenes (GAS) in SDSD isolated from the milk of bovine udder with mastitis. The isolates SDSD VSD5 and VSD13 could adhere and internalize human primary keratinocyte cells, suggesting a possible human infection potential of bovine isolates. In this work, the in vitro and in vivo potential of SDSD to internalize/adhere human cells of the respiratory track and zebrafish as biological models was evaluated. Our results showed that, in vitro, bovine SDSD strains could interact and internalize human respiratory cell lines and that this internalization was dependent on an active transport mechanism and that, in vivo, SDSD are able to cause invasive infections producing zebrafish morbidity and mortality. The infectious potential of these isolates showed to be isolate-specific and appeared to be independent of the presence or absence of GAS phage-encoded virulence genes. Although the infection ability of the bovine SDSD strains was not as strong as the human pathogenic S. pyogenes in the zebrafish model, results suggested that these SDSD isolates are able to interact with human cells and infect zebrafish, a vertebrate infectious model, emerging as pathogens with zoonotic capability. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  8. First Report of Human Fascioliasis in an Endemic Region of Bovine Fascioliasis in Caldas-Colombia

    Science.gov (United States)

    Giraldo-Pinzon, Etna Julieth; Aguilar-Marín, Sandra

    2016-01-01

    Abstract Fascioliasis causes significant economic losses to the cattle industry and is considered a reemerging zoonosis. In Caldas-Colombia, an increase of bovine fascioliasis was detected at the Manizales Municipal Slaughterhouse, which is a potential risk to public health. The ecoepidemiology of human fascioliasis was analyzed in a region of bovine fascioliasis in Caldas-Colombia. The risk factors were studied. Samples were taken from 111 people who were directly related to the bovine milk production process. The immunoglobulin G frequency of Fasciola hepatica was determined in serum. A seriate stool test and a molecular analysis were conducted on those with positive results to look for parasite eggs and DNA, respectively. 6.3% of the samples were positive for the presence of antibodies; none was positive for the presence of eggs, while two samples showed a weak amplification band of the 124-bp DNA fragment of F. hepatica. Fifty-seven percent of the positive samples came from places located at 2026 meters above sea level (masl); 71% of people testing positive had been recently dewormed. Also, 86% had been in contact with cattle and handled grass and excrement. They eat salads and drink untreated water from the springs or ravines of the area. An outbreak of human fascioliasis was detected in Caldas, associated with risk factors for the disease. Clinical trials to confirm the presence of the parasite and implement public health control measures are required. PMID:27045315

  9. First Report of Human Fascioliasis in an Endemic Region of Bovine Fascioliasis in Caldas-Colombia.

    Science.gov (United States)

    Perez-C, Jorge Enrique; Giraldo-Pinzon, Etna Julieth; Aguilar-Marín, Sandra

    2016-06-01

    Fascioliasis causes significant economic losses to the cattle industry and is considered a reemerging zoonosis. In Caldas-Colombia, an increase of bovine fascioliasis was detected at the Manizales Municipal Slaughterhouse, which is a potential risk to public health. The ecoepidemiology of human fascioliasis was analyzed in a region of bovine fascioliasis in Caldas-Colombia. The risk factors were studied. Samples were taken from 111 people who were directly related to the bovine milk production process. The immunoglobulin G frequency of Fasciola hepatica was determined in serum. A seriate stool test and a molecular analysis were conducted on those with positive results to look for parasite eggs and DNA, respectively. 6.3% of the samples were positive for the presence of antibodies; none was positive for the presence of eggs, while two samples showed a weak amplification band of the 124-bp DNA fragment of F. hepatica. Fifty-seven percent of the positive samples came from places located at 2026 meters above sea level (masl); 71% of people testing positive had been recently dewormed. Also, 86% had been in contact with cattle and handled grass and excrement. They eat salads and drink untreated water from the springs or ravines of the area. An outbreak of human fascioliasis was detected in Caldas, associated with risk factors for the disease. Clinical trials to confirm the presence of the parasite and implement public health control measures are required.

  10. Hepatic Diacylglycerol-Associated Protein Kinase Cε Translocation Links Hepatic Steatosis to Hepatic Insulin Resistance in Humans

    NARCIS (Netherlands)

    ter Horst, Kasper W.; Gilijamse, Pim W.; Versteeg, Ruth I.; Ackermans, Mariette T.; Nederveen, Aart J.; la Fleur, Susanne E.; Romijn, Johannes A.; Nieuwdorp, Max; Zhang, Dongyan; Samuel, Varman T.; Vatner, Daniel F.; Petersen, Kitt F.; Shulman, Gerald I.; Serlie, Mireille J.

    2017-01-01

    Hepatic lipid accumulation has been implicated in the development of insulin resistance, but translational evidence in humans is limited. We investigated the relationship between liver fat and tissue-specific insulin sensitivity in 133 obese subjects. Although the presence of hepatic steatosis in

  11. Breed differences in development of anti-insulin antibodies in diabetic dogs and investigation of the role of dog leukocyte antigen (DLA) genes.

    Science.gov (United States)

    Holder, Angela L; Kennedy, Lorna J; Ollier, William E R; Catchpole, Brian

    2015-10-15

    Administration of insulin for treatment of diabetes mellitus in dogs can stimulate an immune response, with a proportion of animals developing anti-insulin antibodies (AIA). For an IgG antibody response to occur, this would require B cell presentation of insulin peptides by major histocompatibility complex (MHC) class II molecules, encoded by dog leukocyte antigen (DLA) genes, in order to receive T-cell help for class switching. DLA genes are highly polymorphic in the dog population and vary from breed to breed. The aim of the present study was to evaluate AIA reactivity in diabetic dogs of different breeds and to investigate whether DLA genes influence AIA status. Indirect ELISA was used to determine serological reactivity to insulin in diabetic dogs, treated with either a porcine or bovine insulin preparation. DLA haplotypes for diabetic dogs were determined by sequence-based typing of DLA-DRB1, -DQA1 and -DQB1 loci. Significantly greater insulin reactivity was seen in treated diabetic dogs (n=942) compared with non-diabetic dogs (n=100). Relatively few newly diagnosed diabetic dogs (3/109) were found to be AIA positive, although this provides evidence that insulin autoantibodies might be involved in the pathogenesis of the disease in some cases. Of the diabetic dogs treated with a bovine insulin preparation, 52.3% (182/348) were AIA positive, compared with 12.6% (75/594) of dogs treated with a porcine insulin preparation, suggesting that bovine insulin is more immunogenic. Breeds such as dachshund, Cairn terrier, miniature schnauzer and Tibetan terrier were more likely to develop AIA, whereas cocker spaniels were less likely to develop AIA, compared with crossbreed dogs. In diabetic dogs, DLA haplotype DRB1*0015--DQA1*006--DQB1*023 was associated with being AIA positive, whereas the haplotype DLA-DRB1*006--DQA1*005--DQB1*007 showed an association with being AIA negative. These research findings suggest that DLA genes influence AIA responses in treated diabetic

  12. Monoclonal antibodies directed to human insulin-like growth factor I (IGF I)

    International Nuclear Information System (INIS)

    Laubli, U.K.; Baier, W.; Celio, M.R.; Binz, H.; Humbel, R.E.

    1982-01-01

    Mouse hybridomas secreting antibodies to human insulin-like growth factor I (IGF I) were produced by fusion of spleen cells of hyperimmunised mice with FO mouse-myeloma cells. Eight clones producing antibodies against human IGF I have been isolated, two of which have been characterised. One was used in a radioimmunoassay, the other for immunopurification of IGF. (Auth.)

  13. Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells.

    Science.gov (United States)

    Lee, Jonghyeob; Sugiyama, Takuya; Liu, Yinghua; Wang, Jing; Gu, Xueying; Lei, Ji; Markmann, James F; Miyazaki, Satsuki; Miyazaki, Jun-Ichi; Szot, Gregory L; Bottino, Rita; Kim, Seung K

    2013-11-19

    Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001.

  14. Internalization and localization of basal insulin peglispro in cells.

    Science.gov (United States)

    Moyers, Julie S; Volk, Catherine B; Cao, Julia X C; Zhang, Chen; Ding, Liyun; Kiselyov, Vladislav V; Michael, M Dodson

    2017-10-15

    Basal insulin peglispro (BIL) is a novel, PEGylated insulin lispro that has a large hydrodynamic size compared with insulin lispro. It has a prolonged duration of action, which is related to a delay in insulin absorption and a reduction in clearance. Given the different physical properties of BIL compared with native insulin and insulin lispro, it is important to assess the cellular internalization characteristics of the molecule. Using immunofluorescent confocal imaging, we compared the cellular internalization and localization patterns of BIL, biosynthetic human insulin, and insulin lispro. We assessed the effects of BIL on internalization of the insulin receptor (IR) and studied cellular clearance of BIL. Co-localization studies using antibodies to either insulin or PEG, and the early endosomal marker EEA1 showed that the overall internalization and subcellular localization pattern of BIL was similar to that of human insulin and insulin lispro; all were rapidly internalized and co-localized with EEA1. During ligand washout for 4 h, concomitant loss of insulin, PEG methoxy group, and PEG backbone immunostaining was observed for BIL, similar to the loss of insulin immunostaining observed for insulin lispro and human insulin. Co-localization studies using an antibody to the lysosomal marker LAMP1 did not reveal evidence of lysosomal localization for insulin lispro, human insulin, BIL, or PEG using either insulin or PEG immunostaining reagents. BIL and human insulin both induced rapid phosphorylation and internalization of human IR. Our findings show that treatment of cells with BIL stimulates internalization and localization of IR to early endosomes. Both the insulin and PEG moieties of BIL undergo a dynamic cellular process of rapid internalization and transport to early endosomes followed by loss of cellular immunostaining in a manner similar to that of insulin lispro and human insulin. The rate of clearance for the insulin lispro portion of BIL was slower than

  15. Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells

    Science.gov (United States)

    Zalzman, Michal; Gupta, Sanjeev; Giri, Ranjit K.; Berkovich, Irina; Sappal, Baljit S.; Karnieli, Ohad; Zern, Mark A.; Fleischer, Norman; Efrat, Shimon

    2003-06-01

    Beta-cell replacement is considered to be the most promising approach for treatment of type 1 diabetes. Its application on a large scale is hindered by a shortage of cells for transplantation. Activation of insulin expression, storage, and regulated secretion in stem/progenitor cells offers novel ways to overcome this shortage. We explored whether fetal human progenitor liver cells (FH) could be induced to differentiate into insulin-producing cells after expression of the pancreatic duodenal homeobox 1 (Pdx1) gene, which is a key regulator of pancreatic development and insulin expression in beta cells. FH cells possess a considerable replication capacity, and this was further extended by introduction of the gene for the catalytic subunit of human telomerase. Immortalized FH cells expressing Pdx1 activated multiple beta-cell genes, produced and stored considerable amounts of insulin, and released insulin in a regulated manner in response to glucose. When transplanted into hyperglycemic immunodeficient mice, the cells restored and maintained euglycemia for prolonged periods. Quantitation of human C-peptide in the mouse serum confirmed that the glycemia was normalized by the transplanted human cells. This approach offers the potential of a novel source of cells for transplantation into patients with type 1 diabetes.

  16. Generation of glucose-responsive, insulin-producing cells from human umbilical cord blood-derived mesenchymal stem cells.

    Science.gov (United States)

    Prabakar, Kamalaveni R; Domínguez-Bendala, Juan; Molano, R Damaris; Pileggi, Antonello; Villate, Susana; Ricordi, Camillo; Inverardi, Luca

    2012-01-01

    We sought to assess the potential of human cord blood-derived mesenchymal stem cells (CB-MSCs) to derive insulin-producing, glucose-responsive cells. We show here that differentiation protocols based on stepwise culture conditions initially described for human embryonic stem cells (hESCs) lead to differentiation of cord blood-derived precursors towards a pancreatic endocrine phenotype, as assessed by marker expression and in vitro glucose-regulated insulin secretion. Transplantation of these cells in immune-deficient animals shows human C-peptide production in response to a glucose challenge. These data suggest that human cord blood may be a promising source for regenerative medicine approaches for the treatment of diabetes mellitus.

  17. Insulin redirects differentiation from cardiogenic mesoderm and endoderm to neuroectoderm in differentiating human embryonic stem cells.

    NARCIS (Netherlands)

    Freund, C.M.A.H.; Ward-van Oostwaard, D.; Monshouwer-Kloots, J.; van den Brink, S.; van Rooijen, M.A.; Xu, X.; Zweigerdt, R.; Mummery, C.L.; Passier, R.

    2008-01-01

    Human embryonic stem cells (hESC) can proliferate indefinitely while retaining the capacity to form derivatives of all three germ layers. We have reported previously that hESC differentiate into cardiomyocytes when cocultured with a visceral endoderm-like cell line (END-2). Insulin/insulin-like

  18. Thermo-responsive human α-elastin self-assembled nanoparticles for protein delivery.

    Science.gov (United States)

    Kim, Jae Dong; Jung, Youn Jae; Woo, Chang Hee; Choi, Young Chan; Choi, Ji Suk; Cho, Yong Woo

    2017-01-01

    Self-assembled nanoparticles based on PEGylated human α-elastin were prepared as a potential vehicle for sustained protein delivery. The α-elastin was extracted from human adipose tissue and modified with methoxypolyethyleneglycol (mPEG) to control particle size and enhance the colloidal stability. The PEGylated human α-elastin showed sol-to-particle transition with a lower critical solution temperature (LCST) of 25°C-40°C in aqueous media. The PEGylated human α-elastin nanoparticles (PhENPs) showed a narrow size distribution with an average diameter of 330±33nm and were able to encapsulate significant amounts of insulin and bovine serum albumin (BSA) upon simple mixing at low temperature in water and subsequent heating to physiological temperature. The release profiles of insulin and BSA showed sustained release for 72h. Overall, the thermo-responsive self-assembled PhENPs provide a useful tool for a range of protein delivery and tissue engineering applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Aspects of the regulation of long-chain fatty acid oxidation in bovine liver

    International Nuclear Information System (INIS)

    Jesse, B.W.; Emery, R.S.; Thomas, J.W.

    1986-01-01

    Factors involved in regulation of bovine hepatic fatty acid oxidation were examined using liver slices. Fatty acid oxidation was measured as the conversion of l-[ 14 C] palmitate to 14 CO 2 and total [ 14 C] acid-soluble metabolites. Extended (5 to 7 d) fasting of Holstein cows had relatively little effect on palmitate oxidation to acid-soluble metabolites by liver slices, although oxidation to CO 2 was decreased. Feeding a restricted roughage, high concentrate ration to lactating cows resulted in inhibition of palmitate oxidation. Insulin, glucose, and acetate inhibited palmitate oxidation by bovine liver slices. The authors suggest the regulation of bovine hepatic fatty acid oxidation may be less dependent on hormonally induced alterations in enzyme activity as observed in rat liver and more dependent upon action of rumen fermentation products or their metabolites on enzyme systems involved in fatty acid oxidation

  20. Complete sequence-specific 1H NMR assignments for human insulin

    International Nuclear Information System (INIS)

    Kline, A.D.; Justice, R.M. Jr.

    1990-01-01

    Solvent conditions where human insulin could be studied by high-resolution NMR were determined. Both low pH and addition of acetonitrile were required to overcome the protein's self-association and to obtain useful spectra. Two hundred eighty-six 1 H resonances were located and assigned to specific sites on the protein by using two-dimensional NMR methods. The presence and position of numerous d NN sequential NOE's indicate that the insulin conformation seen in crystallographic studies is largely retained under these solution conditions. Slowly exchanging protons were observed for seven backbone amide protons and were assigned to positions A15 and A16 and to positions B15-B19. These amides all occur within helical regions of the protein

  1. Bovine Necrotic Vulvovaginitis Associated with Porphyromonas levii

    Science.gov (United States)

    Friedgut, Orly; Alpert, Nir; Stram, Yehuda; Lahav, Dan; Tiomkin, Doron; Avramson, Miriam; Grinberg, Kalia; Bernstein, Michael

    2004-01-01

    An outbreak of bovine necrotic vulvovaginitis associated with Porphyromonas levii, an emerging animal and human pathogen, affected 32 cows on a dairy farm in the northeast of Israel. Five animals had to be culled. This report appears to be the first that associates P. levii with bovine necrotic vulvovagnitis. PMID:15109423

  2. Bovine lactoferricin is anti-inflammatory and anti-catabolic in human articular cartilage and synovium.

    Science.gov (United States)

    Yan, Dongyao; Chen, Di; Shen, Jie; Xiao, Guozhi; van Wijnen, Andre J; Im, Hee-Jeong

    2013-02-01

    Bovine lactoferricin (LfcinB) is a multi-functional peptide derived from proteolytic cleavage of bovine lactoferrin. LfcinB was found to antagonize the biological effects mediated by angiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF-2) in endothelial cells. However, the effect of LfcinB on human articular cartilage remained unknown. Here, our findings demonstrate that LfcinB restored the proteoglycan loss promoted by catabolic factors (interleukin-1β) IL-1β and FGF-2 in vitro and ex vivo. Mechanistically, LfcinB attenuated the effects of IL-1β and FGF-2 on the expression of cartilage-degrading enzymes (MMP-1, MMP-3, and MMP-13), destructive cytokines (IL-1β and IL-6), and inflammatory mediators (iNOS and TLR2). LfcinB induced protective cytokine expression (IL-4 and IL-10), and downregulated aggrecanase basal expression. LfcinB specifically activated ERK MAPK and Akt signaling pathways, which may account for its anti-inflammatory activity. We also revealed that LfcinB exerted similar protective effects on human synovial fibroblasts challenged by IL-1β, with minimal cytotoxicity. Collectively, our results suggest that LfcinB possesses potent anti-catabolic and anti-inflammatory bioactivities in human articular tissues, and may be utilized for the prevention and/or treatment of OA in the future. Copyright © 2012 Wiley Periodicals, Inc.

  3. Preparative isolation by high performance liquid chromatography of human insulin B chain produced in escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Cruz, N.; Antonio, S.; De Anda, R.; Gosset, G.; Bolivar, F. (Centro de Investigacion sobre Ingenieria Genetica y Biotecnologia, Universidad Nacional Autonoma de Mexico, Apdo. Postal 510-3 Cuernavaca, Mor. 62271 (MX))

    1990-01-01

    This paper reports on a simple method developed for the analytical and preparative purification of human insulin B chain from recombinant origin. Three solvent systems: acetonitrile, isopropanol and methanol, were studied to determine their capacity to resolve the insulin B chain from a mixture of cyanogen bromide generated bacterial peptides. Using a {mu}Bondapak C18 column, it was possible to resolve the insulin B chain in all three systems. On a preparative scale, using a PrePak 500 C18 column with the isopropanol system, it was possible to purify insulin B chain and to obtain a 95% protein recovery.

  4. Human and rodent muscle Na(+)-K(+)-ATPase in diabetes related to insulin, starvation, and training

    DEFF Research Database (Denmark)

    Schmidt, T A; Hasselbalch, S; Farrell, P A

    1994-01-01

    cerebral cortex Na(+)-K(+)-ATPase concentration as a result of diabetes, semistarvation, or insulin treatment. In human subjects, Na(+)-K(+)-ATPase concentration in vastus lateralis muscle biopsies was 17 and 22% greater (P dependent diabetes...... mellitus (n = 24) and insulin-dependent diabetes mellitus (n = 7) than in control subjects (n = 8). A positive linear correlation between muscle Na(+)-K(+)-ATPase and plasma insulin concentrations was observed (r = 0.50, P = 0.006; n = 29). Thus, insulin seems a regulator of muscle Na......(+)-K(+)-ATPase concentration, reduction of muscle Na(+)-K(+)-ATPase concentration with untreated diabetes bears similarities with undernourishment, and physical conditioning may ameliorate the muscle Na(+)-K(+)-ATPase concentration decrease induced by diabetes....

  5. Expression of human choline kinase in NIH 3T3 fibroblasts increases the mitogenic potential of insulin and insulin-like growth factor I.

    Science.gov (United States)

    Chung, T; Huang, J S; Mukherjee, J J; Crilly, K S; Kiss, Z

    2000-05-01

    In mammalian cells, growth factors, oncogenes, and carcinogens stimulate phosphocholine (PCho) synthesis by choline kinase (CK), suggesting that PCho may regulate cell growth. To validate the role of PCho in mitogenesis, we determined the effects of insulin, insulin-like growth factor I (IGF-I), and other growth factors on DNA synthesis in NIH 3T3 fibroblast sublines highly expressing human choline kinase (CK) without increasing phosphatidylcholine synthesis. In serum-starved CK expressor cells, insulin and IGF-I stimulated DNA synthesis, p70 S6 kinase (p70 S6K) activity, phosphatidylinositol 3-kinase (PI3K) activity, and activating phosphorylation of p42/p44 mitogen-activated protein kinases (MAPK) to greater extents than in the corresponding vector control cells. Furthermore, the CK inhibitor hemicholinium-3 (HC-3) inhibited insulin- and IGF-I-induced DNA synthesis in the CK overexpressors, but not in the vector control cells. The results indicate that high cellular levels of PCho potentiate insulin- and IGF-I-induced DNA synthesis by MAPK- and p70 S6K-regulated mechanisms.

  6. Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation?

    Science.gov (United States)

    Burnouf, Thierry; Strunk, Dirk; Koh, Mickey B C; Schallmoser, Katharina

    2016-01-01

    The essential physiological role of platelets in wound healing and tissue repair builds the rationale for the use of human platelet derivatives in regenerative medicine. Abundant growth factors and cytokines stored in platelet granules can be naturally released by thrombin activation and clotting or artificially by freeze/thaw-mediated platelet lysis, sonication or chemical treatment. Human platelet lysate prepared by the various release strategies has been established as a suitable alternative to fetal bovine serum as culture medium supplement, enabling efficient propagation of human cells under animal serum-free conditions for a multiplicity of applications in advanced somatic cell therapy and tissue engineering. The rapidly increasing number of studies using platelet derived products for inducing human cell proliferation and differentiation has also uncovered a considerable variability of human platelet lysate preparations which limits comparability of results. The main variations discussed herein encompass aspects of donor selection, preparation of the starting material, the possibility for pooling in plasma or additive solution, the implementation of pathogen inactivation and consideration of ABO blood groups, all of which can influence applicability. This review outlines the current knowledge about human platelet lysate as a powerful additive for human cell propagation and highlights its role as a prevailing supplement for human cell culture capable to replace animal serum in a growing spectrum of applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Effects of the beta-carbolines, harmane and pinoline, on insulin secretion from isolated human islets of Langerhans.

    Science.gov (United States)

    Cooper, E Jane; Hudson, Alan L; Parker, Christine A; Morgan, Noel G

    2003-12-15

    It is well known that certain imidazoline compounds can stimulate insulin secretion and this has been attributed to the activation of imidazoline I(3) binding sites in the pancreatic beta-cell. Recently, it has been proposed that beta-carbolines may be endogenous ligands having activity at imidazoline sites and we have, therefore, studied the effects of beta-carbolines on insulin secretion. The beta-carbolines harmane, norharmane and pinoline increased insulin secretion two- to threefold from isolated human islets of Langerhans. The effects of harmane and pinoline were dose-dependent (EC(50): 5 and 25 microM, respectively) and these agents also blocked the inhibitory effects of the potassium channel agonist, diazoxide, on glucose-induced insulin release. Stimulation of insulin secretion by harmane was glucose-dependent but, unlike the imidazoline I(3) receptor agonist efaroxan, it increased the rate of insulin release beyond that elicited by 20 mM glucose (20 mM glucose alone: 253+/-34% vs. basal; 20 mM glucose plus 100 microM harmane: 327+/-15%; P<0.01). Stimulation of insulin secretion by harmane was attenuated by the imidazoline I(3) receptor antagonist KU14R (2 (2-ethyl 2,3-dihydro-2-benzofuranyl)-2-imidazole) and was reduced when islets were treated with efaroxan for 18 h, prior to the addition of harmane. The results reveal that beta-carbolines can potentiate the rate of insulin secretion from human islets and suggest that these agents may be useful prototypes for the development of novel insulin secretagogues.

  8. Exogenous insulin antibody syndrome (EIAS): a clinical syndrome associated with insulin antibodies induced by exogenous insulin in diabetic patients.

    Science.gov (United States)

    Hu, Xiaolei; Chen, Fengling

    2018-01-01

    Insulin has been used for diabetes therapy and has achieved significant therapeutic effect. In recent years, the use of purified and recombinant human insulin preparations has markedly reduced, but not completely suppressed, the incidence of insulin antibodies (IAs). IAs induced by exogenous insulin in diabetic patients is associated with clinical events, which is named exogenous insulin antibody syndrome (EIAS). The present review is based on our research and summarizes the characterization of IAs, the factors affecting IA development, the clinical significance of IAs and the treatments for EIAS. © 2018 The authors.

  9. Comparative analysis in continuous expansion of bovine and human primary nucleus pulposus cells for tissue repair applications

    Directory of Open Access Journals (Sweden)

    DH Rosenzweig

    2017-03-01

    Full Text Available Autologous NP cell implantation is a potential therapeutic avenue for intervertebral disc (IVD degeneration. However, monolayer expansion of cells isolated from surgical samples may negatively impact matrix production by way of dedifferentiation. Previously, we have used a continuous expansion culture system to successfully preserve a chondrocyte phenotype. In this work, we hypothesised that continuous expansion culture could also preserve nucleus pulposus (NP phenotype. We confirmed that serial passaging drove NP dedifferentiation by significantly decreasing collagen type II, aggrecan and chondroadherin (CHAD gene expression, compared to freshly isolated cells. Proliferation, gene expression profile and matrix production in both culture conditions were compared using primary bovine NP cells. Both standard culture and continuous culture produced clinically relevant cell populations. However, continuous culture cells maintained significantly higher collagen type II, aggrecan and CHAD transcript expression levels. Also, continuous expansion cells generated greater amounts of proteoglycan, collagen type II and aggrecan protein deposition in pellet cultures. To our surprise, continuous expansion of human intervertebral disc cells – isolated from acute herniation tissue – produced less collagen type II, aggrecan and CHAD genes and proteins, compared to standard culture. Also, continuous culture of cells isolated from young non-degenerate tissue did not preserve gene and protein expression, compared to standard culture. These data indicated that primary bovine and human NP cells responded differently to continuous culture, where the positive effects observed for bovine cells did not translate to human cells. Therefore, caution must be exercised when choosing animal models and cell sources for pre-clinical studies.

  10. Determining Human Clot Lysis Time (in vitro with Plasminogen/Plasmin from Four Species (Human, Bovine, Goat, and Swine

    Directory of Open Access Journals (Sweden)

    Omaira Cañas Bermúdez

    2015-05-01

    Full Text Available Cardiovascular disease is the leading cause of death worldwide, including failures in the plasminogen/plasmin system which is an important factor in poor lysis of blood clots. This article studies the fibrinolytic system in four species of mammals, and it identifies human plasminogen with highest thrombolysis efficiency. It examines plasminogen from four species (human, bovine, goat, and swine and identifies the most efficient one in human clot lysis in vitro. All plasminogens were identically purified by affinity chromatography. Human fibrinogen was purified by fractionation with ethanol. The purification of both plasminogen and fibrinogen was characterized by one-dimensional SDS-PAGE (10%. Human clot formation in vitro and its dissolution by plasminogen/plasmin consisted of determining lysis time from clot formation to its dilution. Purification of proteins showed greater than 95% purity, human plasminogen showed greater ability to lyse clot than animal plasminogen. The article concludes that human plasminogen/plasmin has the greatest catalysis and efficiency, as it dissolves human clot up to three times faster than that of irrational species.

  11. Insulin and the PI3K/AKT Signaling Pathway Regulate Ribonuclease 7 Expression in the Human Urinary Tract

    Science.gov (United States)

    Eichler, Tad; Becknell, Brian; Easterling, Robert S.; Ingraham, Susan E.; Cohen, Daniel M.; Schwaderer, Andrew; Hains, David S.; Li, Birong; Cohen, Ariel; Metheny, Jackie; Trindandapani, Susheela; Spencer, John David

    2017-01-01

    Diabetes mellitus is a systemic disease associated with a deficiency of insulin production or action. Diabetic patients have an increased susceptibility to infection with the urinary tract being the most common site of infection. Recent studies suggest that Ribonuclease 7 (RNase 7) is a potent antimicrobial peptide that plays an important role in protecting the urinary tract from bacterial insult. The impact of diabetes on RNase 7 expression and function are unknown. Here, we investigate the effects of insulin on RNase 7. Using human urine specimens, we measured urinary RNase 7 concentrations in healthy control patients and insulin-deficient type 1 diabetics before and after starting insulin therapy. Compared to controls, diabetic patients had suppressed urinary RNase 7 concentrations, which increased with insulin. Using primary human urothelial cells, we explored the mechanisms by which insulin induces RNase 7. Insulin induces RNase 7 production via the phosphatidylinositide 3-kinase signaling pathway (PI3K/AKT) to shield urothelial cells from uropathogenic E. coli. In contrast, we show that uropathogenic E. coli suppresses PI3K/AKT and RNase 7. Together, these results indicate that insulin and PI3K/AKT signaling are essential for RNase 7 expression. They also suggest that increased infection risks in diabetic patients may be secondary to suppressed RNase 7 production. These data may provide unique insight into novel UTI therapeutic strategies in at risk populations. PMID:27401534

  12. Bariatric surgery in morbidly obese insulin resistant humans normalises insulin signalling but not insulin-stimulated glucose disposal.

    Directory of Open Access Journals (Sweden)

    Mimi Z Chen

    Full Text Available Weight-loss after bariatric surgery improves insulin sensitivity, but the underlying molecular mechanism is not clear. To ascertain the effect of bariatric surgery on insulin signalling, we examined glucose disposal and Akt activation in morbidly obese volunteers before and after Roux-en-Y gastric bypass surgery (RYGB, and compared this to lean volunteers.The hyperinsulinaemic euglycaemic clamp, at five infusion rates, was used to determine glucose disposal rates (GDR in eight morbidly obese (body mass index, BMI=47.3 ± 2.2 kg/m(2 patients, before and after RYGB, and in eight lean volunteers (BMI=20.7 ± 0.7 kg/m2. Biopsies of brachioradialis muscle, taken at fasting and insulin concentrations that induced half-maximal (GDR50 and maximal (GDR100 GDR in each subject, were used to examine the phosphorylation of Akt-Thr308, Akt-473, and pras40, in vivo biomarkers for Akt activity.Pre-operatively, insulin-stimulated GDR was lower in the obese compared to the lean individuals (P<0.001. Weight-loss of 29.9 ± 4 kg after surgery significantly improved GDR50 (P=0.004 but not GDR100 (P=0.3. These subjects still remained significantly more insulin resistant than the lean individuals (p<0.001. Weight loss increased insulin-stimulated skeletal muscle Akt-Thr308 and Akt-Ser473 phosphorylation, P=0.02 and P=0.03 respectively (MANCOVA, and Akt activity towards the substrate PRAS40 (P=0.003, MANCOVA, and in contrast to GDR, were fully normalised after the surgery (obese vs lean, P=0.6, P=0.35, P=0.46, respectively.Our data show that although Akt activity substantially improved after surgery, it did not lead to a full restoration of insulin-stimulated glucose disposal. This suggests that a major defect downstream of, or parallel to, Akt signalling remains after significant weight-loss.

  13. Human histologic evaluation of anorganic bovine bone mineral combined with recombinant human platelet-derived growth factor BB in maxillary sinus augmentation: case series study.

    Science.gov (United States)

    Nevins, Myron; Garber, David; Hanratty, James J; McAllister, Bradley S; Nevins, Marc L; Salama, Maurice; Schupbach, Peter; Wallace, Steven; Bernstein, Simon M; Kim, David M

    2009-12-01

    The objective of this proof-of-principle study was to examine the potential for improved bone regenerative outcomes in maxillary sinus augmentation procedures when recombinant human platelet-derived growth factor BB (0.3 mg/mL) is combined with particulate anorganic bovine bone mineral. The surgical outcomes in all treated sites were uneventful at 6 to 8 months, with sufficient regenerated bone present to allow successful placement of maxillary posterior implants. Large areas of dense, well-formed lamellar bone were seen throughout the intact core specimens in more than half of the grafted sites. Abundant numbers of osteoblasts were noted in concert with significant osteoid in all sites, indicating ongoing osteogenesis. A number of cores demonstrated efficient replacement of the normally slowly resorbing anorganic bovine bone mineral matrix particles with newly formed bone when the matrix was saturated with recombinant human platelet-derived growth factor BB.

  14. Redifferentiation of insulin-secreting cells after in vitro expansion of adult human pancreatic islet tissue

    International Nuclear Information System (INIS)

    Lechner, Andreas; Nolan, Anna L.; Blacken, Robyn A.; Habener, Joel F.

    2005-01-01

    Cellular replacement therapy holds promise for the treatment of diabetes mellitus but donor tissue is severely limited. Therefore, we investigated whether insulin-secreting cells could be differentiated in vitro from a monolayer of cells expanded from human donor pancreatic islets. We describe a three-step culture protocol that allows for the efficient generation of insulin-producing cell clusters from in vitro expanded, hormone-negative cells. These clusters express insulin at levels of up to 34% that of average freshly isolated human islets and secrete C-peptide upon membrane depolarization. They also contain cells expressing the other major islet hormones (glucagon, somatostatin, and pancreatic polypeptide). The source of the newly differentiated endocrine cells could either be indigenous stem/progenitor cells or the proliferation-associated dedifferentiation and subsequent redifferentiation of mature endocrine cells. The in vitro generated cell clusters may be efficacious in providing islet-like tissue for transplantation into diabetic recipients

  15. Characterization of Insulin-Immunoreactive Cells and Endocrine Cells Within the Duct System of the Adult Human Pancreas.

    Science.gov (United States)

    Li, Rong; Zhang, Xiaoxi; Yu, Lan; Zou, Xia; Zhao, Hailu

    2016-01-01

    The adult pancreatic duct system accommodates endocrine cells that have the potential to produce insulin. Here we report the characterization and distribution of insulin-immunoreactive cells and endocrine cells within the ductal units of adult human pancreas. Sequential pancreas sections from 12 nondiabetic adults were stained with biomarkers of ductal epithelial cells (cytokeratin 19), acinar cells (amylase), endocrine cells (chromogranin A; neuron-specific enolase), islet hormones (insulin, glucagon, somatostatin, pancreatic polypeptide), cell proliferation (Ki-67), and neogenesis (CD29). The number of islet hormone-immunoreactive cells increased from large ducts to the terminal branches. The insulin-producing cells outnumbered endocrine cells reactive for glucagon, somatostatin, or pancreatic polypeptide. The proportions of insulin-immunoreactive count compared with local islets (100% as a baseline) were 1.5% for the main ducts, 7.2% for interlobular ducts, 24.8% for intralobular ducts, 67.9% for intercalated ducts, and 348.9% for centroacinar cells. Both Ki-67- and CD29-labeled cells were predominantly localized in the terminal branches around the islets. The terminal branches also showed cells coexpressing islet hormones and cytokeratin 19. The adult human pancreatic ducts showed islet hormone-producing cells. The insulin-reactive cells predominantly localized in terminal branches where they may retain potential capability for β-cell neogenesis.

  16. In nondiabetic, human immunodeficiency virus-infected patients with lipodystrophy, hepatic insulin extraction and posthepatic insulin clearance rate are decreased in proportion to insulin resistance

    DEFF Research Database (Denmark)

    Haugaard, Steen B; Andersen, Ove; Hansen, Birgitte R

    2005-01-01

    In healthy, nondiabetic individuals with insulin resistance, fasting insulin is inversely correlated to the posthepatic insulin clearance rate (MCRi) and the hepatic insulin extraction (HEXi). We investigated whether similar early mechanisms to facilitate glucose homeostasis exist in nondiabetic...... endogenous insulin secretion, which was estimated by deconvolution of C-peptide concentrations. Hepatic extraction of insulin was calculated as 1 minus the ratio of fasting posthepatic insulin delivery rate to fasting endogenous insulin secretion rate. Compared with controls, LIPO displayed increased fasting...... insulin (130%, P Hepatic extraction of insulin was similar between groups (LIPO, 55%; controls, 57%; P > .8). In LIPO, HEXi and MCRi correlated inversely with fasting insulin (r = -0.56, P

  17. Studies of the structure of insulin fibrils by Fourier transform infrared (FTIR) spectroscopy and electron microscopy.

    Science.gov (United States)

    Nielsen, L; Frokjaer, S; Carpenter, J F; Brange, J

    2001-01-01

    Fibril formation (aggregation) of insulin was investigated in acid media by visual inspection, transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopy. Insulin fibrillated faster in hydrochloric acid than in acetic acid at elevated temperatures, whereas the fibrillation tendencies were reversed at ambient temperatures. Electron micrographs showed that bovine insulin fibrils consisted of long fibers with a diameter of 5 to 10 nm and lengths of several microns. The fibrils appeared either as helical filaments (in hydrochloric acid) or arranged laterally in bundles (in acetic acid, NaCl). Freeze-thawing cycles broke the fibrils into shorter segments. FTIR spectroscopy showed that the native secondary structure of insulin was identical in hydrochloric acid and acetic acid, whereas the secondary structure of fibrils formed in hydrochloric acid was different from that formed in acetic acid. Fibrils of bovine insulin prepared by heating or agitating an acid solution of insulin showed an increased content of beta-sheet (mostly intermolecular) and a decrease in the intensity of the alpha-helix band. In hydrochloric acid, the frequencies of the beta-sheet bands depended on whether the fibrillation was induced by heating or agitation. This difference was not seen in acetic acid. Freeze-thawing cycles of the fibrils in hydrochloric acid caused an increase in the intensity of the band at 1635 cm(-1) concomitant with reduction of the band at 1622 cm(-1). The results showed that the structure of insulin fibrils is highly dependent on the composition of the acid media and on the treatment. Copyright 2001 Wiley-Liss Inc. and the American Pharmaceutical Association J Pharm Sci 90: 29-37, 2001

  18. miR-375 induces human decidua basalis-derived stromal cells to become insulin-producing cells.

    Science.gov (United States)

    Shaer, Anahita; Azarpira, Negar; Vahdati, Akbar; Karimi, Mohammad Hosein; Shariati, Mehrdad

    2014-09-01

    This paper focuses on the development of renewable sources of isletreplacement tissue for the treatment of type I diabetes mellitus. Placental tissue-derived mesenchymal stem cells (MSCs) are a promising source for regenerative medicine due to their plasticity and easy availability. They have the potential to differentiate into insulin-producing cells. miR-375 is a micro RNA that is expressed in the pancreas and involved in islet development. Human placental decidua basalis MSCs (PDB-MSCs) were cultured from full-term human placenta. The immunophenotype of the isolated cells was checked for CD90, CD105, CD44, CD133 and CD34 markers. The MSCs (P3) were chemically transfected with hsa-miR-375. Total RNA was extracted 4 and 6 days after transfection. The expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, and glucagon genes were evaluated using real-time qPCR. On day 6, we tested the potency of the clusters in response to the high glucose challenge and assessed the presence of insulin and NGN3 proteins via immunocytochemistry. Flow cytometry analysis confirmed that more than 90% of the cells were positive for CD90, CD105 and CD44 and negative for CD133 and CD34. Morphological changes were followed from day 2. Cell clusters formed during day 6. Insulin-producing clusters showed a deep red color with DTZ. The expression of pancreatic-specific transcription factors increased remarkably during the four days after transfection and significantly increased on day 7. The clusters were positive for insulin and NGN3 proteins, and C-peptide and insulin secretion increased in response to changes in the glucose concentration (2.8 mM and 16.7 mM). In conclusion, the MSCs could be programmed into functional insulin-producing cells by transfection of miR-375.

  19. Insulin-producing cells generated from dedifferentiated human pancreatic beta cells expanded in vitro.

    Directory of Open Access Journals (Sweden)

    Holger A Russ

    Full Text Available Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT. Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells.Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2 using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation.These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug

  20. Tau hyperphosphorylation induces oligomeric insulin accumulation and insulin resistance in neurons.

    Science.gov (United States)

    Rodriguez-Rodriguez, Patricia; Sandebring-Matton, Anna; Merino-Serrais, Paula; Parrado-Fernandez, Cristina; Rabano, Alberto; Winblad, Bengt; Ávila, Jesús; Ferrer, Isidre; Cedazo-Minguez, Angel

    2017-12-01

    Insulin signalling deficiencies and insulin resistance have been directly linked to the progression of neurodegenerative disorders like Alzheimer's disease. However, to date little is known about the underlying molecular mechanisms or insulin state and distribution in the brain under pathological conditions. Here, we report that insulin is accumulated and retained as oligomers in hyperphosphorylated tau-bearing neurons in Alzheimer's disease and in several of the most prevalent human tauopathies. The intraneuronal accumulation of insulin is directly dependent on tau hyperphosphorylation, and follows the tauopathy progression. Furthermore, cells accumulating insulin show signs of insulin resistance and decreased insulin receptor levels. These results suggest that insulin retention in hyperphosphorylated tau-bearing neurons is a causative factor for the insulin resistance observed in tauopathies, and describe a novel neuropathological concept with important therapeutic implications. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. Exogenous insulin antibody syndrome (EIAS: a clinical syndrome associated with insulin antibodies induced by exogenous insulin in diabetic patients

    Directory of Open Access Journals (Sweden)

    Xiaolei Hu

    2018-01-01

    Full Text Available Insulin has been used for diabetes therapy and has achieved significant therapeutic effect. In recent years, the use of purified and recombinant human insulin preparations has markedly reduced, but not completely suppressed, the incidence of insulin antibodies (IAs. IAs induced by exogenous insulin in diabetic patients is associated with clinical events, which is named exogenous insulin antibody syndrome (EIAS. The present review is based on our research and summarizes the characterization of IAs, the factors affecting IA development, the clinical significance of IAs and the treatments for EIAS.

  2. Immature transformed rat islet beta-cells differentially express C-peptides derived from the genes coding for insulin I and II as well as a transfected human insulin gene

    DEFF Research Database (Denmark)

    Blume, N; Petersen, J S; Andersen, L C

    1992-01-01

    is induced in the transformed heterogeneous rat islet cell clone, NHI-6F, by transient in vivo passage. During this process a transfected human insulin gene is coactivated with the endogenous nonallelic rat insulin I and II genes. Newly established cultures from NHI-6F insulinomas having a high frequency...

  3. Central insulin-like growth factor-1 (IGF-1) restores whole-body insulin action in a model of age-related insulin resistance and IGF-1 decline.

    Science.gov (United States)

    Huffman, Derek M; Farias Quipildor, Gabriela; Mao, Kai; Zhang, Xueying; Wan, Junxiang; Apontes, Pasha; Cohen, Pinchas; Barzilai, Nir

    2016-02-01

    Low insulin-like growth factor-1 (IGF-1) signaling is associated with improved longevity, but is paradoxically linked with several age-related diseases in humans. Insulin-like growth factor-1 has proven to be particularly beneficial to the brain, where it confers protection against features of neuronal and cognitive decline. While aging is characterized by central insulin resistance in the face of hyperinsulinemia, the somatotropic axis markedly declines in older humans. Thus, we hypothesized that increasing IGF-1 in the brain may prove to be a novel therapeutic alternative to overcome central insulin resistance and restore whole-body insulin action in aging. Utilizing hyperinsulinemic-euglycemic clamps, we show that old insulin-resistant rats with age-related declines in IGF-1 level demonstrate markedly improved whole-body insulin action, when treated with central IGF-1, as compared to central vehicle or insulin (P IGF-1, but not insulin, suppressed hepatic glucose production and increased glucose disposal rates in aging rats (P IGF-1 action in the brain and periphery provides a 'balance' between its beneficial and detrimental actions. Therefore, we propose that strategies aimed at 'tipping the balance' of IGF-1 action centrally are the optimal approach to achieve healthy aging and longevity in humans. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  4. A Comparison of the Anorexic Effects of Chicken, Porcine, Human and Bovine Insulin on the Central Nervous System of Chicks

    Science.gov (United States)

    The aim of the present study was to determine if some naturally-occurring substitutions of amino acid residues of insulin could act differentially within the central nervous system (CNS) of neonatal chicks to control ingestive behavior. Intracerebroventricular (ICV) administration of chicken insuli...

  5. Metformin and insulin receptors

    International Nuclear Information System (INIS)

    Vigneri, R.; Gullo, D.; Pezzino, V.

    1984-01-01

    The authors evaluated the effect of metformin (N,N-dimethylbiguanide), a biguanide known to be less toxic than phenformin, on insulin binding to its receptors, both in vitro and in vivo. Specific 125 I-insulin binding to cultured IM-9 human lymphocytes and MCF-7 human breast cancer cells was determined after preincubation with metformin. Specific 125 I-insulin binding to circulating monocytes was also evaluated in six controls, eight obese subjects, and six obese type II diabetic patients before and after a short-term treatment with metformin. Plasma insulin levels and blood glucose were also measured on both occasions. Metformin significantly increased insulin binding in vitro to both IM-9 lymphocytes and MCF-7 cells; the maximum increment was 47.1% and 38.0%, respectively. Metformin treatment significantly increased insulin binding in vivo to monocytes of obese subjects and diabetic patients. Scatchard analysis indicated that the increased binding was mainly due to an increase in receptor capacity. Insulin binding to monocytes of normal controls was unchanged after metformin as were insulin levels in all groups; blood glucose was significantly reduced after metformin only in diabetic patients. These data indicate that metformin increases insulin binding to its receptors in vitro and in vivo. The effect in vivo is observed in obese subjects and in obese type II diabetic patients, paralleling the clinical effectiveness of this antidiabetic agent, and is not due to receptor regulation by circulating insulin, since no variation in insulin levels was recorded

  6. Pancreatic Transdifferentiation and Glucose-Regulated Production of Human Insulin in the H4IIE Rat Liver Cell Line

    Directory of Open Access Journals (Sweden)

    Binhai Ren

    2016-04-01

    Full Text Available Due to the limitations of current treatment regimes, gene therapy is a promising strategy being explored to correct blood glucose concentrations in diabetic patients. In the current study, we used a retroviral vector to deliver either the human insulin gene alone, the rat NeuroD1 gene alone, or the human insulin gene and rat NeuroD1 genes together, to the rat liver cell line, H4IIE, to determine if storage of insulin and pancreatic transdifferentiation occurred. Stable clones were selected and expanded into cell lines: H4IIEins (insulin gene alone, H4IIE/ND (NeuroD1 gene alone, and H4IIEins/ND (insulin and NeuroD1 genes. The H4IIEins cells did not store insulin; however, H4IIE/ND and H4IIEins/ND cells stored 65.5 ± 5.6 and 1475.4 ± 171.8 pmol/insulin/5 × 106 cells, respectively. Additionally, several β cell transcription factors and pancreatic hormones were expressed in both H4IIE/ND and H4IIEins/ND cells. Electron microscopy revealed insulin storage vesicles in the H4IIE/ND and H4IIEins/ND cell lines. Regulated secretion of insulin to glucose (0–20 mmol/L was seen in the H4IIEins/ND cell line. The H4IIEins/ND cells were transplanted into diabetic immunoincompetent mice, resulting in normalization of blood glucose. This data shows that the expression of NeuroD1 and insulin in liver cells may be a useful strategy for inducing islet neogenesis and reversing diabetes.

  7. Radiographic Comparison of Bovine Bone Substitute Alone versus Bovine Bone Substitute and Simvastatin for Human Maxillary Sinus Augmentation

    Directory of Open Access Journals (Sweden)

    Amir Ali Reza Rasouli Ghahroudi

    2018-01-01

    Full Text Available Objectives: The aim of this study was to compare the efficacy of bovine bone substitute (Compact Bone B. ® alone versus bovine bone substitute and simvastatin for human maxillary sinus augmentation.Materials and Methods: This study was conducted on 16 sinuses in eight patients. Radiographic assessments were done preoperatively (T0, immediately (T1 and at nine months after sinus grafting (T2. Alveolar bone height and density were assessed on cone beam computed tomography (CBCT scans using Planmeca Romexis™ Imaging Software 2.2.Results: The change in alveolar bone height and density between T0, T1 and T2 was significant in both groups. Alveolar bone height (h0, h1, h2 and vertical height of the grafted bone (g1, g2 in three lines (anterior, middle and posterior were not significantly different between groups. The grafted bone height shrinkage (% in the anterior, middle and posterior limits of the augmented area were not significantly different between groups. The existing alveolar and grafted bone density increased significantly in both groups between T1 and T2, except for the existing alveolar bone density in the control group. There were no statistically significant differences between the alveolar bone density values obtained in TI and T2 between groups, except for the existing alveolar bone density at T1.Conclusions: This study did not show any significant positive effect for simvastatin in maxillary sinus augmentation based on radiographic examination.

  8. Effect of training on insulin sensitivity of glucose uptake and lipolysis in human adipose tissue

    DEFF Research Database (Denmark)

    Stallknecht, B; Larsen, J J; Mikines, K J

    2000-01-01

    Training increases insulin sensitivity of both whole body and muscle in humans. To investigate whether training also increases insulin sensitivity of adipose tissue, we performed a three-step hyperinsulinemic, euglycemic clamp in eight endurance-trained (T) and eight sedentary (S) young men...... (glucose only). Adipose tissue blood flow was measured by (133)Xe washout. In the basal state, adipose tissue blood flow tended to be higher in T compared with S subjects, and in both groups blood flow was constant during the clamp. The change from basal in arterial-interstitial glucose concentration......-time: T, 44 +/- 9 min (n = 7); S, 102 +/- 23 min (n = 5); P training enhances insulin sensitivity of glucose uptake in subcutaneous adipose tissue and in skeletal muscle. Furthermore, interstitial glycerol data suggest that training also increases insulin sensitivity of lipolysis...

  9. Aspiration lung disorders in bovines: a case report and review.

    Science.gov (United States)

    Shakespeare, Anthony S

    2012-11-01

    Lung aspiration disorders in bovines are invariably diagnosed as infectious aspiration pneumonias. There is a distinct differentiation between aspiration pneumonia and aspiration pneumonitis in humans that can be applied to bovines. The nature and quantity of the aspirate can result in differing pathogeneses which can require differing therapeutic approaches. Whilst blood gases were important in detecting and prognosticating lung problems, changes in barometric pressure with altitude have to be considered when interpreting partial pressures of oxygen. Anatomical differences in the lungs of bovines can explain why this species is more prone to certain pneumonic problems. Pulmonary physiotherapy is important in treating lung disorders in humans and should be considered as an adjunct therapy in bovine respiratory conditions. A case work-up was used to highlight some of the points discussed in this article.

  10. Cephalic phase secretion of insulin and other enteropancreatic hormones in humans

    DEFF Research Database (Denmark)

    Veedfald, Simon; Plamboeck, Astrid; Deacon, Carolyn F

    2016-01-01

    Enteropancreatic hormone secretion is thought to include a cephalic phase, but the evidence in humans is ambiguous. We studied vagally induced gut hormone responses with and without muscarinic blockade in 10 glucose-clamped healthy men (age: 24.5 ± 0.6 yr, means ± SE; body mass index: 24.0 ± 0.5 kg...... and abolished the MSF response. Neither insulin, C-peptide, glucose-dependent insulinotropic polypeptide (GIP), nor glucagon-like peptide-1 (GLP-1) levels changed in response to MSF or atropine. Glucagon and ghrelin levels were markedly attenuated by atropine prior to and during the clamp: at t = 105 min...... and 3.7 ± 21 pg/ml (means ± SE), P phase response was absent for insulin, glucagon, GLP-1, GIP, and ghrelin....

  11. Methicillin resistant staphylococci associated with bovine mastitis and their zoonotic importance

    Directory of Open Access Journals (Sweden)

    S. Vishnupriya

    2014-06-01

    Full Text Available Aim: The present study was conducted to determine the zoonotic importance of methicillin resistant staphylococci associated with bovine mastitis and their potential role in transmission to animal handlers. Materials and Methods: A total of 158 milk samples from bovine mastitis cases and 126 nasal swabs from the animal handlers were sampled in and around Pondicherry (Southern India. The Presence of Staphylococcal organism was confirmed by PCR amplification using the genus specific primers and among the isolated Staphylococci; methicillin resistance was identified by genetic amplification of mec A methicillin resistant gene. Then the amplified gene from the bacteria expressing the mecA gene (PBP2a (~2kb fragment was further sequenced using four sets of primer pairs and aligned for determining their genetic relatedness between the sequences. Both phenotypic and genotypic analysis was carried out for the six MRS isolates (three bovine and three human in this study. Results: Out of 158 mastitis milk samples; 96 and 19 bovine isolates were found to be positive for Staphylococcal genus specific PCR and methicillin resistant (mecA gene PCR, respectively. Similarly, Out of 126 human nasal swabs, 64 and 13 human isolates were found to be positive for Staphylococcal genus specific PCR and mec A gene PCR, respectively. Among the 160 staphylococcal isolates (Bovine and Human origin; 51 were identified as coagulase-positive staphylococci (CPS and remaining as coagulase-negative staphylococci (CONS. The results obtained in this study revealed the presence of many species of Staphylococci but the predominant species were Staphylococcus aureus and S. epidermidis. The Sequence analysis of the mec A gene of human isolates obtained in this study had a maximum identity (99% -100% with the bovine isolates. Conclusion: The phenotypic and genotypic analysis carried out for the six MRS (Methicillin Resistant Staphylococci isolates in this study were indistinguishable

  12. PREVALENCE OF BOVINE HERPESVIRUS-1,PARAINFLUENZA-3,BOVINE ROTAVIRUS, BOVINE VIRAL DIARRHEA, BOVINE ADENOVIRUS-7,BOVINE LEUKEMIA VIRUS AND BLUETONGUE VIRUS ANTIBODIES IN CATTLE IN MEXICO

    OpenAIRE

    SUZAN, Victor M.; ONUMA, Misao; AGUILAR, Romero E.; MURAKAMI, Yosuke

    1983-01-01

    Sera were collected from dairy and beef cattle in 19 different states of Mexico. These sera were tested for bovine herpesvirus-1 (BHV-1), parainfluenza-3 virus (PIV-3), bovine rotavirus (BRV), bovine leukemia virus (BLV), bovine adenovirus-7 (BAV-7), bluetongue virus (BTV) and bovine viral diarrhea virus (BVDV). Seropositive rates for each virus for dairy cattle tested were 158/277(57.0%) for BHV-1,217/286(75.0%) for PIV-3,541/1498(36.1%) for BLV, 134/144(93.1%) for BRV, 39/90(43.3%) for BTV,...

  13. BOVINE TUBERCULOSIS (BTB) AS A RISK FACTOR FOR DEVELOPING TUBERCULOSIS IN HUMANS IN THE RURAL COMMUNITY OF ETHIOPIA: A CASE-CONTROL STUDY.

    Science.gov (United States)

    Mengistu, Araya; Enquselassi, Fikre; Aseffa, Abraham; Beyen, Demissew

    2015-01-01

    The current study aimed at assessing BTB as a possible risk factor for human TB in the rural community of North Eastern and Western parts of Ethiopia. A case-control design was conducted among cattle owning households with TB and without TB. Comparative cervical intradermal test using purified protein derivatives were used to test cattle. Reading of the reaction was done 72 ± 4hrs after antigen injection. Based on the skin test reaction measurement, cattle categorized as negative, doubtful and positive. Questionnaires were used to collect the required factors. Thirty-five with TB and 105 households without TB participated in this study of which 49.3% and 61.4% had the habit of drinking raw milk and eating uncooked meat, respectively. About 70.7% knew about the chance of disease transmission from animals to humans. Among the TB households 31.43% shared their house with their cattle. Of the attendants, approximately 38% shared utensil. Based on > 2mms as a cutoff value 23.6% an overall apparent bovine tuberculosis (BTB) and 48.6% apparent BTB in households with TB were recorded. The odds for households having bovine TB in their cattle to get tuberculosis was more than 8 times (95% CI; 2.82-24.60) higher than those owned by households without TB. Bovine TB has been seen as an exposure to human pulmonary TB occurrence. A separate house for cattle should be constructed to minimize the fear of cross infections and further study regarding the possible infection of cattle with M. tuberculosis is suggested. Key wordsi bovine tuberculosis, households, human TB, M. tuberculosis, risk.

  14. In nondiabetic, human immunodeficiency virus-infected patients with lipodystrophy, hepatic insulin extraction and posthepatic insulin clearance rate are decreased in proportion to insulin resistance

    DEFF Research Database (Denmark)

    Haugaard, Steen B; Andersen, Ove; Hansen, Birgitte R

    2005-01-01

    In healthy, nondiabetic individuals with insulin resistance, fasting insulin is inversely correlated to the posthepatic insulin clearance rate (MCRi) and the hepatic insulin extraction (HEXi). We investigated whether similar early mechanisms to facilitate glucose homeostasis exist in nondiabetic...... > .1). Our data suggest that HEXi and MCRi are decreased in proportion to the degree of insulin resistance in nondiabetic HIV-infected patients with lipodystrophy....... insulin clearance rate was estimated as the ratio of posthepatic insulin appearance rate to steady-state plasma insulin concentration during a euglycemic hyperinsulinemic clamp (40 mU.m-2 .min-1). Posthepatic insulin appearance rate during the clamp was calculated, taking into account the remnant...

  15. Insulin acutely improves mitochondrial function of rat and human skeletal muscle by increasing coupling efficiency of oxidative phosphorylation.

    Science.gov (United States)

    Nisr, Raid B; Affourtit, Charles

    2014-02-01

    Insulin is essential for the regulation of fuel metabolism and triggers the uptake of glucose by skeletal muscle. The imported glucose is either stored or broken down, as insulin stimulates glycogenesis and ATP synthesis. The mechanism by which ATP production is increased is incompletely understood at present and, generally, relatively little functional information is available on the effect of insulin on mitochondrial function. In this paper we have exploited extracellular flux technology to investigate insulin effects on the bioenergetics of rat (L6) and human skeletal muscle myoblasts and myotubes. We demonstrate that a 20-min insulin exposure significantly increases (i) the cell respiratory control ratio, (ii) the coupling efficiency of oxidative phosphorylation, and (iii) the glucose sensitivity of anaerobic glycolysis. The improvement of mitochondrial function is explained by an insulin-induced immediate decrease of mitochondrial proton leak. Palmitate exposure annuls the beneficial mitochondrial effects of insulin. Our data improve the mechanistic understanding of insulin-stimulated ATP synthesis, and reveal a hitherto undisclosed insulin sensitivity of cellular bioenergetics that suggests a novel way of detecting insulin responsiveness of cells. © 2013.

  16. Insulin acutely improves mitochondrial function of rat and human skeletal muscle by increasing coupling efficiency of oxidative phosphorylation☆

    Science.gov (United States)

    Nisr, Raid B.; Affourtit, Charles

    2014-01-01

    Insulin is essential for the regulation of fuel metabolism and triggers the uptake of glucose by skeletal muscle. The imported glucose is either stored or broken down, as insulin stimulates glycogenesis and ATP synthesis. The mechanism by which ATP production is increased is incompletely understood at present and, generally, relatively little functional information is available on the effect of insulin on mitochondrial function. In this paper we have exploited extracellular flux technology to investigate insulin effects on the bioenergetics of rat (L6) and human skeletal muscle myoblasts and myotubes. We demonstrate that a 20-min insulin exposure significantly increases (i) the cell respiratory control ratio, (ii) the coupling efficiency of oxidative phosphorylation, and (iii) the glucose sensitivity of anaerobic glycolysis. The improvement of mitochondrial function is explained by an insulin-induced immediate decrease of mitochondrial proton leak. Palmitate exposure annuls the beneficial mitochondrial effects of insulin. Our data improve the mechanistic understanding of insulin-stimulated ATP synthesis, and reveal a hitherto undisclosed insulin sensitivity of cellular bioenergetics that suggests a novel way of detecting insulin responsiveness of cells. PMID:24212054

  17. Resveratrol ameliorates the chemical and microbial induction of inflammation and insulin resistance in human placenta, adipose tissue and skeletal muscle.

    Science.gov (United States)

    Tran, Ha T; Liong, Stella; Lim, Ratana; Barker, Gillian; Lappas, Martha

    2017-01-01

    Gestational diabetes mellitus (GDM), which complicates up to 20% of all pregnancies, is associated with low-grade maternal inflammation and peripheral insulin resistance. Sterile inflammation and infection are key mediators of this inflammation and peripheral insulin resistance. Resveratrol, a stilbene-type phytophenol, has been implicated to exert beneficial properties including potent anti-inflammatory and antidiabetic effects in non-pregnant humans and experimental animal models of GDM. However, studies showing the effects of resveratrol on inflammation and insulin resistance associated with GDM in human tissues have been limited. In this study, human placenta, adipose (omental and subcutaneous) tissue and skeletal muscle were stimulated with pro-inflammatory cytokines TNF-α and IL-1β, the bacterial product lipopolysaccharide (LPS) and the synthetic viral dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) to induce a GDM-like model. Treatment with resveratrol significantly reduced the expression and secretion of pro-inflammatory cytokines IL-6, IL-1α, IL-1β and pro-inflammatory chemokines IL-8 and MCP-1 in human placenta and omental and subcutaneous adipose tissue. Resveratrol also significantly restored the defects in the insulin signalling pathway and glucose uptake induced by TNF-α, LPS and poly(I:C). Collectively, these findings suggest that resveratrol reduces inflammation and insulin resistance induced by chemical and microbial products. Resveratrol may be a useful preventative therapeutic for pregnancies complicated by inflammation and insulin resistance, like GDM.

  18. Effects of inhibition of interleukin-6 signalling on insulin sensitivity and lipoprotein (a levels in human subjects with rheumatoid diseases.

    Directory of Open Access Journals (Sweden)

    Olaf Schultz

    2010-12-01

    Full Text Available Interleukin-6 (IL-6 is a pro-inflammatory cytokine that has been found to be increased in type 2 diabetic subjects. However, it still remains unclear if these elevated IL-6 levels are co-incidental or if this cytokine is causally related to the development of insulin resistance and type 2 diabetes in humans. Therefore, in the present study we examined insulin sensitivity, serum adipokine levels and lipid parameters in human subjects before and after treatment with the IL-6 receptor antibody Tocilizumab.11 non-diabetic patients with rheumatoid disease were included in the study. HOMA-IR was calculated and serum levels for leptin, adiponectin, triglycerides, LDL-cholesterol, HDL-cholesterol and lipoprotein (a (Lp (a were measured before as well as one and three months after Tocilizumab treatment. The HOMA index for insulin resistance decreased significantly. While leptin concentrations were not altered by inhibition of IL-6 signalling, adiponectin concentrations significantly increased. Thus the leptin to adiponectin ratio, a novel marker for insulin resistance, exhibited a significant decrease. Serum triglycerides, LDL-cholesterol and HDL-cholesterol tended to be increased whereas Lp (a levels significantly decreased.Inhibition of IL-6 signalling improves insulin sensitivity in humans with immunological disease suggesting that elevated IL-6 levels in type 2 diabetic subjects might be causally involved in the pathogenesis of insulin resistance. Furthermore, our data indicate that inhibition of IL-6 signalling decreases Lp (a serum levels, which might reduce the cardiovascular risk of human subjects.

  19. Effect of starvation on human muscle protein metabolism and its response to insulin

    International Nuclear Information System (INIS)

    Fryburg, D.A.; Barrett, E.J.; Louard, R.J.; Gelfand, R.A.

    1990-01-01

    To assess the effect of fasting on muscle protein turnover in the basal state and in response to insulin, we measured forearm amino acid kinetics, using [3H]phenylalanine (Phe) and [14C]leucine (Leu) infused systemically, in eight healthy subjects after 12 (postabsorptive) and 60 h of fasting. After a 150-min basal period, forearm local insulin concentration was selectively raised by approximately 25 muU/ml for 150 min by intra-arterial insulin infusion (0.02 mU.kg-1. min-1). The 60-h fast increased urine nitrogen loss and whole body Leu flux and oxidation (by 50-75%, all P less than 0.02). Post-absorptively, forearm muscle exhibited a net release of Phe and Leu, which increased two- to threefold after the 60-h fast (P less than 0.05); this effect was mediated exclusively by accelerated local rates of amino acid appearance (Ra), with no reduction in rates of disposal (Rd). Local hyperinsulinemia in the postabsorptive condition caused a twofold increase in forearm glucose uptake (P less than 0.01) and completely suppressed the net forearm output of Phe and Leu (P less than 0.02). After the 60-h fast, forearm glucose disposal was depressed basally and showed no response to insulin; in contrast, insulin totally abolished the accelerated net forearm release of Phe and Leu. The action of insulin to reverse the augmented net release of Phe and Leu was mediated exclusively by approximately 40% suppression of Ra (P less than 0.02) rather than a stimulation of Rd. We conclude that in short-term fasted humans (1) muscle amino acid output accelerates due to increased proteolysis rather than reduced protein synthesis, and (2) despite its catabolic state and a marked impairment in insulin-mediated glucose disposal, muscle remains sensitive to insulin's antiproteolytic action

  20. Effect of starvation on human muscle protein metabolism and its response to insulin

    Energy Technology Data Exchange (ETDEWEB)

    Fryburg, D.A.; Barrett, E.J.; Louard, R.J.; Gelfand, R.A. (Yale Univ. School of Medicine, New Haven, CT (USA))

    1990-10-01

    To assess the effect of fasting on muscle protein turnover in the basal state and in response to insulin, we measured forearm amino acid kinetics, using (3H)phenylalanine (Phe) and (14C)leucine (Leu) infused systemically, in eight healthy subjects after 12 (postabsorptive) and 60 h of fasting. After a 150-min basal period, forearm local insulin concentration was selectively raised by approximately 25 muU/ml for 150 min by intra-arterial insulin infusion (0.02 mU.kg-1. min-1). The 60-h fast increased urine nitrogen loss and whole body Leu flux and oxidation (by 50-75%, all P less than 0.02). Post-absorptively, forearm muscle exhibited a net release of Phe and Leu, which increased two- to threefold after the 60-h fast (P less than 0.05); this effect was mediated exclusively by accelerated local rates of amino acid appearance (Ra), with no reduction in rates of disposal (Rd). Local hyperinsulinemia in the postabsorptive condition caused a twofold increase in forearm glucose uptake (P less than 0.01) and completely suppressed the net forearm output of Phe and Leu (P less than 0.02). After the 60-h fast, forearm glucose disposal was depressed basally and showed no response to insulin; in contrast, insulin totally abolished the accelerated net forearm release of Phe and Leu. The action of insulin to reverse the augmented net release of Phe and Leu was mediated exclusively by approximately 40% suppression of Ra (P less than 0.02) rather than a stimulation of Rd. We conclude that in short-term fasted humans (1) muscle amino acid output accelerates due to increased proteolysis rather than reduced protein synthesis, and (2) despite its catabolic state and a marked impairment in insulin-mediated glucose disposal, muscle remains sensitive to insulin's antiproteolytic action.

  1. [Solid state isotope hydrogen exchange for deuterium and tritium in human gene-engineered insulin].

    Science.gov (United States)

    Zolotarev, Yu A; Dadayan, A K; Kozik, V S; Gasanov, E V; Nazimov, I V; Ziganshin, R Kh; Vaskovsky, B V; Murashov, A N; Ksenofontov, A L; Haribin, O N; Nikolaev, E N; Myasoedov, N F

    2014-01-01

    The reaction of high temperature solid state catalytic isotope exchange in peptides and proteins under the action of catalyst-activated spillover hydrogen was studied. The reaction of human gene-engineered insulin with deuterium and tritium was conducted at 120-140° C to produce insulin samples containing 2-6 hydrogen isotope atoms. To determine the distribution of the isotope label over tritium-labeled insulin's amino acid residues, oxidation of the S-S bonds of insulin by performic acid was performed and polypeptide chains isolated; then their acid hydrolysis, amino acid analysis and liquid scintillation counts of tritium in the amino acids were conducted. The isotope label was shown to be incorporated in all amino acids of the protein, with the peptide fragment FVNQHLCGSHLVE of the insulin β-chain showing the largest incorporation. About 45% of the total protein isotope label was incorporated in His5 and His10 of this fragment. For the analysis of isotope label distribution in labeled insulin's peptide fragments, the recovery of the S-S bonds by mercaptoethanol, the enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius and HPLC division of the resulting peptides were carried out. Attribution of the peptide fragments formed due to hydrolysis at the Glu-X bond in the β-chain was accomplished by mass spectrometry. Mass spectrometry analysis data of the deuterium-labeled insulin samples' isotopomeric composition showed that the studied solid state isotope exchange reaction equally involved all the protein molecules. Biological studying of tritium-labeled insulin showed its physiological activity to be completely retained.

  2. Zinc stimulates glucose oxidation and glycemic control by modulating the insulin signaling pathway in human and mouse skeletal muscle cell lines.

    Science.gov (United States)

    Norouzi, Shaghayegh; Adulcikas, John; Sohal, Sukhwinder Singh; Myers, Stephen

    2018-01-01

    Zinc is a metal ion that is an essential cell signaling molecule. Highlighting this, zinc is an insulin mimetic, activating cellular pathways that regulate cellular homeostasis and physiological responses. Previous studies have linked dysfunctional zinc signaling with several disease states including cancer, obesity, cardiovascular disease and type 2 diabetes. The present study evaluated the insulin-like effects of zinc on cell signaling molecules including tyrosine, PRSA40, Akt, ERK1/2, SHP-2, GSK-3β and p38, and glucose oxidation in human and mouse skeletal muscle cells. Insulin and zinc independently led to the phosphorylation of these proteins over a 60-minute time course in both mouse and human skeletal muscle cells. Similarly, utilizing a protein array we identified that zinc could active the phosphorylation of p38, ERK1/2 and GSK-3B in human and ERK1/2 and GSK-3B in mouse skeletal muscle cells. Glucose oxidation assays were performed on skeletal muscle cells treated with insulin, zinc, or a combination of both and resulted in a significant induction of glucose consumption in mouse (pzinc alone. Insulin, as expected, increased glucose oxidation in mouse (pzinc and insulin did not augment glucose consumption in these cells. Zinc acts as an insulin mimetic, activating key molecules implicated in cell signaling to maintain glucose homeostasis in mouse and human skeletal muscle cells. Zinc is an important metal ion implicated in several biological processes. The role of zinc as an insulin memetic in activating key signaling molecules involved in glucose homeostasis could provide opportunities to utilize this ion therapeutically in treating disorders associated with dysfunctional zinc signaling.

  3. Reduced malonyl-CoA content in recovery from exercise correlates with improved insulin-stimulated glucose uptake in human skeletal muscle

    DEFF Research Database (Denmark)

    Frøsig, Christian; Roepstorff, Carsten; Brandt, Nina

    2009-01-01

    This study evaluated whether improved insulin-stimulated glucose uptake in recovery from acute exercise coincides with reduced malonyl-CoA (MCoA) content in human muscle. Furthermore, we investigated whether a high-fat diet [65 energy-% (Fat)] would alter the content of MCoA and insulin action...... to be compromised, although to a minor extent, by the Fat diet. Collectively, this study indicates that reduced muscle MCoA content in recovery from exercise may be part of the adaptive response leading to improved insulin action on glucose uptake after exercise in human muscle....

  4. Phospholipase C δ-type consists of three isozymes: bovine PLCδ2 is a homologue of human/mouse PLCδ4

    International Nuclear Information System (INIS)

    Irino, Yasuhiro; Cho, Hiroyuki; Nakamura, Yoshikazu; Nakahara, Masamichi; Furutani, Masahiro; Suh, Pann-Ghill; Takenawa, Tadaomi; Fukami, Kiyoko

    2004-01-01

    To date, 12 phospholipase C (PLC) isozymes have been identified in mammals, and they are divided into five classes, β-, γ-, δ-, ε-, and ζ-type. PLCδ-type is reported to be composed of four isozymes, PLCδ1-δ4. Here we report that a screening for mouse PLCδ2 from a BAC library with primers that amplify a specific region of bovine PLCδ2 resulted in isolation of one clone containing the mouse PLCδ4 gene. Furthermore, a database search revealed that there is only one gene corresponding to PLCδ2 and PLCδ4 in the mouse and human genomes, indicating that bovine PLCδ2 is a homologue of human and mouse PLCδ4. However, PLCδ2 Western blot analysis with a widely used commercial anti-PLCδ2 antibody showed an expression pattern distinct from that of PLCδ4 in wild-type mice. In addition, an 80-kDa band, which was recognized by antibody against PLCδ2, was smaller than an 85-kDa band detected by anti-PLCδ4 antibody, and the 80-kDa band was detectable in lysates of brain, testis, and spleen from PLCδ4-deficient mice. We also found that immunoprecipitates from brain lysates with this PLCδ2 antibody contained no PLC activity. From these data, we conclude that bovine PLCδ2 is a homologue of human and mouse PLCδ4, and that three isozymes (δ1, δ3, and δ4) exist in the PLCδ family

  5. Similar efficacy of human banked milk and bovine colostrum to decrease incidence of necrotizing enterocolitis in preterm piglets

    DEFF Research Database (Denmark)

    Jensen, Michael L.; Sangild, Per Torp; Lykke, Mikkel

    2013-01-01

    Preterm birth and formula feeding predispose to necrotizing enterocolitis (NEC) in infants. As mother's milk is often absent following preterm delivery, infant formula (IF) and human donor milk (HM) are frequently used as alternatives. We have previously shown that porcine and bovine colostrum (BC...

  6. Two Cases of Allergy to Insulin in Gestational Diabetes

    Directory of Open Access Journals (Sweden)

    Gi Jun Kim

    2015-09-01

    Full Text Available Allergic reaction to insulin is uncommon since the introduction of human recombinant insulin preparations and is more rare in pregnant than non-pregnant females due to altered immune reaction during pregnancy. Herein, we report two cases of allergic reaction to insulin in gestational diabetes that were successfully managed. One case was a 33-year-old female using isophane-neutral protamine Hagedorn human insulin and insulin lispro. She experienced dyspnea, cough, urticaria and itching sensation at the sites of insulin injection immediately after insulin administration. We discontinued insulin therapy and started oral hypoglycemic agents with metformin and glibenclamide. The other case was a 32-year-old female using insulin lispro and insulin detemer. She experienced pruritus and burning sensation and multiple nodules at the sites of insulin injection. We changed the insulin from insulin lispro to insulin aspart. Assessments including immunoglobulin E (IgE, IgG, eosinophil, insulin antibody level and skin biopsy were performed. In the two cases, the symptoms were resolved after changing the insulin to oral agents or other insulin preparations. We report two cases of allergic reaction to human insulin in gestational diabetes due to its rarity.

  7. Insulin analogues with improved absorption characteristics.

    Science.gov (United States)

    Brange, J; Hansen, J F; Langkjaer, L; Markussen, J; Ribel, U; Sørensen, A R

    1992-01-01

    The insulin preparations available today are not ideal for therapy as s.c. injection does not provide a physiological insulin profile. With the aim to improve the absorption properties recombinant DNA technology has been utilized to design novel insulin molecules with changed physico-chemical characteristics and hence altered subcutaneous absorption kinetics. Soluble, long-acting human insulin analogues in which the isoelectric point has been increased from 5.4 to approx. 7 are absorbed very slowly, providing a more constant basal insulin delivery with lower day-to-day variation than present protracted preparations. In addition they have better storage stability. Rapid-acting human insulin analogues with largely reduced self-association are absorbed substantially faster from subcutaneous tissue than current regular insulin and thus are better suited for bolus injection. The absorption kinetics of these analogues have been able to explain the mechanism behind the dose effect on insulin absorption rate.

  8. Novel covalently linked insulin dimer engineered to investigate the function of insulin dimerization

    DEFF Research Database (Denmark)

    Vinther, Tine N.; Norrman, Mathias; Strauss, Holger M.

    2012-01-01

    An ingenious system evolved to facilitate insulin binding to the insulin receptor as a monomer and at the same time ensure sufficient stability of insulin during storage. Insulin dimer is the cornerstone of this system. Insulin dimer is relatively weak, which ensures dissociation into monomers...... in the circulation, and it is stabilized by hexamer formation in the presence of zinc ions during storage in the pancreatic ß-cell. Due to the transient nature of insulin dimer, direct investigation of this important form is inherently difficult. To address the relationship between insulin oligomerization...... and insulin stability and function, we engineered a covalently linked insulin dimer in which two monomers were linked by a disulfide bond. The structure of this covalent dimer was identical to the self-association dimer of human insulin. Importantly, this covalent dimer was capable of further oligomerization...

  9. Human pituitary and placental hormones control human insulin-like growth factor II secretion in human granulosa cells

    International Nuclear Information System (INIS)

    Ramasharma, K.; Li, C.H.

    1987-01-01

    Human granulosa cells cultured with calf serum actively proliferated for 18-20 generations and secreted progesterone into the medium; progesterone levels appeared to decline with increase in generation number. Cells cultured under serum-free conditions secreted significant amounts of progesterone and insulin-like growth factor II (IGF-II). The progesterone secretion was enhanced by the addition of human follitropin, lutropin, and chorionic gonadotropin but not by growth hormone. These cells, when challenged to varying concentrations of human growth hormone, human chorionic somatomammotropin, human prolactin, chorionic gonadotropin, follitropin, and lutropin, secreted IGF-II into the medium as measured by specific IGF-II RIA. Among these human hormones, chorionic gonadotropin, follitropin, and lutropin were most effective in inducing IGF-II secretion from these cells. When synthetic lutropin-releasing hormone and α-inhibin-92 were tested, only lutropin-releasing hormone was effective in releasing IGF-II. The results described suggest that cultured human granulosa cells can proliferate and actively secrete progesterone and IGF-II into the medium. IGF-II production in human granulosa cells was influenced by a multi-hormonal complex including human growth hormone, human chorionic somatomammotropin, and prolactin

  10. Insulin analogues: have they changed insulin treatment and improved glycaemic control?

    DEFF Research Database (Denmark)

    Madsbad, Sten

    2002-01-01

    To improve insulin therapy, new insulin analogues have been developed. Two fast-acting analogues with a more rapid onset of effect and a shorter duration of action combined with a low day-to-day variation in absorption rate are now available. Despite this favourable time-action profile most studies....... This is probably the main explanation for the absence of improvement in overall glycaemic control when compared with regular human insulin. A tendency to a reduction in hypoglycaemic events during treatment with fast-acting analogues has been observed in most studies. Recent studies have indicated that NPH insulin...... administered several times daily at mealtimes can improve glycaemic control without increasing the risk of hypoglycaemia. The fast-acting analogues are now also available as insulin mixed with NPH. Insulin glargine is a new long-acting insulin which is soluble and precipitates after injection, resulting...

  11. Interaction of bovine peripheral blood polymorphonuclear cells and Leptospira species; innate responses in the natural bovine reservoir host.

    Directory of Open Access Journals (Sweden)

    Jennifer H Wilson-Welder

    2016-07-01

    Full Text Available Cattle are the reservoir hosts of Leptospira borgpetersenii serovar Hardjo, and can also be reservoir hosts of other Leptospira species such as L. kirschneri, and L. interrogans. As a reservoir host, cattle shed Leptospira, infecting other animals, including humans. Previous studies with human and murine neutrophils have shown activation of neutrophil extracellular trap or NET formation, and upregulation of inflammatory mediators by neutrophils in the presence of Leptospira. Humans, companion animals and most widely studied models of Leptospirosis are of acute infection, hallmarked by systemic inflammatory response, neutrophilia and septicemia. In contrast, cattle exhibit chronic infection with few outward clinical signs aside from reproductive failure. Taking into consideration that there is host species variation in innate immunity, especially in pathogen recognition and response, the interaction of bovine peripheral blood polymorphonuclear cells (PMNs and several Leptospira strains was evaluated. Studies including bovine-adapted strains, human pathogen strains, a saprophyte and inactivated organisms. Incubation of PMNs with Leptospira did induce slight activation of neutrophil NETs, greater than unstimulated cells but less than the quantity from E. coli P4 stimulated PMNs. Very low but significant from non-stimulated, levels of reactive oxygen peroxides were produced in the presence of all Leptospira strains and E. coli P4. Similarly, significant levels of reactive nitrogen intermediaries (NO2 was produced from PMNs when incubated with the Leptospira strains and greater quantities in the presence of E. coli P4. PMNs incubated with Leptospira induced RNA transcripts of IL-1β, MIP-1α, and TNF-α, with greater amounts induced by live organisms when compared to heat-inactivated leptospires. Transcript for inflammatory cytokine IL-8 was also induced, at similar levels regardless of Leptospira strain or viability. However, incubation of

  12. Effect of Technological Treatments on Human-Like Leptin Level in Bovine Milk for Human Consumption

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    Damiano Magistrelli

    2014-07-01

    Full Text Available In this experiment, raw milk and commercially available full-cream UHT milk, semi-skimmed UHT milk, skimmed UHT milk, full-cream pasteurized milk, semi-skimmed pasteurized milk and infant formulas for babies between 6 and 12 months of age were analyzed by RIA, with a method using an antibody directed against human leptin and human leptin as reference standard. Raw milk and full-cream UHT milk did not differ for human-like leptin. Leptin content of full-cream pasteurized milk was not different to that of full-cream UHT milk, but it was 14% lower (p < 0.05 than that observed in raw milk. Human-like leptin level of semi-skimmed UHT milk was not different to that of semi-skimmed pasteurized milk, but it was 30% lower (p < 0.0001 than those of full-cream UHT and full-cream pasteurized milks. In skimmed UHT milk, leptin was 40% lower (p < 0.0001 than in full-cream UHT milk. Leptin was correlated (p < 0.001 with lipid content. Leptin level of infant formulas was not different to that of skimmed milks. Results suggest that the heat treatment (pasteurization or UHT is not a modifier of human-like leptin content of edible commercial bovine milks, whereas the skimming process significantly reduces milk leptin level.

  13. Aspiration lung disorders in bovines: A case report and review

    Directory of Open Access Journals (Sweden)

    Anthony S. Shakespeare

    2012-04-01

    Full Text Available Lung aspiration disorders in bovines are invariably diagnosed as infectious aspiration pneumonias. There is a distinct differentiation between aspiration pneumonia and aspiration pneumonitis in humans that can be applied to bovines. The nature and quantity of the aspirate can result in differing pathogeneses which can require differing therapeutic approaches. Whilst blood gases were important in detecting and prognosticating lung problems, changes in barometric pressure with altitude have to be considered when interpreting partial pressures of oxygen. Anatomical differences in the lungs of bovines can explain why this species is more prone to certain pneumonic problems. Pulmonary physiotherapy is important in treating lung disorders in humans and should be considered as an adjunct therapy in bovine respiratory conditions. A case work-up was used to highlight some of the points discussed in this article.

  14. Aspiration lung disorders in bovines: A case report and review

    Directory of Open Access Journals (Sweden)

    Anthony S. Shakespeare

    2012-11-01

    Full Text Available Lung aspiration disorders in bovines are invariably diagnosed as infectious aspiration pneumonias. There is a distinct differentiation between aspiration pneumonia and aspiration pneumonitis in humans that can be applied to bovines. The nature and quantity of the aspirate can result in differing pathogeneses which can require differing therapeutic approaches. Whilst blood gases were important in detecting and prognosticating lung problems, changes in barometric pressure with altitude have to be considered when interpreting partial pressures of oxygen. Anatomical differences in the lungs of bovines can explain why this species is more prone to certain pneumonic problems. Pulmonary physiotherapy is important in treating lung disorders in humans and should be considered as an adjunct therapy in bovine respiratory conditions. A case work-up was used to highlight some of the points discussed in this article.

  15. Characterization of Bovine 5′-flanking Region during Differentiation of Mouse Embryonic Stem Cells

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    Hye-Jeong Jang

    2015-12-01

    Full Text Available Embryonic stem cells (ESCs have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (−420/+181 bovine NANOG 5′-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (−420/+181 promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells.

  16. Effect of potassium and hypomagnesemia on insulin in the bovine

    Energy Technology Data Exchange (ETDEWEB)

    Lentz, D.E.; Madsen, F.C.; Miller, J.K.; Hansard, S.L.

    1976-01-01

    Grass tetany in cattle has been associated with the consumption of early spring forages high in potassium (K) and low in magnesium (Mg). Alterations in serum Mg and K may affect intermediary carbohydrate metabolism, resulting in hypoglycemia and ketosis that often accompany grass tetany. We investigated these interrelationships by infusing potassium chloride (KCl) intravenously in normal (plasma Mg greater than 2.1 mg/100 ml) and Mg-deficient (plasma Mg less than .7 mg/100 ml) 9-month-old Holstein bull calves and intraruminally into nonpregnant, nonlactating Holstein cows. Plasma levels of both K and immunoreactive insulin (IRI) were elevated (P less than .01) by 1.14, 2, and 3 percent KCl (51, 64, and 135 mg K/kg) in calves and by 550 g KCl (440 mg K/kg body weight) in cows. Plasma K was lower (P less than .01) and IRI higher (P less than .01) in Mg-deficient calves than in normal calves during 2 percent KCl infusion. These results suggest that prolonged elevation of K and insulin in ruminants could lead to a series of metabolic disturbances that may play an important role in the etiology of grass tetany.

  17. LukMF′ is the major secreted leukocidin of bovine Staphylococcus aureus and is produced in vivo during bovine mastitis

    Science.gov (United States)

    Vrieling, Manouk; Boerhout, Eveline M.; van Wigcheren, Glenn F.; Koymans, Kirsten J.; Mols-Vorstermans, Tanja G.; de Haas, Carla J. C.; Aerts, Piet C.; Daemen, Ineke J. J. M.; van Kessel, Kok P. M.; Koets, Ad P.; Rutten, Victor P. M. G.; Nuijten, Piet J.M.; van Strijp, Jos A. G.; Benedictus, Lindert

    2016-01-01

    Staphylococcus aureus is a major human and animal pathogen and a common cause of mastitis in cattle. S. aureus secretes several leukocidins that target bovine neutrophils, crucial effector cells in the defence against bacterial pathogens. In this study, we investigated the role of staphylococcal leukocidins in the pathogenesis of bovine S. aureus disease. We show that LukAB, in contrast to the γ-hemolysins, LukED, and LukMF′, was unable to kill bovine neutrophils, and identified CXCR2 as a bovine receptor for HlgAB and LukED. Furthermore, we assessed functional leukocidin secretion by bovine mastitis isolates and observed that, although leukocidin production was strain dependent, LukMF′ was most abundantly secreted and the major toxin killing bovine neutrophils. To determine the role of LukMF′ in bovine mastitis, cattle were challenged with high (S1444) or intermediate (S1449, S1463) LukMF′-producing isolates. Only animals infected with S1444 developed severe clinical symptoms. Importantly, LukM was produced in vivo during the course of infection and levels in milk were associated with the severity of mastitis. Altogether, these findings underline the importance of LukMF′ as a virulence factor and support the development of therapeutic approaches targeting LukMF′ to control S. aureus mastitis in cattle. PMID:27886237

  18. Interacting with the Human Insulin Receptor

    DEFF Research Database (Denmark)

    Kidmose, Rune Thomas; Andersen, Gregers Rom

    2016-01-01

    Insulin is an essential regulator of glucose homeostasis. In this issue of Structure, Croll et al. (2016) reports a significantly improved model of the Fab-complexed IR ectodomain refined against a dataset extending to 3.3 Å.......Insulin is an essential regulator of glucose homeostasis. In this issue of Structure, Croll et al. (2016) reports a significantly improved model of the Fab-complexed IR ectodomain refined against a dataset extending to 3.3 Å....

  19. From the Cover: Cell-replacement therapy for diabetes: Generating functional insulin-producing tissue from adult human liver cells

    Science.gov (United States)

    Sapir, Tamar; Shternhall, Keren; Meivar-Levy, Irit; Blumenfeld, Tamar; Cohen, Hamutal; Skutelsky, Ehud; Eventov-Friedman, Smadar; Barshack, Iris; Goldberg, Iris; Pri-Chen, Sarah; Ben-Dor, Lya; Polak-Charcon, Sylvie; Karasik, Avraham; Shimon, Ilan; Mor, Eytan; Ferber, Sarah

    2005-05-01

    Shortage in tissue availability from cadaver donors and the need for life-long immunosuppression severely restrict the large-scale application of cell-replacement therapy for diabetic patients. This study suggests the potential use of adult human liver as alternate tissue for autologous beta-cell-replacement therapy. By using pancreatic and duodenal homeobox gene 1 (PDX-1) and soluble factors, we induced a comprehensive developmental shift of adult human liver cells into functional insulin-producing cells. PDX-1-treated human liver cells express insulin, store it in defined granules, and secrete the hormone in a glucose-regulated manner. When transplanted under the renal capsule of diabetic, immunodeficient mice, the cells ameliorated hyperglycemia for prolonged periods of time. Inducing developmental redirection of adult liver offers the potential of a cell-replacement therapy for diabetics by allowing the patient to be the donor of his own insulin-producing tissue. pancreas | transdifferentiation

  20. Increased interaction with insulin receptor substrate 1, a novel abnormality in insulin resistance and type 2 diabetes

    DEFF Research Database (Denmark)

    Caruso, Michael; Ma, Danjun; Msallaty, Zaher

    2014-01-01

    Insulin receptor substrate 1 (IRS1) is a key mediator of insulin signal transduction. Perturbations involving IRS1 complexes may lead to the development of insulin resistance and type 2 diabetes (T2D). Surprisingly little is known about the proteins that interact with IRS1 in humans under health...... in obesity and T2D in humans, provides new insights into the molecular mechanism of insulin resistance and identifies new targets for T2D drug development....... and disease conditions. We used a proteomic approach to assess IRS1 interaction partners in skeletal muscle from lean healthy control subjects (LCs), obese insulin-resistant nondiabetic control subjects (OCs), and participants with T2D before and after insulin infusion. We identified 113 novel endogenous IRS1...

  1. Human Liver Cells Expressing Albumin and Mesenchymal Characteristics Give Rise to Insulin-Producing Cells

    Directory of Open Access Journals (Sweden)

    Irit Meivar-Levy

    2011-01-01

    Full Text Available Activation of the pancreatic lineage in the liver has been suggested as a potential autologous cell replacement therapy for diabetic patients. Transcription factors-induced liver-to-pancreas reprogramming has been demonstrated in numerous species both in vivo and in vitro. However, human-derived liver cells capable of acquiring the alternate pancreatic repertoire have never been characterized. It is yet unknown whether hepatic-like stem cells or rather adult liver cells give rise to insulin-producing cells. Using an in vitro experimental system, we demonstrate that proliferating adherent human liver cells acquire mesenchymal-like characteristics and a considerable level of cellular plasticity. However, using a lineage-tracing approach, we demonstrate that insulin-producing cells are primarily generated in cells enriched for adult hepatic markers that coexpress both albumin and mesenchymal markers. Taken together, our data suggest that adult human hepatic tissue retains a substantial level of developmental plasticity, which could be exploited in regenerative medicine approaches.

  2. Ghrelin- and GH-induced insulin resistance

    DEFF Research Database (Denmark)

    Vestergaard, Esben Thyssen; Krag, Morten B; Poulsen, Morten M

    2013-01-01

    Supraphysiological levels of ghrelin and GH induce insulin resistance. Serum levels of retinol-binding protein-4 (RBP4) correlate inversely with insulin sensitivity in patients with type 2 diabetes. We aimed to determine whether ghrelin and GH affect RBP4 levels in human subjects.......Supraphysiological levels of ghrelin and GH induce insulin resistance. Serum levels of retinol-binding protein-4 (RBP4) correlate inversely with insulin sensitivity in patients with type 2 diabetes. We aimed to determine whether ghrelin and GH affect RBP4 levels in human subjects....

  3. Monomeric insulins and their experimental and clinical implications.

    Science.gov (United States)

    Brange, J; Owens, D R; Kang, S; Vølund, A

    1990-09-01

    Due to the inherent pharmacokinetic properties of available insulins, normoglycemia is rarely, if ever, achieved in insulin-dependent diabetic patients without compromising their quality of life. Subcutaneous insulin absorption is influenced by many factors, among which the associated state of insulin (hexameric) in pharmaceutical formulation may be of importance. This review describes the development of a series of human insulin analogues with reduced tendency to self-association that, because of more rapid absorption, are better suited to meal-related therapy. DNA technology has made it possible to prepare insulins that remain dimeric or even monomeric at high concentration by introducing one or a few amino acid substitutions into human insulin. These analogues were characterized and used for elucidating the mechanisms involved in subcutaneous absorption and were investigated in preliminary clinical studies. Their relative receptor binding and in vitro potency (free-fat cell assay), ranging from 0.05 to 600% relative to human insulin, were strongly correlated (r = 0.97). In vivo, most of the analogues exhibited approximately 100% activity, explainable by a dominating receptor-mediated clearance. This was confirmed by clamp studies in which correlation between receptor binding and clearance was observed. Thus, an analogue with reduced binding and clearance gives higher circulating concentrations, counterbalancing the reduced potency at the cellular level. Absorption studies in pigs revealed a strong inverse correlation (r = 0.96) between the rate of subcutaneous absorption and the mean association state of the insulin analogues. These studies also demonstrated that monomeric insulins were absorbed three times faster than human insulin. In healthy subjects, rates of disappearance from subcutis were two to three times faster for dimeric and monomeric analogues than for human insulin. Concomitantly, a more rapid rise in plasma insulin concentration and an earlier

  4. Acute pain induces insulin resistance in humans

    DEFF Research Database (Denmark)

    Greisen, J.; Juhl, C.B.; Grøfte, Thorbjørn

    2001-01-01

    Background: Painful trauma results in a disturbed metabolic state with impaired insulin sensitivity, which is related to the magnitude of the trauma. The authors explored whether pain per se influences hepatic and extrahepatic actions of insulin. Methods: Ten healthy male volunteers underwent two...... randomly sequenced hyperinsulinemic–euglycemic (insulin infusion rate, 0.6 mU · kg-1 · min-1 for 180 min) clamp studies 4 weeks apart. Self-controlled painful electrical stimulation was applied to the abdominal skin for 30 min, to a pain intensity of 8 on a visual analog scale of 0–10, just before...... the clamp procedure (study P). In the other study, no pain was inflicted (study C). Results: Pain reduced whole-body insulin-stimulated glucose uptake from 6.37 ± 1.87 mg · kg-1 · min-1 (mean ± SD) in study C to 4.97 ± 1.38 mg · kg-1 · min-1 in study P (P

  5. 1H NMR spectrum of the native human insulin monomer. Evidence for conformational differences between the monomer and aggregated forms.

    Science.gov (United States)

    Roy, M; Lee, R W; Brange, J; Dunn, M F

    1990-04-05

    The effects of high dilution on the 1H Fourier transform NMR spectrum of native human insulin at pH* 8.0 and 9.3 have been examined at 500 MHz resolution. The dependence of the spectrum on concentration and comparison with the spectrum of a biologically highly potent monomeric insulin mutant (SerB9----Asp) establish that at 36 microM (pH* 9.3) or 18 microM (pH* 8) and no added buffer or salts, human insulin is monomeric. Under these conditions of dilution, ionic strength, and pH*, human insulin and the SerB9----Asp mutant exhibit nearly identical 1H NMR spectra. At higher concentrations (i.e. greater than 36 microM to 0.91 mM), native human insulin dimerizes, and this aggregation causes a change in insulin conformation. Although there are many changes in the spectrum, the TyrB26 ring H3,5 proton signals located at 6.63 ppm and the methyl signal located at 0.105 ppm (characteristics of monomeric insulin) are particularly distinct signatures of the conformation change that accompanies dimerization. Magnetization transfer experiments show that the 0.105 ppm methyl signal shifts downfield to a new position at 0.45 ppm. We conclude that the 0.105 ppm methyl signal is due to a conformation in which a Leu methyl group is centered over and in van der Waals contact with the ring of an aromatic side chain. Dimerization causes a conformation change that alters this interaction, thereby causing the downfield shift. Nuclear Overhauser studies indicate that the methyl group involved is located within a cluster of aromatic side chains and that the closest ring-methyl group interaction is with the ring of PheB24.

  6. Skeletal muscle phosphatidylcholine and phosphatidylethanolamine are related to insulin sensitivity and respond to acute exercise in humans.

    Science.gov (United States)

    Newsom, Sean A; Brozinick, Joseph T; Kiseljak-Vassiliades, Katja; Strauss, Allison N; Bacon, Samantha D; Kerege, Anna A; Bui, Hai Hoang; Sanders, Phil; Siddall, Parker; Wei, Tao; Thomas, Melissa; Kuo, Ming Shang; Nemkov, Travis; D'Alessandro, Angelo; Hansen, Kirk C; Perreault, Leigh; Bergman, Bryan C

    2016-06-01

    Several recent reports indicate that the balance of skeletal muscle phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is a key determinant of muscle contractile function and metabolism. The purpose of this study was to determine relationships between skeletal muscle PC, PE and insulin sensitivity, and whether PC and PE are dynamically regulated in response to acute exercise in humans. Insulin sensitivity was measured via intravenous glucose tolerance in sedentary obese adults (OB; n = 14), individuals with type 2 diabetes (T2D; n = 15), and endurance-trained athletes (ATH; n = 15). Vastus lateralis muscle biopsies were obtained at rest, immediately after 90 min of cycle ergometry at 50% maximal oxygen consumption (V̇o2 max), and 2-h postexercise (recovery). Skeletal muscle PC and PE were measured via infusion-based mass spectrometry/mass spectrometry analysis. ATH had greater levels of muscle PC and PE compared with OB and T2D (P insulin sensitivity (both P insulin sensitivity among the entire cohort (r = -0.43, P = 0.01). Muscle PC and PE were altered by exercise, particularly after 2 h of recovery, in a highly group-specific manner. However, muscle PC:PE ratio remained unchanged in all groups. In summary, total muscle PC and PE are positively related to insulin sensitivity while PC:PE ratio is inversely related to insulin sensitivity in humans. A single session of exercise significantly alters skeletal muscle PC and PE levels, but not PC:PE ratio. Copyright © 2016 the American Physiological Society.

  7. Successful transplantation of in vitro expanded human cadaver corneal endothelial precursor cells on to a cadaver bovine's eye using a nanocomposite gel sheet.

    Science.gov (United States)

    Parikumar, Periyasamy; Haraguchi, Kazutoshi; Ohbayashi, Akira; Senthilkumar, Rajappa; Abraham, Samuel J K

    2014-05-01

    In vitro expansion of human corneal endothelial precursor (HCEP) cells has been reported via production of cell aggregated spheres. However, to translate this procedure in human patients warrants maintaining the position of the eyeballs facing down for 36 h, which is not feasible. In this study, we report a method using a nanocomposite (NC) gel sheet to accomplish the integration of HCEP cells to the endothelium of cadaver bovine's eyes. HCEP cells were isolated from the corneal endothelium of a cadaver human eye and then expanded using a thermoreversible gelation polymer (TGP) as reported earlier. For the study, three cadaver bovine eyes were used. The NC gel sheets were inserted into the bovine eyes', aligned and suture-fixed in position under the host endothelium. HCEP cells previously expanded in the TGP were harvested and injected using a 26-gauge syringe between the endothelium and the NC gel sheet. The eyes were left undisturbed for three hours following which the NC gel sheets were gently removed. The corneas were harvested and subjected to histopathological studies. Histopathological studies showed that all the three corneas used for NC gel sheet implantation showed the presence of engrafted HCEP cells, seen as multi-layered cells over the native endothelium of the bovine cornea. Examination of the NC gel sheets used for implantation showed that only very few corneal endothelial cells remained on the sheets amounting to what could be considered negligible. The use of the NC gel sheet makes HCEP cell transplantation feasible for human patients. Further in vitro basic studies followed by translational studies are necessary to bring this method for clinical application in appropriate indications.

  8. Comparison of Knoop and Vickers surface microhardness and transverse microradiography for the study of early caries lesion formation in human and bovine enamel.

    Science.gov (United States)

    Lippert, F; Lynch, R J M

    2014-07-01

    The aims of the present laboratory study were twofold: a) to investigate the suitability of Knoop and Vickers surface microhardness (SMH) in comparison to transverse microradiography (TMR) to investigate early enamel caries lesion formation; b) to compare the kinetics of caries lesion initiation and progression between human and bovine enamel. Specimens (90×bovine and 90×human enamel) were divided into six groups (demineralization times of 8/16/24/32/40/48h) of 15 per enamel type and demineralized using a partially saturated lactic acid solution. SMH was measured before and after demineralization and changes in indentation length (ΔIL) calculated. Lesions were characterized using TMR. Data were analyzed (two-way ANOVA) and Pearson correlation coefficients calculated. ΔIL increased with increasing demineralization times but plateaued after 40h, whereas lesion depth (L) and integrated mineral loss (ΔZ) increased almost linearly throughout. No differences between Knoop and Vickers SMH in their ability to measure enamel demineralization were observed as both correlated strongly. Overall, ΔIL correlated strongly with ΔZ and L but only moderately with the degree of surface zone mineralization, whereas ΔZ and L correlated strongly. Bovine demineralized faster than human enamel (all techniques). Lesions in bovine formed faster than in human enamel, although the resulting lesions were almost indistinguishable in their mineral distribution characteristics. Early caries lesion demineralization can be sufficiently studied by SMH, but its limitations on the assessment of the mineral status of more demineralized lesions must be considered. Ideally, complementary techniques to assess changes in both physical and chemical lesion characteristics would be employed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Blood-based biomarkers of age-associated epigenetic changes in human islets associate with insulin secretion and diabetes

    DEFF Research Database (Denmark)

    Bacos, Karl; Gillberg, Linn; Volkov, Petr

    2016-01-01

    identified in human islets (for example, KLF14, FHL2, ZNF518B and FAM123C) and some associate with insulin secretion and T2D. DNA methylation correlates with islet expression of multiple genes, including FHL2, ZNF518B, GNPNAT1 and HLTF. Silencing these genes in β-cells alter insulin secretion. Together, we...

  10. Immunodetection and quantification of insulin-like antigens in sprouts: development of an efficient functional food

    Directory of Open Access Journals (Sweden)

    Manju Pathak

    2011-11-01

    Full Text Available Background: Hormone Insulin is a drug used for the treatment of type 1 and type 2 Diabetes Mellitus. Insulin used in this experiment is derived from bovine and pork pancreas, as well as through recombinant technology. Patients with diabetes mellitus face an inability to utilize glucose from blood due to either less secretion of insulin, or the inability of the insulin to act; As a result of this glucose levels in the blood rise. The prevention and treatment of type 2 Diabetes Mellitus one is world’s major public health issues. Natural alternatives have a big role to play in this field. This study aims at discovering functional foods rich in Insulin like proteins. Here we are reporting Insulin-like proteins synthesizing during the embryo development stage of Glycine max: soybean, Vigna radiata: moong and Vigna unguiculata: cowpea seeds. Hence, germination transforms these seeds containing human insulin like proteins.Methods: In our investigation we have provided protein extraction with Enzyme-linked immunosorbent assay (ELISA. The plant materials weighing 1g were crushed in mortar and pestle, and the protein from the plant materials was extracted with 20 ml of 0.05 M sodium phosphate buffer (pH 7.6. The suspensions were centrifuged at 6000 rpm for 15 min, and the clear supernatants were subjected to Enzyme linked immunosorbent assay (ELISA for the detection of insulin-like proteins. We have used USDA nutritional data sources for the analysis of new products. Results: Our results demonstrate that Insulin is not expressed in dry mature dormant seeds, but is expressed only during the embryo development stage. Dry mature dormant seeds and the seeds germinated for 24 hours, 48 hours, 72 hours, and 96 hours of Glycine max, Vigna radiata and Vigna unguiculata, were investigated for expression of insulin through immunodetection using anti-insulin antibodies. Dry dormant seeds of all the three seeds showed zero expression at 450 nm for insulin, while

  11. Circulating ApoJ is closely associated with insulin resistance in human subjects.

    Science.gov (United States)

    Seo, Ji A; Kang, Min-Cheol; Ciaraldi, Theodore P; Kim, Sang Soo; Park, Kyong Soo; Choe, Charles; Hwang, Won Min; Lim, Dong Mee; Farr, Olivia; Mantzoros, Christos; Henry, Robert R; Kim, Young-Bum

    2018-01-01

    Insulin resistance is a major risk factor for type 2 diabetes. ApolipoproteinJ (ApoJ) has been implicated in altered pathophysiologic states including cardiovascular and Alzheimer's disease. However, the function of ApoJ in regulation of glucose homeostasis remains unclear. This study sought to determine whether serum ApoJ levels are associated with insulin resistance in human subjects and if they change after interventions that improve insulin sensitivity. Serum ApoJ levels and insulin resistance status were assessed in nondiabetic (ND) and type 2 diabetic (T2D) subjects. The impacts of rosiglitazone or metformin therapy on serum ApoJ levels and glucose disposal rate (GDR) during a hyperinsulinemic/euglycemic clamp were evaluated in a separate cohort of T2D subjects. Total ApoJ protein or that associated with the HDL and LDL fractions was measured by immunoblotting or ELISA. Fasting serum ApoJ levels were greatly elevated in T2D subjects (ND vs T2D; 100±8.3 vs. 150.6±8.5AU, Pinsulin, HOMA-IR, and BMI. ApoJ levels were significantly and independently associated with HOMA-IR, even after adjustment for age, sex, and BMI. Rosiglitazone treatment in T2D subjects resulted in a reduction in serum ApoJ levels (before vs. after treatment; 100±13.9 vs. 77±15.2AU, P=0.015), whereas metformin had no effect on ApoJ levels. The change in ApoJ levels during treatment was inversely associated with the change in GDR. Interestingly, ApoJ content in the LDL fraction was inversely associated with HOMA-IR. Serum ApoJ levels are closely correlated with the magnitude of insulin resistance regardless of obesity, and decrease along with improvement of insulin resistance in response only to rosiglitazone in type 2 diabetes. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. High-throughput genotyping assay for the large-scale genetic characterization of Cryptosporidium parasites from human and bovine samples.

    Science.gov (United States)

    Abal-Fabeiro, J L; Maside, X; Llovo, J; Bello, X; Torres, M; Treviño, M; Moldes, L; Muñoz, A; Carracedo, A; Bartolomé, C

    2014-04-01

    The epidemiological study of human cryptosporidiosis requires the characterization of species and subtypes involved in human disease in large sample collections. Molecular genotyping is costly and time-consuming, making the implementation of low-cost, highly efficient technologies increasingly necessary. Here, we designed a protocol based on MALDI-TOF mass spectrometry for the high-throughput genotyping of a panel of 55 single nucleotide variants (SNVs) selected as markers for the identification of common gp60 subtypes of four Cryptosporidium species that infect humans. The method was applied to a panel of 608 human and 63 bovine isolates and the results were compared with control samples typed by Sanger sequencing. The method allowed the identification of species in 610 specimens (90·9%) and gp60 subtype in 605 (90·2%). It displayed excellent performance, with sensitivity and specificity values of 87·3 and 98·0%, respectively. Up to nine genotypes from four different Cryptosporidium species (C. hominis, C. parvum, C. meleagridis and C. felis) were detected in humans; the most common ones were C. hominis subtype Ib, and C. parvum IIa (61·3 and 28·3%, respectively). 96·5% of the bovine samples were typed as IIa. The method performs as well as the widely used Sanger sequencing and is more cost-effective and less time consuming.

  13. Bovine cysticercosis and human taeniasis: Animal-human health and economic approach with treatment trends in Kombolcha Town, Wollo, Ethiopia

    Directory of Open Access Journals (Sweden)

    Aragaw Tegegne

    2018-03-01

    Full Text Available Background and Aim: Bovine cysticercosis and human taeniasis accounted for parasitic zoonotic implications with economic losses from organ contamination and treatment cost. The disease is common where hygienic conditions are poor and the inhabitants traditionally eat raw or insufficiently cooked meat under inadequate community awareness on the associated risk factors for the occurrences of infections in developing countries such as Ethiopia. The aim of this study was to assess bovine cysticercosis and Taenia saginata human taeniasis considering animal-human health and economic approach with treatment trends in Kombolcha Town, Wollo, Ethiopia. Materials and Methods: A cross-sectional study was conducted in cattle slaughtered at Kombolcha ELFORA abattoir from November 2016 to April 2017. A questionnaire survey was applied for community awareness, exposure risk, and treatment trends for taeniasis assessment in Kombolcha Town with economic losses from organ condemnation, and drug cost for taeniasis treatments were estimated. Results: Of the 234 examined carcasses, 21 (8.97% were found infected with bovine cysticercosis. Organ distribution of the cysts showed highest proportions in liver 40 (29.2%, followed by heart 26 (18.9%, tongue 22 (16.1%, masseter muscle 20 (14.6%, triceps 15 (10.9%, diaphragm 9 (6.7%, and lung 5 (3.6%. Both male, i.e., 15 (6.4%, and female, i.e., 6 (12.8%, were infected. Regardless of sample size, Cysticercus bovis infection was found 8 (21.62% in adults and 13 (6.60% older aged. Of 110 interviewed individuals, about 31.8% aware of taeniasis and they also have exposure risk for taeniasis with no differences (p>0.05 within studied demography. The majorities (54.3% of exposed groups use pharmaceutical drugs, while 28.6% use herbal medicine, but 17.2% use both for treatment. Of 31,469 clinical cases in Kombolcha Town, 22 (0.07% were positive for taeniasis over the year 2016. An inventory of pharmaceutical shops revealed the

  14. Determination of human insulin in dog plasma by a selective liquid chromatography-tandem mass spectrometry method: Application to a pharmacokinetic study.

    Science.gov (United States)

    Dong, Shiqi; Zeng, Yong; Wei, Guangli; Si, Duanyun; Liu, Changxiao

    2018-03-01

    A simple, sensitive and selective LC-MS/MS method for quantitative analysis of human insulin was developed and validated in dog plasma. Insulin glargine was used as the internal standard. After a simple step of solid-phase extraction, the chromatographic separation of human insulin was achieved by using InertSustain Bio C18 column with a mobile phase of acetonitrile containing 1% formic acid (A)-water containing 1% formic acid (B). The detection was performed by positive ion electrospray ionization in multiple-reaction monitoring (MRM) mode. Good linearity was observed in the concentration range of 1-1000 μIU/mL (r 2  > 0.99), and the lower limit of quantification was 1 μIU/mL (equal to 38.46 pg/mL). The intra- and inter-day precision (expressed as relative standard deviation, RSD) of human insulin were ≤12.1% and ≤13.0%, respectively, and the accuracy (expressed as relative error, RE) was in the range of -7.23-11.9%. The recovery and matrix effect were both within acceptable limits. This method was successfully applied for the pharmacokinetic study of human insulin in dogs after subcutaneous administration. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Calcification of human vascular smooth muscle cells: associations with osteoprotegerin expression and acceleration by high-dose insulin

    DEFF Research Database (Denmark)

    Olesen, Ping; Knudsen, Kirsten Quyen Nguyen; Wogensen, Lise

    2007-01-01

    Arterial medial calcifications occur often in diabetic individuals as part of the diabetic macroangiopathy. The pathogenesis is unknown, but the presence of calcifications predicts risk of cardiovascular events. We examined the effects of insulin on calcifying smooth muscle cells in vitro...... and measured the expression of the bone-related molecule osteoprotegerin (OPG). Human vascular smooth muscle cells (VSMCs) were grown from aorta from kidney donors. Induction of calcification was performed with beta-glycerophosphate. The influence of insulin (200 microU/ml or 1,000 microU/ml) on calcification...... calcification in human smooth muscle cells from a series of donors after variable time in culture. Decreased OPG amounts were observed from the cells during the accelerated calcification phase. High dose of insulin (1,000 microU/ml) accelerated the calcification, whereas lower concentrations (200 microU/ml) did...

  16. Somatic insulin signaling regulates a germline starvation response in Drosophila egg chambers

    Science.gov (United States)

    Burn, K. Mahala; Shimada, Yuko; Ayers, Kathleen; Lu, Feiyue; Hudson, Andrew M.; Cooley, Lynn

    2014-01-01

    Egg chambers from starved Drosophila females contain large aggregates of processing (P) bodies and cortically enriched microtubules. As this response to starvation is rapidly reversed upon re-feeding females or culturing egg chambers with exogenous bovine insulin, we examined the role of endogenous insulin signaling in mediating the starvation response. We found that systemic Drosophila insulin-like peptides (dILPs) activate the insulin pathway in follicle cells, which then regulate both microtubule and P body organization in the underlying germline cells. This organization is modulated by the motor proteins Dynein and Kinesin. Dynein activity is required for microtubule and P body organization during starvation, while Kinesin activity is required during nutrient-rich conditions. Blocking the ability of egg chambers to form P body aggregates in response to starvation correlated with reduced progeny survival. These data suggest a potential mechanism to maximize fecundity even during periods of poor nutrient availability, by mounting a protective response in immature egg chambers. PMID:25481758

  17. Regulation of lipoprotein lipase in primary cultures of isolated human adipocytes

    International Nuclear Information System (INIS)

    Kern, P.A.; Marshall, S.; Eckel, R.H.

    1985-01-01

    To study the regulation of adipose tissue lipoprotein lipase (LPL) in human adipocytes, omental adipose tissue was obtained from healthy subjects and digested in collagenase. The isolated adipocytes thus obtained were suspended in Medium 199 and cultured at 37 degrees C. Cell viability was demonstrated in adipocytes cultured for up to 72 h by constancy of cell number, cell size, trypan-blue exclusion, and specific 125 I-insulin binding. In addition, chloroquine induced an increase in cell-associated 125 I-insulin at 24, 48, and 72 h after preparation. Thus, isolated adipocytes retained their ability to bind, internalize, and degrade insulin. LPL was measured as activity secreted into the culture medium (CM), released from cells by heparin (HR), and extracted from cell digests. A broad range of heparin concentrations produced a prompt release of LPL from a rapidly replenishable pool of cellular activity. When cells were cultured in medium containing 10% fetal bovine serum, there was a marked stimulation of CM and HR. The secretory response to serum (CM) correlated strongly with HR 24 h after preparation. In addition, HR was found to correlate logarithmically and inversely with body mass index. Insulin, at 400 ng/ml only, increased HR by 36 +/- 10%, an effect simulated by lower concentrations of insulin-like growth factor-1 (IGF1). Thus, LPL is produced and regulated in isolated human adipocytes. The degree of adiposity and serum are important regulators of HR activity, whereas insulin is stimulatory only at a pharmacologic concentration. This effect of insulin may be mediated through the IGF1 receptor. Isolated human adipocytes represent a novel and useful system for the study of LPL and lipid metabolism as well as for other aspects of adipocyte biology

  18. Effect of insulin catheter wear-time on subcutaneous adipose tissue blood flow and insulin absorption in humans

    DEFF Research Database (Denmark)

    Clausen, Trine Schnedler; Kaastrup, Peter; Stallknecht, Bente

    2009-01-01

    blood flow (ATBF) and absorption of the rapid-acting insulin analog insulin aspart over a period of 4 days. METHODS: Teflon insulin catheters (Medtronic, Minneapolis, MN) were inserted into the abdominal SAT of 10 healthy men without diabetes (mean +/- SEM age, 23.0 +/- 1.1 years; body mass index, 22...... +/- 3 min on day 0 to 45 +/- 4 min on day 4 (P = 0.019). Neither peak plasma concentration nor area under the curve of insulin aspart changed significantly. CONCLUSIONS: Insertion of a Teflon insulin catheter into the SAT results in increased ATBF and faster absorption of insulin aspart in a period of 4...

  19. UV-light exposure of insulin: pharmaceutical implications upon covalent insulin dityrosine dimerization and disulphide bond photolysis.

    Science.gov (United States)

    Correia, Manuel; Neves-Petersen, Maria Teresa; Jeppesen, Per Bendix; Gregersen, Søren; Petersen, Steffen B

    2012-01-01

    In this work we report the effects of continuous UV-light (276 nm, ~2.20 W.m(-2)) excitation of human insulin on its absorption and fluorescence properties, structure and functionality. Continuous UV-excitation of the peptide hormone in solution leads to the progressive formation of tyrosine photo-product dityrosine, formed upon tyrosine radical cross-linkage. Absorbance, fluorescence emission and excitation data confirm dityrosine formation, leading to covalent insulin dimerization. Furthermore, UV-excitation of insulin induces disulphide bridge breakage. Near- and far-UV-CD spectroscopy shows that UV-excitation of insulin induces secondary and tertiary structure losses. In native insulin, the A and B chains are held together by two disulphide bridges. Disruption of either of these bonds is likely to affect insulin's structure. The UV-light induced structural changes impair its antibody binding capability and in vitro hormonal function. After 1.5 and 3.5 h of 276 nm excitation there is a 33.7% and 62.1% decrease in concentration of insulin recognized by guinea pig anti-insulin antibodies, respectively. Glucose uptake by human skeletal muscle cells decreases 61.7% when the cells are incubated with pre UV-illuminated insulin during 1.5 h. The observations presented in this work highlight the importance of protecting insulin and other drugs from UV-light exposure, which is of outmost relevance to the pharmaceutical industry. Several drug formulations containing insulin in hexameric, dimeric and monomeric forms can be exposed to natural and artificial UV-light during their production, packaging, storage or administration phases. We can estimate that direct long-term exposure of insulin to sunlight and common light sources for indoors lighting and UV-sterilization in industries can be sufficient to induce irreversible changes to human insulin structure. Routine fluorescence and absorption measurements in laboratory experiments may also induce changes in protein

  20. UV-light exposure of insulin: pharmaceutical implications upon covalent insulin dityrosine dimerization and disulphide bond photolysis.

    Directory of Open Access Journals (Sweden)

    Manuel Correia

    Full Text Available In this work we report the effects of continuous UV-light (276 nm, ~2.20 W.m(-2 excitation of human insulin on its absorption and fluorescence properties, structure and functionality. Continuous UV-excitation of the peptide hormone in solution leads to the progressive formation of tyrosine photo-product dityrosine, formed upon tyrosine radical cross-linkage. Absorbance, fluorescence emission and excitation data confirm dityrosine formation, leading to covalent insulin dimerization. Furthermore, UV-excitation of insulin induces disulphide bridge breakage. Near- and far-UV-CD spectroscopy shows that UV-excitation of insulin induces secondary and tertiary structure losses. In native insulin, the A and B chains are held together by two disulphide bridges. Disruption of either of these bonds is likely to affect insulin's structure. The UV-light induced structural changes impair its antibody binding capability and in vitro hormonal function. After 1.5 and 3.5 h of 276 nm excitation there is a 33.7% and 62.1% decrease in concentration of insulin recognized by guinea pig anti-insulin antibodies, respectively. Glucose uptake by human skeletal muscle cells decreases 61.7% when the cells are incubated with pre UV-illuminated insulin during 1.5 h. The observations presented in this work highlight the importance of protecting insulin and other drugs from UV-light exposure, which is of outmost relevance to the pharmaceutical industry. Several drug formulations containing insulin in hexameric, dimeric and monomeric forms can be exposed to natural and artificial UV-light during their production, packaging, storage or administration phases. We can estimate that direct long-term exposure of insulin to sunlight and common light sources for indoors lighting and UV-sterilization in industries can be sufficient to induce irreversible changes to human insulin structure. Routine fluorescence and absorption measurements in laboratory experiments may also induce changes

  1. Insulin-increased L-arginine transport requires A(2A adenosine receptors activation in human umbilical vein endothelium.

    Directory of Open Access Journals (Sweden)

    Enrique Guzmán-Gutiérrez

    Full Text Available Adenosine causes vasodilation of human placenta vasculature by increasing the transport of arginine via cationic amino acid transporters 1 (hCAT-1. This process involves the activation of A(2A adenosine receptors (A(2AAR in human umbilical vein endothelial cells (HUVECs. Insulin increases hCAT-1 activity and expression in HUVECs, and A(2AAR stimulation increases insulin sensitivity in subjects with insulin resistance. However, whether A(2AAR plays a role in insulin-mediated increase in L-arginine transport in HUVECs is unknown. To determine this, we first assayed the kinetics of saturable L-arginine transport (1 minute, 37°C in the absence or presence of nitrobenzylthioinosine (NBTI, 10 µmol/L, adenosine transport inhibitor and/or adenosine receptors agonist/antagonists. We also determined hCAT-1 protein and mRNA expression levels (Western blots and quantitative PCR, and SLC7A1 (for hCAT-1 reporter promoter activity. Insulin and NBTI increased the extracellular adenosine concentration, the maximal velocity for L-arginine transport without altering the apparent K(m for L-arginine transport, hCAT-1 protein and mRNA expression levels, and SLC7A1 transcriptional activity. An A2AAR antagonist ZM-241385 blocked these effects. ZM241385 inhibited SLC7A1 reporter transcriptional activity to the same extent in cells transfected with pGL3-hCAT-1(-1606 or pGL3-hCAT-1(-650 constructs in the presence of NBTI + insulin. However, SLC7A1 reporter activity was increased by NBTI only in cells transfected with pGL3-hCAT-1(-1606, and the ZM-241385 sensitive fraction of the NBTI response was similar in the absence or in the presence of insulin. Thus, insulin modulation of hCAT-1 expression and activity requires functional A(2AAR in HUVECs, a mechanism that may be applicable to diseases associated with fetal insulin resistance, such as gestational diabetes.

  2. Influence of the fetal bovine serum proteins on the growth of human osteoblast cells on graphene

    Czech Academy of Sciences Publication Activity Database

    Kalbáčová, M.; Brož, A.; Kalbáč, Martin

    100A, č. 11 (2012), s. 3001-3007 ISSN 1549-3296 R&D Projects: GA AV ČR IAA400400911; GA AV ČR KAN200100801; GA ČR GAP204/10/1677; GA ČR(CZ) GAP208/12/1062; GA MŠk ME09060 Institutional support: RVO:61388955 Keywords : human osteoblast * graphene * fetal bovine serum Subject RIV: CG - Electrochemistry Impact factor: 2.834, year: 2012

  3. A randomized trial of insulin aspart with intensified basal NPH insulin supplementation in people with Type 1 diabetes

    NARCIS (Netherlands)

    DeVries, J. H.; Lindholm, A.; Jacobsen, J. L.; Heine, R. J.; Home, P. D.

    2003-01-01

    Aims Insulin aspart has been shown to improve post-prandial and overall glycaemic control in people with Type 1 diabetes. We hypothesized that insulin aspart with intensified basal NPH insulin supplementation would result in better overall glycaemic control than human regular insulin with standard

  4. A New Method for Generating Insulin-Secreting Cells from Human Pancreatic Epithelial Cells After Islet Isolation Transformed by NeuroD1

    Science.gov (United States)

    Shimoda, Masayuki; Chen, Shuyuan; Noguchi, Hirofumi; Takita, Morihito; Sugimoto, Koji; Itoh, Takeshi; Chujo, Daisuke; Iwahashi, Shuichi; Naziruddin, Bashoo; Levy, Marlon F.

    2014-01-01

    Abstract The generation of insulin-secreting cells from nonendocrine pancreatic epithelial cells (NEPEC) has been demonstrated for potential clinical use in the treatment of diabetes. However, previous methods either had limited efficacy or required viral vectors, which hinder clinical application. In this study, we aimed to establish an efficient method of insulin-secreting cell generation from NEPEC without viral vectors. We used nonislet fractions from both research-grade human pancreata from brain-dead donors and clinical pancreata after total pancreatectomy with autologous islet transplantation to treat chronic pancreatitis. It is of note that a few islets could be mingled in the nonislet fractions, but their influence could be limited. The NeuroD1 gene was induced into NEPEC using an effective triple lipofection method without viral vectors to generate insulin-secreting cells. The differentiation was promoted by adding a growth factor cocktail into the culture medium. Using the research-grade human pancreata, the effective method showed high efficacy in the differentiation of NEPEC into insulin-positive cells that secreted insulin in response to a glucose challenge and improved diabetes after being transplanted into diabetic athymic mice. Using the clinical pancreata, similar efficacy was obtained, even though those pancreata suffered chronic pancreatitis. In conclusion, our effective differentiation protocol with triple lipofection method enabled us to achieve very efficient insulin-secreting cell generation from human NEPEC without viral vectors. This method offers the potential for supplemental insulin-secreting cell transplantation for both allogeneic and autologous islet transplantation. PMID:24845703

  5. Intraportal injection of insulin-producing cells generated from human bone marrow mesenchymal stem cells decreases blood glucose level in diabetic rats.

    Science.gov (United States)

    Tsai, Pei-Jiun; Wang, Hwai-Shi; Lin, Chi-Hung; Weng, Zen-Chung; Chen, Tien-Hua; Shyu, Jia-Fwu

    2014-01-01

    We studied the process of trans-differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) into insulin-producing cells. Streptozotocin (STZ)-induced diabetic rat model was used to study the effect of portal vein transplantation of these insulin-producing cells on blood sugar levels. The BM-MSCs were differentiated into insulin-producing cells under defined conditions. Real-time PCR, immunocytochemistry and glucose challenge were used to evaluate in vitro differentiation. Flow cytometry showed that hBM-MSCs were strongly positive for CD44, CD105 and CD73 and negative for hematopoietic markers CD34, CD38 and CD45. Differentiated cells expressed C-peptide as well as β-cells specific genes and hormones. Glucose stimulation increased C-peptide secretion in these cells. The insulin-producing, differentiated cells were transplanted into the portal vein of STZ-induced diabetic rats using a Port-A catheter. The insulin-producing cells were localized in the liver of the recipient rat and expressed human C-peptide. Blood glucose levels were reduced in diabetic rats transplanted with insulin-producing cells. We concluded that hBM-MSCs could be trans-differentiated into insulin-producing cells in vitro. Portal vein transplantation of insulin-producing cells alleviated hyperglycemia in diabetic rats.

  6. Survival and Diversity of Human Homologous Dietary MicroRNAs in Conventionally Cooked Top Sirloin and Dried Bovine Tissue Extracts.

    Directory of Open Access Journals (Sweden)

    Joseph T Dever

    Full Text Available Dietary microRNAs (miRNAs, notably those found in milk, are currently being investigated for their potential to elicit biological effects via canonical binding to human messenger RNA targets once ingested. Besides milk, beef and other bovine tissue-derived ingredients could also be a relevant source of potentially bioactive dietary miRNAs. In this study, we characterized the human homologous miRNA profiles in food-grade, bovine-sourced sirloin, heart and adrenal tissue (raw, cooked, and pasteurized, freeze-dried extracts via deep-sequencing and quantitative reverse transcription PCR (RT-qPCR. A total of 198 human homologous miRNAs were detected at 10 or more normalized reads in all replicates (n = 3 of at least one preparation method. Tissue origin rather than preparation method was the major differentiating factor of miRNA profiles, and adrenal-based miRNA profiles were the most distinct. The ten most prevalent miRNAs in each tissue represented 71-93% of the total normalized counts for all annotated miRNAs. In cooked sirloin, the most abundant miRNAs were miR-10b-5p, (48.8% of total annotated miRNA reads along with the muscle-specific miR-1 (24.1% and miR-206 (4.8%. In dried heart extracts, miR-1 (17.0%, miR-100-5p (16.1% and miR-99a-5p (11.0% gave the highest normalized read counts. In dried adrenal extracts, miR-10b-5p (71.2% was the most prominent followed by miR-143-3p (7.1% and 146b-5p (3.7%. Sequencing results for five detected and two undetected miRNAs were successfully validated by RT-qPCR. We conclude that edible, bovine tissues contain unique profiles of human homologous dietary miRNAs that survive heat-based preparation methods.

  7. Insulin aspart pharmacokinetics

    DEFF Research Database (Denmark)

    Rasmussen, Christian Hove; Roge, Rikke Meldgaard; Ma, Zhulin

    2014-01-01

    Background: Insulin aspart (IAsp) is used by many diabetics as a meal-time insulin to control postprandial glucose levels. As is the case with many other insulin types, the pharmacokinetics (PK), and consequently the pharmacodynamics (PD), is associated with clinical variability, both between...... to investigate and quantify the properties of the subcutaneous depot. Data from Brange et al. (1990) are used to determine the effects of insulin chemistry in subcutis on the absorption rate. Intravenous (i.v.) bolus and infusion PK data for human insulin are used to understand and quantify the systemic...... distribution and elimination (Porksen et al., 1997; Sjostrand et al., 2002). PK and PD profiles for type 1 diabetics from Chen et al. (2005) are analyzed to demonstrate the effects of IAsp antibodies in terms of bound and unbound insulin. PK profiles from Thorisdottir et al. (2009) and Ma et al. (2012b...

  8. The road to the first, fully active and more stable human insulin variant with an additional disulfide bond

    DEFF Research Database (Denmark)

    Vinther, Tine N.; Kjeldsen, Thomas B.; Jensen, Knud Jørgen

    2015-01-01

    Insulin, a small peptide hormone, is crucial in maintaining blood glucose homeostasis. The stability and activity of the protein is directed by an intricate system involving disulfide bonds to stabilize the active monomeric species and by their non-covalent oligomerization. All known insulin...... variants in vertebrates consist of two peptide chains and have six cysteine residues, which form three disulfide bonds, two of them link the two chains and a third is an intra-chain bond in the A-chain. This classical insulin fold appears to have been conserved over half a billion years of evolution. We...... addressed the question whether a human insulin variant with four disulfide bonds could exist and be fully functional. In this review, we give an overview of the road to engineering four-disulfide bonded insulin analogs. During our journey, we discovered several active four disulfide bonded insulin analogs...

  9. DNA Synthesis during Endomitosis Is Stimulated by Insulin via the PI3K/Akt and TOR Signaling Pathways in the Silk Gland Cells of Bombyx mori

    Directory of Open Access Journals (Sweden)

    Yaofeng Li

    2015-03-01

    Full Text Available Silk gland cells undergo multiple endomitotic cell cycles during silkworm larval ontogeny. Our previous study demonstrated that feeding is required for continued endomitosis in the silk gland cells of silkworm larvae. Furthermore, the insulin signaling pathway is closely related to nutritional signals. To investigate whether the insulin signaling pathway is involved in endomitosis in silk gland cells, in this study, we initially analyzed the effects of bovine insulin on DNA synthesis in endomitotic silk gland cells using 5-bromo-2'-deoxyuridine (BrdU labeling technology, and found that bovine insulin can stimulate DNA synthesis. Insulin signal transduction is mainly mediated via phosphoinositide 3-kinase (PI3K/Akt, the target of rapamycin (TOR and the extracellular signal-regulated kinase (ERK pathways in vertebrates. We ascertained that these three pathways are involved in DNA synthesis in endomitotic silk gland cells using specific inhibitors against each pathway. Moreover, we investigated whether these three pathways are involved in insulin-stimulated DNA synthesis in endomitotic silk gland cells, and found that the PI3K/Akt and TOR pathways, but not the ERK pathway, are involved in this process. These results provide an important theoretical foundation for the further investigations of the mechanism underlying efficient endomitosis in silk gland cells.

  10. The Role of MicroRNAs in Bovine Infection and Immunity

    Directory of Open Access Journals (Sweden)

    Nathan eLawless

    2014-11-01

    Full Text Available MicroRNAs (miRNAs are a class of small, non-coding RNAs that are recognised as critical regulators of immune gene expression during infection. Many immunologically significant human miRNAs have been found to be conserved in agriculturally important species, including cattle. Discovering how bovine miRNAs mediate the immune defence during infection is critical to understanding the aetiology of the most prevalent bovine diseases. Here, we review current knowledge of miRNAs in the bovine genome, and discuss the advances in understanding of miRNAs as regulators of immune cell function, and bovine immune response activation, regulation, and resolution. Finally, we consider the future perspectives on miRNAs in bovine viral disease, their role as potential biomarkers and in therapy.

  11. Early detection of degraded A14-125I-insulin in human fibroblasts by the use of high performance liquid chromatography

    International Nuclear Information System (INIS)

    Stentz, F.B.; Harris, H.L.; Kitabchi, A.E.

    1983-01-01

    We studied the metabolism of A14-125I-insulin in intact human fibroblasts using high performance liquid chromatography (HPLC) to detect and separate its early degradation products. The high resolving power of HPLC enabled us to separate what has been considered ''intact insulin'' by Sephadex G-50 chromatography or TCA precipitability into two additional peaks that had decreased biochemical properties with respect to immunoprecipitability and receptor binding but not decreased TCA precipitability. We conclude that human fibroblast is capable of metabolizing insulin within 2 min at 37 degrees C into intermediate molecules that can be detected by HPLC but not by TCA precipitability or molecular sieve chromatography

  12. Activation of bovine neutrophils by Brucella spp.

    Science.gov (United States)

    Keleher, Lauren L; Skyberg, Jerod A

    2016-09-01

    Brucellosis is a globally important zoonotic infectious disease caused by gram negative bacteria of the genus Brucella. While many species of Brucella exist, Brucella melitensis, Brucella abortus, and Brucella suis are the most common pathogens of humans and livestock. The virulence of Brucella is largely influenced by its ability to evade host factors, including phagocytic killing mechanisms, which are critical for the host response to infection. The aim of this study was to characterize the bovine neutrophil response to virulent Brucella spp. Here, we found that virulent strains of smooth B. abortus, B. melitensis, B. suis, and virulent, rough, strains of Brucella canis possess similar abilities to resist killing by resting, or IFN-γ-activated, bovine neutrophils. Bovine neutrophils responded to infection with a time-dependent oxidative burst that varied little between Brucella spp. Inhibition of TAK1, or SYK kinase blunted the oxidative burst of neutrophils in response to Brucella infection. Interestingly, Brucella spp. did not induce robust death of bovine neutrophils. These results indicate that bovine neutrophils respond similarly to virulent Brucella spp. In addition, virulent Brucella spp., including naturally rough strains of B. canis, have a conserved ability to resist killing by bovine neutrophils. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Partial rescue of in vivo insulin signalling in skeletal muscle by impaired insulin clearance in heterozygous carriers of a mutation in the insulin receptor gene

    DEFF Research Database (Denmark)

    Højlund, K.; Wojtaszewski, Jørgen; Birk, Jesper Bratz

    2006-01-01

    AIMS/HYPOTHESIS: Recently we reported the coexistence of postprandial hypoglycaemia and moderate insulin resistance in heterozygous carriers of the Arg1174Gln mutation in the insulin receptor gene (INSR). Controlled studies of in vivo insulin signalling in humans with mutant INSR are unavailable,...

  14. Variation in macrophage migration inhibitory factor [MIF] immunoreactivity during bovine gestation

    DEFF Research Database (Denmark)

    Paulesu, L.; Pfarrer, C.; Romagnoli, R.

    2012-01-01

    and hemochorial human and mouse placentae. Here we studied the bovine placenta being multiplex, villous and synepitheliochorial with a low degree of invasion, to see if MIF could be involved. Placental tissues sampled from 12 cows at 9 stages of gestation (days 18-250), and endometrial tissues from two non......-pregnant animals were processed for immunohistochemistry. Bovine MIF was detected by Western blot using anti-human MIF monoclonal antibodies. An immunoreactive band of approximately 12kDa confirmed similarities between bovine and human MIFs. Compared to the non-pregnant stage with very faint staining...... both caruncular and trophoblast epithelium of the placentomes were positive with different intensity in relation to the gestational stage. In the uterine glands, some strongly stained cells were present. The mature binucleated trophoblast giant cells were negative throughout pregnancy. During...

  15. Protein Kinase-C Beta Contributes to Impaired Endothelial Insulin Signaling in Humans with Diabetes Mellitus

    Science.gov (United States)

    Tabit, Corey E; Shenouda, Sherene M; Holbrook, Monica; Fetterman, Jessica L; Kiani, Soroosh; Frame, Alissa A; Kluge, Matthew A; Held, Aaron; Dohadwala, Mustali; Gokce, Noyan; Farb, Melissa; Rosenzweig, James; Ruderman, Neil; Vita, Joseph A; Hamburg, Naomi M

    2013-01-01

    Background Abnormal endothelial function promotes atherosclerotic vascular disease in diabetes. Experimental studies indicate that disruption of endothelial insulin signaling through the activity of protein kinase C-β (PKCβ) and nuclear factor κB (NFκB) reduces nitric oxide availability. We sought to establish whether similar mechanisms operate in the endothelium in human diabetes mellitus. Methods and Results We measured protein expression and insulin response in freshly isolated endothelial cells from patients with Type 2 diabetes mellitus (n=40) and non-diabetic controls (n=36). Unexpectedly, we observed 1.7-fold higher basal endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 in patients with diabetes (P=0.007) without a difference in total eNOS expression. Insulin stimulation increased eNOS phosphorylation in non-diabetic subjects but not in diabetic patients (P=0.003) consistent with endothelial insulin resistance. Nitrotyrosine levels were higher in diabetic patients indicating endothelial oxidative stress. PKCβ expression was higher in diabetic patients and was associated with lower flow-mediated dilation (r=−0.541, P=0.02) Inhibition of PKCβ with LY379196 reduced basal eNOS phosphorylation and improved insulin-mediated eNOS activation in patients with diabetes. Endothelial NFκB activation was higher in diabetes and was reduced with PKCβ inhibition. Conclusions We provide evidence for the presence of altered eNOS activation, reduced insulin action and inflammatory activation in the endothelium of patients with diabetes. Our findings implicate PKCβ activity in endothelial insulin resistance. PMID:23204109

  16. Understanding the structural differences between spherical and rod-shaped human insulin nanoparticles produced by supercritical fluids precipitation.

    Science.gov (United States)

    Park, Yeonju; Seo, Yongil; Chae, Boknam; Pyo, Dongjin; Chung, Hoeil; Hwang, Hyonseok; Jung, Young Mee

    2015-02-02

    In this study, the thermal denaturation mechanism and secondary structures of two types of human insulin nanoparticles produced by a process of solution-enhanced dispersion by supercritical fluids using dimethyl sulfoxide (DMSO) and ethanol (EtOH) solutions of insulin are investigated using spectroscopic approaches and molecular dynamics calculations. First, the temperature-dependent IR spectra of spherical and rod-shaped insulin nanoparticles prepared from DMSO and EtOH solution, respectively, are analyzed using principal component analysis (PCA) and 2D correlation spectroscopy to obtain a deeper understanding of the molecular structures and thermal behavior of the two insulin particle shapes. All-atom molecular dynamics (AAMD) calculations are performed to investigate the influence of the solvent molecules on the production of the insulin nanoparticles and to elucidate the geometric differences between the two types of nanoparticles. The results of the PCA, the 2D correlation spectroscopic analysis, and the AAMD calculations clearly reveal that the thermal denaturation mechanisms and the degrees of hydrogen bonding in the spherical and rod-shaped insulin nanoparticles are different. The polarity of the solvent might not alter the structure or function of the insulin produced, but the solvent polarity does influence the synthesis of different shapes of insulin nanoparticles. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Novel covalently linked insulin dimer engineered to investigate the function of insulin dimerization.

    Directory of Open Access Journals (Sweden)

    Tine N Vinther

    Full Text Available An ingenious system evolved to facilitate insulin binding to the insulin receptor as a monomer and at the same time ensure sufficient stability of insulin during storage. Insulin dimer is the cornerstone of this system. Insulin dimer is relatively weak, which ensures dissociation into monomers in the circulation, and it is stabilized by hexamer formation in the presence of zinc ions during storage in the pancreatic β-cell. Due to the transient nature of insulin dimer, direct investigation of this important form is inherently difficult. To address the relationship between insulin oligomerization and insulin stability and function, we engineered a covalently linked insulin dimer in which two monomers were linked by a disulfide bond. The structure of this covalent dimer was identical to the self-association dimer of human insulin. Importantly, this covalent dimer was capable of further oligomerization to form the structural equivalent of the classical hexamer. The covalently linked dimer neither bound to the insulin receptor, nor induced a metabolic response in vitro. However, it was extremely thermodynamically stable and did not form amyloid fibrils when subjected to mechanical stress, underlining the importance of oligomerization for insulin stability.

  18. Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

    Science.gov (United States)

    Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

    2014-02-01

    Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  19. Effect of magnesium supplementation on insulin resistance in humans: A systematic review.

    Science.gov (United States)

    Morais, Jennifer Beatriz Silva; Severo, Juliana Soares; de Alencar, Geórgia Rosa Reis; de Oliveira, Ana Raquel Soares; Cruz, Kyria Jayanne Clímaco; Marreiro, Dilina do Nascimento; Freitas, Betânia de Jesus E Silva de Almendra; de Carvalho, Cecília Maria Resende; Martins, Maria do Carmo de Carvalho E; Frota, Karoline de Macedo Gonçalves

    2017-06-01

    Recent studies have demonstrated that minerals play a role in glucose metabolism disorders in humans. Magnesium, in particular, is an extensively studied mineral that has been shown to function in the management of hyperglycemia, hyperinsulinemia, and insulin resistance (IR) action. The aim of this study was to investigate the effect of magnesium supplementation on IR in humans via systematic review of the available clinical trials. This review was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations. A survey was conducted to select clinical trials related to the effects of this mineral in insulin sensitivity using the following databases: PubMed, SciVerse Scopus, ScienceDirect, and SciVerse Cochrane. After the selection process, 12 articles were identified as eligible, representing different clinical conditions and being free of restriction with regard to sex, age, ethnicity, and differential dosing/shape of magnesium. The results of eight clinical trials showed that supplementation with magnesium influences serum fasting glucose concentrations, and five trials determined an effect on fasting insulin levels. The results of seven studies demonstrated that mineral supplementation reduced homeostasis model assessment for IR values. The data of this systematic review provide evidence as to the benefits of magnesium supplementation in reducing IR in patients with hypomagnesemia presenting IR. However, new intervention studies are needed to elucidate the role of the nutrient in protection against this metabolic disorder, as well as the standardization of the type, dose, and time of magnesium supplementation. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Lack of Aquaporin 3 in bovine erythrocyte membranes correlates with low glycerol permeation.

    Science.gov (United States)

    Campos, Elisa; Moura, Teresa F; Oliva, Abel; Leandro, Paula; Soveral, Graça

    2011-05-13

    In general, erythrocytes are highly permeable to water, urea and glycerol. However, expression of aquaporin isoforms in erythrocytes appears to be species characteristic. In the present study, human (hRBC) and bovine (bRBC) erythrocytes were chosen for comparative studies due to their significant difference in membrane glycerol permeability. Osmotic water permeability (P(f)) at 23°C was (2.89 ± 0.37) × 10(-2) and (5.12 ± 0.61) × 10(-2)cms(-1) for human and bovine cells, respectively, with similar activation energies for water transport. Glycerol permeability (P(gly)) for human ((1.37 ± 0.26) × 10(-5)cms(-1)) differed in three orders of magnitude from bovine erythrocytes ((5.82 ± 0.37) × 10(-8)cms(-1)) that also showed higher activation energy for glycerol transport. When compared to human, bovine erythrocytes showed a similar expression pattern of AQP1 glycosylated forms on immunoblot analysis, though in slight higher levels, which could be correlated with the 1.5-fold larger P(f) found. However, AQP3 expression was not detectable. Immunofluorescence analysis confirmed the absence of AQP3 expression in bovine erythrocyte membranes. In conclusion, lack of AQP3 in bovine erythrocytes points to the lipid pathway as responsible for glycerol permeation and explains the low glycerol permeability and high E(a) for transport observed in ruminants. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Studies on insulin receptor, 1

    International Nuclear Information System (INIS)

    Sakai, Yukio

    1979-01-01

    The present study was designed for the purpose of establishing a method of insulin radioreceptor assay using plasma membranes of guinea pigs as receptor sites. The results obtained are as follows: 1) Insulin receptor in the renal plasma membranes of guinea pigs showed a significantly high affinity to porcine insulin compared with that in the plasma membranes of guinea pig liver or rat kidney and liver. 2) In the insulin radioreceptor assay, an optimum condition was observed by the incubation at 4 0 C for 24 - 48 hours with 100 μg membrane protein of guinea pig kidney and 0.08 ng of 125 I-insulin. This assay method was specific for insulin and showed an accurate biological activity of insulin. 3) The recovery rate of insulin radioreceptor assay was 98.4% and dilution check up to 16 times did not influence on the result. An average of coefficient variation was 3.92% within assay. All of these results indicated the method to be satisfactory. 4) Glucose induced insulin release by perfusion method in isolated Langerhans islets of rats showed an identical pattern of reaction curves between radioreceptor assay and radioimmunoassay, although the values of radioreceptor assay was slightly low. 5) Insulin free serum produced by ultra filtration method was added to the standard assay medium. By this procedure, direct measurement of human serum by radioreceptor assay became possible. 6) The value of human serum insulin receptor binding activity by the radioreceptor assay showed a high correlation with that of insulin radioimmunoassay in sera of normal, borderline or diabetic type defined by glucose tolerance test. (author)

  2. Whole genome analysis of selected human and animal rotaviruses identified in Uganda from 2012 to 2014 reveals complex genome reassortment events between human, bovine, caprine and porcine strains.

    Science.gov (United States)

    Bwogi, Josephine; Jere, Khuzwayo C; Karamagi, Charles; Byarugaba, Denis K; Namuwulya, Prossy; Baliraine, Frederick N; Desselberger, Ulrich; Iturriza-Gomara, Miren

    2017-01-01

    Rotaviruses of species A (RVA) are a common cause of diarrhoea in children and the young of various other mammals and birds worldwide. To investigate possible interspecies transmission of RVAs, whole genomes of 18 human and 6 domestic animal RVA strains identified in Uganda between 2012 and 2014 were sequenced using the Illumina HiSeq platform. The backbone of the human RVA strains had either a Wa- or a DS-1-like genetic constellation. One human strain was a Wa-like mono-reassortant containing a DS-1-like VP2 gene of possible animal origin. All eleven genes of one bovine RVA strain were closely related to those of human RVAs. One caprine strain had a mixed genotype backbone, suggesting that it emerged from multiple reassortment events involving different host species. The porcine RVA strains had mixed genotype backbones with possible multiple reassortant events with strains of human and bovine origin.Overall, whole genome characterisation of rotaviruses found in domestic animals in Uganda strongly suggested the presence of human-to animal RVA transmission, with concomitant circulation of multi-reassortant strains potentially derived from complex interspecies transmission events. However, whole genome data from the human RVA strains causing moderate and severe diarrhoea in under-fives in Uganda indicated that they were primarily transmitted from person-to-person.

  3. Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells

    DEFF Research Database (Denmark)

    Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani

    2015-01-01

    on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins...... and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2.......Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge...

  4. Design of insulin analogues for meal-related therapy.

    Science.gov (United States)

    Brange, J

    1993-01-01

    The human insulin in replacement therapy has a hexameric structure. Hexamerization of the insulin molecule facilitates biosynthesis and beta-cell storage of insulin, but is unnecessary for biologic activity and appears to contribute to delayed absorption of exogenous insulin from the subcutis. Insulin analogues with reduced self-association that are produced through recombinant DNA techniques have been shown to have in vivo activity comparable to that of human insulin and absorption kinetics characterized by higher and more constant rates of disappearance from the subcutaneous injection site. In preliminary studies in patients receiving insulin therapy, monomeric insulin analogues have been found to provide glycemic control in the postprandial period that is at least equivalent to that of human insulin. Findings in these studies suggest that the use of such analogues may provide meal-related insulin effects closer to those observed in the physiologic state by limiting excessive postprandial glucose excursions and decreasing the risk of late hypoglycemia. Banting and Best revolutionized diabetes therapy 70 years ago with the extraction of insulin from animal pancreas glands (J Lab Clin Med 7:464-472, 1922). Since that time, many refinements of the therapeutic properties of pharmaceutical preparations of the hormone have been introduced. Until recently, however, such advances have been limited to improvements in insulin purity, insulin species, and adjustment of the composition of the vehicle with respect to auxiliary substances and other additives. With the advent of recombinant DNA techniques, it has become possible to optimize the insulin molecule itself for purposes of replacement therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. A high-throughput mass spectrometry assay to simultaneously measure intact insulin and C-peptide.

    Science.gov (United States)

    Taylor, Steven W; Clarke, Nigel J; Chen, Zhaohui; McPhaul, Michael J

    2016-04-01

    Measurements of fasting levels of insulin and C-peptide are useful in documenting insulin resistance and may help predict development of diabetes mellitus. However, the specific insulin and C-peptide levels associated with specific degrees of insulin resistance have not been defined, owing to marked variability among immunoassays and lack of standardization. Herein, we describe a multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for intact insulin and C-peptide. Insulin and C-peptide were enriched from patient sera using monoclonal antibodies immobilized on magnetic beads and processed on a robotic liquid handler. Eluted peptides were analyzed by LC-MS/MS. Bovine insulin and a stable isotopically-labeled (13C/15N) C-peptide were utilized as internal standards. The assay had an analytical measurement range of 3 to 320 μIU/ml (18 to 1920 pmol/l) for insulin and 0.11 to 27.2 ng/ml (36 to 9006 pmol/l) for C-peptide. Intra- and inter-day assay variation was less than 11% for both peptides. Of the 5 insulin analogs commonly prescribed to treat diabetes, only the recombinant drug insulin lispro caused significant interference for the determination of endogenous insulin. There were no observed interferences for C-peptide. We developed and validated a high-throughput, quantitative, multiplexed LC-MS/MS assay for intact insulin and C-peptide. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Thermodynamic and kinetic studies of As2O3 toxicological effects on human insulin in generation diabetes mellitus

    Science.gov (United States)

    Mohsennia, Mohsen; Motaharinejad, Atieh; Rafiee-Pour, Hossain-Ali; Torabbeigi, Marzieh

    2017-12-01

    The interaction of arsenic trioxide with human insulin was investigated by circular dichroism (CD), cyclic voltammetry and electrophoresis techniques. The interfacial behavior of insulin in presence of As2O3 onto the Ag electrode surface was studied at 310 K in phosphate buffer solution (PBS). According to Far-UV CD spectroscopy results, As2O3 caused to decrease in structural compactness and variety of alpha helix into beta structures. Near-UV CD indicated that As2O3 dissociates disulfide linkage in insulin structure. The kinetic parameters, including charge-transfer coefficient and apparent heterogeneous electron transfer rate constant were also determined. The thermodynamic parameters of insulin denaturation in presence of arsenic trioxide were calculated and reported. The obtained results indicated strong adsorption of insulin in presence of arsenic trioxide onto the Ag surface via chemisorptions.

  7. Human insulin polymorphism upon ligand binding and pH variation: the case of 4-ethylresorcinol.

    Science.gov (United States)

    Fili, S; Valmas, A; Norrman, M; Schluckebier, G; Beckers, D; Degen, T; Wright, J; Fitch, A; Gozzo, F; Giannopoulou, A E; Karavassili, F; Margiolaki, I

    2015-09-01

    This study focuses on the effects of the organic ligand 4-ethylresorcinol on the crystal structure of human insulin using powder X-ray crystallography. For this purpose, systematic crystallization experiments have been conducted in the presence of the organic ligand and zinc ions within the pH range 4.50-8.20, while observing crystallization behaviour around the isoelectric point of insulin. High-throughput crystal screening was performed using a laboratory X-ray diffraction system. The most representative samples were selected for synchrotron X-ray diffraction measurements, which took place at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS). Four different crystalline polymorphs have been identified. Among these, two new phases with monoclinic symmetry have been found, which are targets for the future development of microcrystalline insulin drugs.

  8. Application of Pulsed-Field Gel Electrophoresis and Binary Typing as Tools in Veterinary Clinical Microbiology and Molecular Epidemiologic Analysis of Bovine and Human Staphylococcus aureus Isolates

    Science.gov (United States)

    Zadoks, Ruth; van Leeuwen, Willem; Barkema, Herman; Sampimon, Otlis; Verbrugh, Henri; Schukken, Ynte Hein; van Belkum, Alex

    2000-01-01

    Thirty-eight bovine mammary Staphylococcus aureus isolates from diverse clinical, temporal, and geographical origins were genotyped by pulsed-field gel electrophoresis (PFGE) after SmaI digestion of prokaryotic DNA and by means of binary typing using 15 strain-specific DNA probes. Seven pulsed-field types and four subtypes were identified, as were 16 binary types. Concordant delineation of genetic relatedness was documented by both techniques, yet based on practical and epidemiological considerations, binary typing was the preferable method. Genotypes of bovine isolates were compared to 55 previously characterized human S. aureus isolates through cluster analysis of binary types. Genetic clusters containing strains of both human and bovine origin were found, but bacterial genotypes were predominantly associated with a single host species. Binary typing proved an excellent tool for comparison of S. aureus strains, including methicillin-resistant S. aureus, derived from different host species and from different databases. For 28 bovine S. aureus isolates, detailed clinical observations in vivo were compared to strain typing results in vitro. Associations were found between distinct genotypes and severity of disease, suggesting strain-specific bacterial virulence. Circumstantial evidence furthermore supports strain-specific routes of bacterial dissemination. We conclude that PFGE and binary typing can be successfully applied for genetic analysis of S. aureus isolates from bovine mammary secretions. Binary typing in particular is a robust and simple method and promises to become a powerful tool for strain characterization, for resolution of clonal relationships of bacteria within and between host species, and for identification of sources and transmission routes of bovine S. aureus. PMID:10790124

  9. Bovine lactoferricin, an antimicrobial peptide, is anti-inflammatory and anti-catabolic in human articular cartilage and synovium

    Science.gov (United States)

    Yan, Dongyao; Chen, Di; Shen, Jie; Xiao, Guozhi; van Wijnen, Andre J; Im, Hee-Jeong

    2012-01-01

    Bovine lactoferricin (LfcinB) is a multi-functional peptide derived from proteolytic cleavage of bovine lactoferrin. LfcinB was found to antagonize the biological effects mediated by angiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF-2) in endothelial cells. However, the effect of LfcinB on human articular cartilage remained unknown. Here, our findings demonstrate that LfcinB restored the proteoglycan loss promoted by catabolic factors (interleukin-1 β) IL-1β and FGF-2 in vitro and ex vivo. Mechanistically, LfcinB attenuated the effects of IL-1β and FGF-2 on the expression of cartilage-degrading enzymes (MMP-1, MMP-3, and MMP-13), destructive cytokines (IL-1β and IL-6), and inflammatory mediators (iNOS and TLR2). LfcinB induced protective cytokine expression (IL-4 and IL-10), and downregulated aggrecanase basal expression. LfcinB specifically activated ERK MAPK and Akt signaling pathways, which may account for its anti-inflammatory activity. We also revealed that LfcinB exerted similar protective effects on human synovial fibroblasts challenged by IL-1β, with minimal cytotoxicity. Collectively, our results suggest that LfcinB possesses potent anti-catabolic and anti-inflammatory bioactivities in human articular tissues, and may be utilized for the prevention and/or treatment of OA in the future. PMID:22740381

  10. Mapping and polymorphism of bovine ghreline gene

    OpenAIRE

    Colinet, Frédéric; Eggen, André; Halleux, Caroline; Arnould, Valérie; Portetelle, Daniel; Renaville, Robert

    2006-01-01

    Bovine ghrelin, a 27-amino-acid peptide has been identified in bovine oxyntic glands of the abomasum. It is an endogenous growth hormone secretagogue. Total mRNA was extracted from abomasum and complete ghrelin mRNA was sequenced by rapid amplification of cDNA ends. The gene contains five exons and four introns with a short noncoding first exon of 17 bp similar to mouse and human ghrelin gene. Using a radiation hybrid panel, the gene was mapped to chromosome 22 near microsat...

  11. Interaction of amphiphilic drugs with human and bovine serum albumins.

    Science.gov (United States)

    Khan, Abbul Bashar; Khan, Javed Masood; Ali, Mohd Sajid; Khan, Rizwan Hasan; Kabir-Ud-Din

    2012-11-01

    To know the interaction of amphiphilic drugs nortriptyline hydrochloride (NOT) and promazine hydrochloride (PMZ) with serum albumins (i.e., human serum albumin (HSA) and bovine serum albumin (BSA)), techniques of UV-visible, fluorescence, and circular dichroism (CD) spectroscopies are used. The binding affinity is more in case of PMZ with both the serum albumins. The quenching rate constant (k(q)) values suggest a static quenching process for all the drug-serum albumin interactions. The UV-visible results show that the change in protein conformation of PMZ-serum albumin interactions are more prominent as compared to NOT-serum albumin interactions. The CD results also explain the conformational changes in the serum albumins on binding with the drugs. The increment in %α-helical structure is slightly more for drug-BSA complexes as compared to drug-HSA complexes. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Direct in vivo characterization of delta 5 desaturase activity in humans by deuterium labeling: Effect of insulin

    International Nuclear Information System (INIS)

    el Boustani, S.; Causse, J.E.; Descomps, B.; Monnier, L.; Mendy, F.; Crastes de Paulet, A.

    1989-01-01

    The conversion of dihomogamma linolenic acid (DHLA) into arachidonic acid (AA) was compared in normal subjects and diabetic patients before and after treatment with insulin. The kinetics of the incorporation of deuterium-labeled DHLA and its conversion product, deuterium-labeled AA, was determined in plasma triglycerides, plasma phospholipids, and platelet lipids of subjects after ingestion of 2 g of the labeled precursor. Analysis was performed by gas liquid chromatography-mass spectrometry using multiple ion detection. In normal subjects, the deuterium-labeled DHLA concentration rose to 24 to 69 mg/L in plasma triglycerides four to nine hours after ingestion and to 20 to 34 mg/L in plasma phospholipids about four hours later. Deuterium-labeled AA appeared at 12 hours, rose to 2.4 to 3.8 mg/L between 48 and 72 hours in plasma phospholipids, but remained at the limit of detection in plasma triglycerides and was undetectable in platelet lipids. In diabetic patients both before and after insulin treatment, the deuterium-labeled DHLA concentration in plasma triglycerides and in plasma phospholipids followed the same pattern as in normal subjects. However, the deuterium-labeled arachidonic acid concentration was below 1 mg/L in plasma phospholipids before insulin. After insulin treatment the patients recovered normal DHLA metabolism because deuterium-labeled AA rose in phospholipids to a mean value of 3.5 mg/L, which is in the same range as that observed in normal subjects (3.2 mg/L). The present data provide direct evidence for the conversion of DHLA into AA in humans. The effect of insulin and the data from the literature of animal studies suggest insulin dependence of delta 5 desaturase in humans

  13. Insulin-like activity in the retina

    International Nuclear Information System (INIS)

    Das, A.

    1986-01-01

    A number of studies have recently demonstrated that insulin or a homologous peptide may be synthesized outside the pancreas also. The present study was designed to investigate whether insulin-like activity exists in the retina, and if it exists, whether it is due to local synthesis of insulin or a similar peptide in the retina. To determine whether the insulin-like immunoreactivity in retinal glial cells is due to binding and uptake or local synthesis of insulin, a combined approach of immunocytochemistry and in situ DNA-RNA hybridization techniques was used on cultured rat retinal glial cells. Insulin-like immunoreactivity was demonstrated in the cytoplasma of these cells. In situ hybridization studies using labeled rat insulin cDNA indicated that these cells contain the mRNA necessary for de novo synthesis of insulin or a closely homologous peptide. Since human retinal cells have, as yet, not been conveniently grown in culture, an ocular tumor cell line, human Y79 retinoblastoma was used as a model to extend these investigations. The presence of insulin-like immunoreactivity as well as insulin-specific mRNA was demonstrated in this cell line. Light microscopic autoradiography following incubation of isolated rat retinal cells with 125 I-insulin showed the presence of insulin binding sites on the photoreceptors and amarcine cells. On the basis of these observations that rat retina glial cells, including Muller cells are sites of synthesis of insulin or a similar peptide, a model for the pathogenesis of dabetic retinopathy is proposed

  14. Pharmacology of bovine and human thyrotropin: an historical perspective.

    Science.gov (United States)

    Robbins, J

    1999-05-01

    Before the induction of a brief period of hypothyroidism became the standard method for inducing 131I uptake in thyroid cancer diagnosis and therapy, several other methods were explored and used. At the dawn of this new era of recombinant human thyrotropin (TSH) it is of interest to reflect briefly on some of this work. Partially purified bovine TSH (bTSH) was supplied for many years by the Armour Company as Thytropar for intramuscular injection and was first used in thyroid cancer 50 years ago at the Montefiore Hospital and at the Memorial Sloan Kettering Cancer Center in New York City. Most of the patients were already hypothyroid and bTSH induced further 131I uptake in only a few. Experience over the next 30 years revealed frequent allergic reactions, occasionally serious ones, and in 1961 it was shown that prolonged use could result in resistance to both bTSH and human TSH. bTSH was, therefore, reserved for thyroid cancer patients unable to increase endogenous TSH when hypothyroid. bTSH also was used widely as a test to distinguish between hypothyroidism caused by thyroid or pituitary failure until it was replaced by thyrotropin-releasing hormone (TRH). In a few studies, TRH was also tested as an adjuvant to increase endogenous TSH and thus help to stimulate function in thyroid cancer, but this attracted little interest. Prolonged hypothyroidism, enhanced by antithyroid drugs, was used early on, but this very effective stimulant of thyroid cancer function was, for multiple reasons, discarded. Beginning interest 15 to 25 years ago in obtaining TSH from human pituitary glands, a byproduct of growth hormone production, was interrupted when this product was found to risk development of Creutzfeld-Jakob disease. Recombinant human TSH, a safe and effective substitute, is now ready for widespread use and development in thyroid cancer management.

  15. Development of a transgenic mouse model to study the immunogenicity of recombinant human insulin

    NARCIS (Netherlands)

    Torosantucci, Riccardo; Brinks, Vera; Kijanka, Grzegorz; Halim, Liem Andhyk; Sauerborn, Melody; Schellekens, Huub; Jiskoot, Wim

    2014-01-01

    Mouse models are commonly used to assess the immunogenicity of therapeutic proteins and to investigate the immunological processes leading to antidrug antibodies. The aim of this work was to develop a transgenic (TG) Balb/c mouse model for evaluating the immunogenicity of recombinant human insulin

  16. An Information Theoretical Analysis of Human Insulin-Glucose System Toward the Internet of Bio-Nano Things.

    Science.gov (United States)

    Abbasi, Naveed A; Akan, Ozgur B

    2017-12-01

    Molecular communication is an important tool to understand biological communications with many promising applications in Internet of Bio-Nano Things (IoBNT). The insulin-glucose system is of key significance among the major intra-body nanonetworks, since it fulfills metabolic requirements of the body. The study of biological networks from information and communication theoretical (ICT) perspective is necessary for their introduction in the IoBNT framework. Therefore, the objective of this paper is to provide and analyze for the first time in the literature, a simple molecular communication model of the human insulin-glucose system from ICT perspective. The data rate, channel capacity, and the group propagation delay are analyzed for a two-cell network between a pancreatic beta cell and a muscle cell that are connected through a capillary. The results point out a correlation between an increase in insulin resistance and a decrease in the data rate and channel capacity, an increase in the insulin transmission rate, and an increase in the propagation delay. We also propose applications for the introduction of the system in the IoBNT framework. Multi-cell insulin glucose system models may be based on this simple model to help in the investigation, diagnosis, and treatment of insulin resistance by means of novel IoBNT applications.

  17. Insulin analogues in pregnancy and specific congenital anomalies

    DEFF Research Database (Denmark)

    de Jong, Josta; Garne, Ester; Wender-Ozegowska, Ewa

    2016-01-01

    Insulin analogues are commonly used in pregnant women with diabetes. It is not known if the use of insulin analogues in pregnancy is associated with any higher risk of congenital anomalies in the offspring compared with use of human insulin. We performed a literature search for studies of pregnant...... women with pregestational diabetes using insulin analogues in the first trimester and information on congenital anomalies. The studies were analysed to compare the congenital anomaly rate among foetuses of mothers using insulin analogues with foetuses of mothers using human insulin. Of 29 studies, we...... samples in the included studies provided insufficient statistical power to identify a moderate increased risk of specific congenital anomalies. Copyright © 2015 John Wiley & Sons, Ltd....

  18. Evidence that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of gene expression from human cytomegalovirus promoter in CHO cells

    OpenAIRE

    Zhang, Yingpei; Katakura, Yoshinori; Seto, Perry; Shirahata, Sanetaka

    1997-01-01

    The signal transduction from insulin to its receptors and Ras has been extensively studied, while little has been reported beyond these steps. We found that the expression of human interleukin 6 gene under the control of immediate early gene promoter of human cytomegalovirus was enhanced by insulin sitmulation in Chinese hamster ovary cells. The induction effect of insulin was not significantly affected by inhibitors or activators of conventional protein kinase C, cAMP dependent protein kinas...

  19. Intranasal insulin modulates intrinsic reward and prefrontal circuitry of the human brain in lean women.

    Science.gov (United States)

    Kullmann, Stephanie; Frank, Sabine; Heni, Martin; Ketterer, Caroline; Veit, Ralf; Häring, Hans-Ulrich; Fritsche, Andreas; Preissl, Hubert

    2013-01-01

    There is accumulating evidence that food consumption is controlled by a wide range of brain circuits outside of the homeostatic system. Activation in these brain circuits may override the homeostatic system and also contribute to the enormous increase of obesity. However, little is known about the influence of hormonal signals on the brain's non-homeostatic system. Thus, selective insulin action in the brain was investigated by using intranasal application. We performed 'resting-state' functional magnetic resonance imaging in 17 healthy lean female subjects to assess intrinsic brain activity by fractional amplitude of low-frequency fluctuations (fALFF) before, 30 and 90 min after application of intranasal insulin. Here, we showed that insulin modulates intrinsic brain activity in the hypothalamus and orbitofrontal cortex. Furthermore, we could show that the prefrontal and anterior cingulate cortex response to insulin is associated with body mass index. This demonstrates that hormonal signals as insulin may reduce food intake by modifying the reward and prefrontal circuitry of the human brain, thereby potentially decreasing the rewarding properties of food. Due to the alarming increase in obesity worldwide, it is of great importance to identify neural mechanisms of interaction between the homeostatic and non-homeostatic system to generate new targets for obesity therapy. Copyright © 2012 S. Karger AG, Basel.

  20. Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study.

    Science.gov (United States)

    García-Álvarez, Laura; Holden, Matthew T G; Lindsay, Heather; Webb, Cerian R; Brown, Derek F J; Curran, Martin D; Walpole, Enid; Brooks, Karen; Pickard, Derek J; Teale, Christopher; Parkhill, Julian; Bentley, Stephen D; Edwards, Giles F; Girvan, E Kirsty; Kearns, Angela M; Pichon, Bruno; Hill, Robert L R; Larsen, Anders Rhod; Skov, Robert L; Peacock, Sharon J; Maskell, Duncan J; Holmes, Mark A

    2011-08-01

    Animals can act as a reservoir and source for the emergence of novel meticillin-resistant Staphylococcus aureus (MRSA) clones in human beings. Here, we report the discovery of a strain of S aureus (LGA251) isolated from bulk milk that was phenotypically resistant to meticillin but tested negative for the mecA gene and a preliminary investigation of the extent to which such strains are present in bovine and human populations. Isolates of bovine MRSA were obtained from the Veterinary Laboratories Agency in the UK, and isolates of human MRSA were obtained from diagnostic or reference laboratories (two in the UK and one in Denmark). From these collections, we searched for mecA PCR-negative bovine and human S aureus isolates showing phenotypic meticillin resistance. We used whole-genome sequencing to establish the genetic basis for the observed antibiotic resistance. A divergent mecA homologue (mecA(LGA251)) was discovered in the LGA251 genome located in a novel staphylococcal cassette chromosome mec element, designated type-XI SCCmec. The mecA(LGA251) was 70% identical to S aureus mecA homologues and was initially detected in 15 S aureus isolates from dairy cattle in England. These isolates were from three different multilocus sequence type lineages (CC130, CC705, and ST425); spa type t843 (associated with CC130) was identified in 60% of bovine isolates. When human mecA-negative MRSA isolates were tested, the mecA(LGA251) homologue was identified in 12 of 16 isolates from Scotland, 15 of 26 from England, and 24 of 32 from Denmark. As in cows, t843 was the most common spa type detected in human beings. Although routine culture and antimicrobial susceptibility testing will identify S aureus isolates with this novel mecA homologue as meticillin resistant, present confirmatory methods will not identify them as MRSA. New diagnostic guidelines for the detection of MRSA should consider the inclusion of tests for mecA(LGA251). Department for Environment, Food and Rural Affairs

  1. Influence of gamma-irradiation upon the chemical and biological properties of insulin

    Energy Technology Data Exchange (ETDEWEB)

    Salemink, P J.M.; Kolkman-Roodbeen, J C; Gribnau, T C.J.; Janssen, P S.L.; Veen, A.J. van der

    1987-06-19

    Partially purified insulin preparations of bovine and porcine origin, were subjected to gamma-irradiation with doses ranging from 1.0 up to 25 kGy at 0/sup 0/C or ambient temperature. The susceptibility of insulin to the irradiation was determined by chromatography, electrophoresis and assay of the biological activity. The sterilizing effect of the gamma-irradiation was investigated for Bacillus pumilus as well as for artificial mixtures of lactose and several bacilli. It is concluded that the sterilizing dose for the investigated insulins was greater than or equal to 2.2 kGy. At doses up to 25 kGy at 0/sup 0/C no specific radiolytic products were detectable, whereas the biological activity was fully retained. The content of dimers and the content of related peptides appeared to increase gradually with the irradiation dose absorbed. No effects of long-term storage could be demonstrated on biological and chemical properties of insulin after 2.2, 4.5 and 7.5 kGy. 21 refs.; 4 figs.; 5 tabs.

  2. Bovine respiratory syncytial virus : immunopathology and vaccine evaluation

    NARCIS (Netherlands)

    Antonis, A.F.G.

    2010-01-01

    Human and bovine RSVs cause severe disease in humans and in cattle respectively. They have been recognised as important respiratory pathogens in the last five decades, and this has resulted in significant research activities on the pathogenesis and intervention strategies around the world.

  3. Discovery and genomic characterization of a novel ovine partetravirus and a new genotype of bovine partetravirus.

    Directory of Open Access Journals (Sweden)

    Herman Tse

    Full Text Available Partetravirus is a recently described group of animal parvoviruses which include the human partetravirus, bovine partetravirus and porcine partetravirus (previously known as human parvovirus 4, bovine hokovirus and porcine hokovirus respectively. In this report, we describe the discovery and genomic characterization of partetraviruses in bovine and ovine samples from China. These partetraviruses were detected by PCR in 1.8% of bovine liver samples, 66.7% of ovine liver samples and 71.4% of ovine spleen samples. One of the bovine partetraviruses detected in the present samples is phylogenetically distinct from previously reported bovine partetraviruses and likely represents a novel genotype. The ovine partetravirus is a novel partetravirus and phylogenetically most related to the bovine partetraviruses. The genome organization is conserved amongst these viruses, including the presence of a putative transmembrane protein encoded by an overlapping reading frame in ORF2. Results from the present study provide further support to the classification of partetraviruses as a separate genus in Parvovirinae.

  4. Insulin analogues and cancer: a note of caution

    Directory of Open Access Journals (Sweden)

    Joseph A.M.J.L. eJanssen

    2014-05-01

    Full Text Available Abstract In view of the lifelong exposure and large patient populations involved, insulin analogues with an increased mitogenic effect in comparison to human insulin may potentially constitute a major health problem, since these analogues may possibly induce the growth of pre-existing neoplasms. At present, the available data suggest that insulin analogues are safe. In line with these findings, we observed that serum of diabetic patients treated with insulin analogues, compared to that of diabetic patients treated with human insulin, did not induce an increased phosphorylation of tyrosine residues of the insulin-like growth factor-I receptor (IGF-IR. However, the classical model of the IGF-IR signaling may be insufficient to explain (all mitogenic effects of insulin analogues since also non-canonical signaling pathways of the IGF-IR may play a major role in this respect. Although phosphorylation of tyrosine residues of the IGF-IR is generally considered to be the initial activation step within the intracellular IGF-IR signaling pathway, it has been found that cells undergo a signaling switch under hyperglycemic conditions. After this switch, a completely different mechanism is utilized to activate the mitogenic (mitogen-activated protein kinase (MAPK pathways of the IGF-IR that is independent from tyrosine phosphorylation of the IGF-IR. At present it is unknown whether activation of this alternative intracellular pathway of the IGF-IR occurs during hyperglycemia in vivo and whether it is stronger in patients treated with (some insulin analogues than in patients treated with human insulin. In addition, it is unknown whether the insulin receptors (IRs also undergo a signaling switch during hyperglycemia. This should be investigated in future studies. Finally, relative overexpression of IR isoform A (IR-A in (pre cancer tissues may play a key role in the development and progression of human cancers during treatment with insulin (analogues. Further

  5. Insulin Resistance and Mitochondrial Dysfunction.

    Science.gov (United States)

    Gonzalez-Franquesa, Alba; Patti, Mary-Elizabeth

    2017-01-01

    Insulin resistance precedes and predicts the onset of type 2 diabetes (T2D) in susceptible humans, underscoring its important role in the complex pathogenesis of this disease. Insulin resistance contributes to multiple tissue defects characteristic of T2D, including reduced insulin-stimulated glucose uptake in insulin-sensitive tissues, increased hepatic glucose production, increased lipolysis in adipose tissue, and altered insulin secretion. Studies of individuals with insulin resistance, both with established T2D and high-risk individuals, have consistently demonstrated a diverse array of defects in mitochondrial function (i.e., bioenergetics, biogenesis and dynamics). However, it remains uncertain whether mitochondrial dysfunction is primary (critical initiating defect) or secondary to the subtle derangements in glucose metabolism, insulin resistance, and defective insulin secretion present early in the course of disease development. In this chapter, we will present the evidence linking mitochondrial dysfunction and insulin resistance, and review the potential for mitochondrial targets as a therapeutic approach for T2D.

  6. Quantitative milk genomics: estimation of variance components and prediction of fatty acids in bovine milk

    DEFF Research Database (Denmark)

    Krag, Kristian

    The composition of bovine milk fat, used for human consumption, is far from the recommendations for human fat nutrition. The aim of this PhD was to describe the variance components and prediction probabilities of individual fatty acids (FA) in bovine milk, and to evaluate the possibilities...

  7. Zn2+ chelation by serum albumin improves hexameric Zn2+-insulin dissociation into monomers after exocytosis.

    Directory of Open Access Journals (Sweden)

    José A G Pertusa

    Full Text Available β-cells release hexameric Zn2+-insulin into the extracellular space, but monomeric Zn2+-free insulin appears to be the only biologically active form. The mechanisms implicated in dissociation of the hexamer remain unclear, but they seem to be Zn2+ concentration-dependent. In this study, we investigate the influence of albumin binding to Zn2+ on Zn2+-insulin dissociation into Zn2+-free insulin and its physiological, methodological and therapeutic relevance. Glucose and K+-induced insulin release were analyzed in isolated mouse islets by static incubation and perifusion experiments in the presence and absence of albumin and Zn2+ chelators. Insulin tolerance tests were performed in rats using different insulin solutions with and without Zn2+ and/or albumin. Albumin-free buffer does not alter quantification by RIA of Zn2+-free insulin but strongly affects RIA measurements of Zn2+-insulin. In contrast, accurate determination of Zn2+-insulin was obtained only when bovine serum albumin or Zn2+ chelators were present in the assay buffer solution. Albumin and Zn2+ chelators do not modify insulin release but do affect insulin determination. Preincubation with albumin or Zn2+ chelators promotes the conversion of "slow" Zn2+-insulin into "fast" insulin. Consequently, insulin diffusion from large islets is ameliorated in the presence of Zn2+ chelators. These observations support the notion that the Zn2+-binding properties of albumin improve the dissociation of Zn2+-insulin into subunits after exocytosis, which may be useful in insulin determination, insulin pharmacokinetic assays and islet transplantation.

  8. Familial hyperinsulinemia associated with secretion of an abnormal insulin, and coexistence of insulin resistance in the propositus.

    Science.gov (United States)

    Vinik, A I; Seino, S; Funakoshi, A; Schwartz, J; Matsumoto, M; Schteingart, D E; Fu, Z Z; Tsai, S T

    1986-04-01

    A 45-yr-old muscular nonobese white man who had a 9-yr history of syncopal episodes was studied on several occasions between April 1979 and August 1984. Fasting glucose concentrations ranged between 74-115 mg/dl, and those of insulin ranged between 14-64 microU/ml. Reactive hypoglycemia 3-4 h after ingestion of glucose occurred in the first 2 yr. Glucose tolerance was impaired in 1979, from February 1982 through September 1983, and again in August 1984. The maximum plasma insulin response to glucose ranged between 475-1630 microU/ml. When studied in November 1982, insulin (0.1 U/kg) caused a fall in blood glucose concentration of only 25% (normal, greater than 50%), and maximal glucose utilization during the euglycemic hyperinsulinemic clamp was 7.5 mg/kg . min (normal, greater than 12 mg/kg . min). Plasma counterregulatory hormone concentrations were normal, and antibodies to insulin and the insulin receptor were absent. Binding of exogenous insulin to the patient's cellular receptors (monocytes, red blood cells, and skin fibroblasts) was normal. Insulin was purified from plasma by immunoaffinity and molecular sieve chromatography and was found to elute later than human insulin on reversed phase high performance liquid chromatography. It was more hydrophobic than normal human insulin and had only 10% of the activity of normal insulin in terms of ability to bind to and stimulate glucose metabolism in isolated rat adipocytes. The abnormal insulin was identified in two of three sons and a sister, but not in the mother, brother, or niece. Sensitivity to insulin was normal in the two sons who had abnormal insulin. These results suggest that in this family the abnormal insulin was due to a biosynthetic defect, inherited as an autosomal dominant trait. The hyperinsulinemia was not associated with diabetes in family members who had no insulin resistance.

  9. Update on insulin treatment for dogs and cats: insulin dosing pens and more

    Directory of Open Access Journals (Sweden)

    Thompson A

    2015-04-01

    Full Text Available Ann Thompson,1 Patty Lathan,2 Linda Fleeman3 1School of Veterinary Science, The University of Queensland, Gatton, QLD, Australia; 2College of Veterinary Medicine Mississippi State University, Starkville, MS, USA; 3Animal Diabetes Australia, Melbourne, VIC, Australia Abstract: Insulin therapy is still the primary therapy for all diabetic dogs and cats. Several insulin options are available for each species, including veterinary registered products and human insulin preparations. The insulin chosen depends on the individual patient's requirements. Intermediate-acting insulin is usually the first choice for dogs, and longer-acting insulin is the first choice for cats. Once the insulin type is chosen, the best method of insulin administration should be considered. Traditionally, insulin vials and syringes have been used, but insulin pen devices have recently entered the veterinary market. Pens have different handling requirements when compared with standard insulin vials including: storage out of the refrigerator for some insulin preparations once pen cartridges are in use; priming of the pen to ensure a full dose of insulin is administered; and holding the pen device in place for several seconds during the injection. Many different types of pen devices are available, with features such as half-unit dosing, large dials for visually impaired people, and memory that can display the last time and dose of insulin administered. Insulin pens come in both reusable and disposable options. Pens have several benefits over syringes, including improved dose accuracy, especially for low insulin doses. Keywords: diabetes, mellitus, canine, feline, NPH, glargine, porcine lente

  10. Effect of Human and Bovine Serum Albumin on kinetic Chemiluminescence of Mn (III-Tetrakis (4-Sulfonatophenyl Porphyrin-Luminol-Hydrogen Peroxide System

    Directory of Open Access Journals (Sweden)

    Sayed Yahya Kazemi

    2012-01-01

    Full Text Available The present work deals with an attempt to study the effect of human and bovine serum albumin on kinetic parameters of chemiluminescence of luminol-hydrogen peroxide system catalyzed by manganese tetrasulfonatophenyl porphyrin (MnTSPP. The investigated parameters involved pseudo-first-order rise and fall rate constant for the chemiluminescence burst, maximum level intensity, time to reach maximum intensity, total light yield, and values of the intensity at maximum CL which were evaluated by nonlinear least square program KINFIT. Because of interaction of metalloporphyrin with proteins, the CL parameters are drastically affected. The systems resulted in Stern-Volmer plots with values of 3.17×105 and 3.7×105M−1 in the quencher concentration range of 1.5×10−6 to 1.5×10−5 M for human serum albumin (HSA and bovine serum albumin (BSA, respectively.

  11. Acute systemic insulin intolerance does not alter the response of the Akt/GSK-3 pathway to environmental hypoxia in human skeletal muscle

    DEFF Research Database (Denmark)

    D'Hulst, Gommaar; Sylow, Lykke; Hespel, Peter

    2015-01-01

    PURPOSE: To investigate how acute environmental hypoxia regulates blood glucose and downstream intramuscular insulin signaling after a meal in healthy humans. METHODS: Fifteen subjects were exposed for 4 h to normoxia (NOR) or to normobaric hypoxia (HYP, FiO2 = 0.11) in a randomized order 40 min ...... insulin intolerance developed independently of defects in conventional insulin signaling in skeletal muscle....

  12. Differentiation of PDX1 gene-modified human umbilical cord mesenchymal stem cells into insulin-producing cells in vitro.

    Science.gov (United States)

    He, Dongmei; Wang, Juan; Gao, Yangjun; Zhang, Yuan

    2011-12-01

    Mesenchymal stem cells (MSCs) have significant advantages over other stem cell types, and greater potential for immediate clinical application. MSCs would be an interesting cellular source for treatment of type 1 diabetes. In this study, MSCs from human umbilical cord were differentiated into functional insulin-producing cells in vitro by introduction of the pancreatic and duodenal homeobox factor 1 (PDX1) and in the presence of induction factors. The expressions of cell surface antigens were detected by flow cytometry. After induction in an adipogenic medium or an osteogenic medium, the cells were observed by Oil Red O staining and alkaline phosphatase staining. Recombinant adenovirus carrying the PDX1 gene was constructed and MSCs were infected by the recombinant adenovirus, then treated with several inducing factors for differentiation into islet β-like cells. The expression of the genes and protein related to islet β-cells was detected by immunocytochemistry, RT-PCR and Western blot analysis. Insulin and C-peptide secretion were assayed. Our results show that the morphology and immunophenotype of MSCs from human umbilical cord were similar to those present in human bone marrow. The MSCs could be induced to differentiate into osteocytes and adipocytes. After induction by recombined adenovirus vector with induction factors, MSCs were aggregated and presented islet-like bodies. Dithizone staining of these cells was positive. The genes' expression related to islet β-cells was found. After induction, insulin and C-peptide secretion in the supernatant were significantly increased. In conclusion, our results demonstrated that PDX1 gene-modified human umbilical cord mesenchymal stem cells could be differentiated into insulin-producing cells in vitro.

  13. Branched chain amino acid suppressed insulin-initiated proliferation of human cancer cells through induction of autophagy.

    Science.gov (United States)

    Wubetu, Gizachew Yismaw; Utsunomiya, Tohru; Ishikawa, Daichi; Ikemoto, Tetsuya; Yamada, Shinichiro; Morine, Yuji; Iwahashi, Shuichi; Saito, Yu; Arakawa, Yusuke; Imura, Satoru; Arimochi, Hideki; Shimada, Mitsuo

    2014-09-01

    Branched chain amino acid (BCAA) dietary supplementation inhibits activation of the insulin-like growth factor (IGF)/IGF-I receptor (IGF-IR) axis in diabetic animal models. However, the in vitro effect of BCAA on human cancer cell lines under hyper-insulinemic conditions remains unclear. Colon (HCT-116) and hepatic (HepG2) tumor cells were treated with varying concentrations of BCAA with or without fluorouracil (5-FU). The effect of BCAA on insulin-initiated proliferation was determined. Gene and protein expression was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. BCAA supplementation had no significant effect on cell proliferation and did not show significant synergistic or antagonistic effects with 5-FU. However, BCAA significantly decreased insulin-initiated proliferation of human colon and hepatic cancer cell lines in vitro. BCAA supplementation caused a marked decrease in activated IGF-IR expression and significantly enhanced both mRNA and protein expression of LC3-II and BECN1 (BECLIN-1). BCAA could be a useful chemopreventive modality for cancer in hyperinsulinemic conditions. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  14. Insulin and insulin-like growth factor receptors and responses

    International Nuclear Information System (INIS)

    Roth, R.A.; Steele-Perkins, G.; Hari, J.; Stover, C.; Pierce, S.; Turner, J.; Edman, J.C.; Rutter, W.J.

    1988-01-01

    Insulin is a member of a family of structurally related hormones with diverse physiological functions. In humans, the best-characterized members of this family include insulin, insulin-like growth factor (IGF)-I, and IGF-II. Each of these three polypeptide hormones has its own distinct receptor. The structures of each of these receptors have now been deduced from analyses of isolated cDNA clones. To study further the responses mediated through these three different receptors, the authors have been studying cells expressing the proteins encoded by these three cDNAs. The isolated cDNAs have been transfected into Chinese hamster ovary (CHO) cells, and the resulting transfected cell lines have been characterized as to the ligand-binding activities and signal-transducing activities of the expressed proteins

  15. Coordinated defects in hepatic long chain fatty acid metabolism and triglyceride accumulation contribute to insulin resistance in non-human primates.

    Directory of Open Access Journals (Sweden)

    Subhash Kamath

    Full Text Available Non-alcoholic fatty liver disease (NAFLD is characterized by accumulation of triglycerides (TG in hepatocytes, which may also trigger cirrhosis. The mechanisms of NAFLD are not fully understood, but insulin resistance has been proposed as a key determinant.To determine the TG content and long chain fatty acyl CoA composition profile in liver from obese non-diabetic insulin resistant (IR and lean insulin sensitive (IS baboons in relation with hepatic and peripheral insulin sensitivity.Twenty baboons with varying grades of adiposity were studied. Hepatic (liver and peripheral (mainly muscle insulin sensitivity was measured with a euglycemic clamp and QUICKI. Liver biopsies were performed at baseline for TG content and LCFA profile by mass spectrometry, and histological analysis. Findings were correlated with clinical and biochemical markers of adiposity and insulin resistance.Obese IR baboons had elevated liver TG content compared to IS. Furthermore, the concentration of unsaturated (LC-UFA was greater than saturated (LC-SFA fatty acyl CoA in the liver. Interestingly, LC-FA UFA and SFA correlated with waist, BMI, insulin, NEFA, TG, QUICKI, but not M/I. Histological findings of NAFLD ranging from focal to diffuse hepatic steatosis were found in obese IR baboons.Liver TG content is closely related with both hepatic and peripheral IR, whereas liver LC-UFA and LC-SFA are closely related only with hepatic IR in non-human primates. Mechanisms leading to the accumulation of TG, LC-UFA and an altered UFA: LC-SFA ratio may play an important role in the pathophysiology of fatty liver disease in humans.

  16. Effects of insulin detemir and NPH insulin on renal handling of sodium, fluid retention and weight in type 2 diabetic patients

    DEFF Research Database (Denmark)

    Hendriksen, K V; Jensen, Tonny Joran; Oturai, P

    2012-01-01

    In type 2 diabetic patients, insulin detemir (B29Lys(ε-tetradecanoyl),desB30 human insulin) induces less weight gain than NPH insulin. Due to the proposed reduction of tubular action by insulin detemir, type 2 diabetic patients should have increased urinary sodium excretion, thereby reducing extr...

  17. Development of a bioassay system for investigating insulin resistance factors of pregnancy

    International Nuclear Information System (INIS)

    Hausman, D.B.; Singh, R.; Martin, R.J.

    1986-01-01

    To determine if late-term pregnant serum and/or placenta could induce insulin resistance in normal adipose cells, the authors have developed an insulin sensitive bioassay system. Cells isolated from epididymal fat pads of 250-275 g Sprague Dawley rats are preincubated for 3 hours at 37 0 in media 199 and serum or placental extract. The cells are washed free of serum and tested for metabolic activity in a 2 hour incubation which measures the conversion of U- 14 C-glucose to 14 CO 2 and to 14 C-triglyceride fatty acids under basal and insulin stimulated conditions. Maximal insulin responsiveness (350-450% basal for CO 2 and 1400-1700% basal for fatty acids) is achieved using Worthington Type II collagenase and a 45-60 minute digestion period for cell isolations and Krebs-Ringer bicarbonate buffer containing 0.5 mM glucose, 2% Armour bovine serum albumin (CRG-7), 1000 μU/ml insulin and 110,000 to 120,000 cells in the 2 hour incubations. Using this bioasssay system the authors have found that insulin responsiveness, in terms of glucose conversion to fatty acids, is unchanged when cells are preincubated with 5% control pig serum but reduced following preincubation with late pregnant (110 day) pig serum. In future experiments the authors hope to further characterize the factor(s) in pregnant serum responsible for inducing this metabolic effect

  18. Investigation of the mechanisms that influence the accretion of bovine intramuscular and subcutaneous adipose tissue

    International Nuclear Information System (INIS)

    Miller, M.F.

    1987-01-01

    The understanding of the mechanisms that differ between breeds of cattle and their ability to deposit intramuscular adipose tissue is imperative to profitable beef production. Thus, the interactions among breeds, metabolic substrates and specific hormones in bovine intramuscular and subcutaneous adipose tissue were investigated. Subcutaneous and intramuscular adipose tissues were obtained from 10 Angus and 9 Santa Gertrudis steers immediately postmortem. The adipose tissues were incubated for 2 h and 48 h with and without 1 mU/ml insulin and 30 mg/ml bovine serum albumin (BSA) to measure the incorporation of 14 C-labeled acetate and glucose into lipid fractions. At the same chronological age, Angus steers had a more youthful lean maturity score, higher USDA marbling scores and higher USDA quality grades than carcasses from Santa Gertrudis steers

  19. Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells.

    Science.gov (United States)

    Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani; Streicher, Werner; Wikström, Mats; Cazzamali, Giuseppe

    2015-04-01

    Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Adipokines and Hepatic Insulin Resistance

    Science.gov (United States)

    Hassan, Waseem

    2013-01-01

    Obesity is a major risk factor for insulin resistance and type 2 diabetes. Adipose tissue is now considered to be an active endocrine organ that secretes various adipokines such as adiponectin, leptin, resistin, tumour necrosis factor-α, and interleukin-6. Recent studies have shown that these factors might provide a molecular link between increased adiposity and impaired insulin sensitivity. Since hepatic insulin resistance plays the key role in the whole body insulin resistance, clarification of the regulatory processes about hepatic insulin resistance by adipokines in rodents and human would seem essential in order to understand the mechanism of type 2 diabetes and for developing novel therapeutic strategies to treat it. PMID:23762871

  1. Insulin sensitivity is independent of lipid binding protein trafficking at the plasma membrane in human skeletal muscle

    DEFF Research Database (Denmark)

    Jordy, Andreas Børsting; Serup, Annette Karen; Karstoft, Kristian

    2014-01-01

    The aim of the present study was to investigate lipid-induced regulation of lipid binding proteins in human skeletal muscle and the impact hereof on insulin sensitivity. Eleven healthy male subjects underwent a 3-day hyper-caloric and high-fat diet regime. Muscle biopsies were taken before......-regulated by increased fatty acid availability. This suggests a time dependency in the up-regulation of FAT/CD36 and FABPpm protein during high availability of plasma fatty acids. Furthermore, we did not detect FATP1 and FATP4 protein in giant sarcolemmal vesicles obtained from human skeletal muscle. In conclusion......, this study shows that a short-term lipid-load increases mRNA content of key lipid handling proteins in human muscle. However, decreased insulin sensitivity after high-fat diet is not accompanied with relocation of FAT/CD36 or FABPpm protein to the sarcolemma. Finally, FATP1 and FATP4 protein could...

  2. Detection of Escherichia coli Shiga toxin-producing in viscera of animals bovine and chicken intended for human consumption

    Directory of Open Access Journals (Sweden)

    Zotta, Claudio Marcelo

    2016-05-01

    Full Text Available Escherichia coli producing-Shiga toxin (STEC is associated with foodborne illness (ETA. It can cause bloody diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. The aim of the study was to detect the presence of STEC in samples of organs (offal of bovine animals and chicken intended for human consumption. Between 2008-2009, 76 samples bovine entrails and 22 chicken viscera samples, were processed and underwent, as screening technique, the polymerase chain reaction (PCR for detection of multiple genes coding for the factors virulence: Shiga toxin (stx1, stx2 and rfbO157 gene coding for capsular O157 lipopolysaccharide LPS. Samples from bovine offal development showed 84.2% for coliform bacteria. These isolates showed no virulence factor that characterized as STEC or Escherichia coli O157. The chicken offal samples showed 95.5% of development for coliform bacteria, being negative for the presence of genes encoding the Shiga toxins 1 and 2 (stx1, stx2 and rfbO157 gene. While this work does not STEC was detected, the presence of coliform bacteria in the samples studied makes these foods should be considered as potentially hazardous to consume undercooked with the consequent possibility of filing ETA.

  3. Effect of 5'-flanking sequence deletions on expression of the human insulin gene in transgenic mice

    DEFF Research Database (Denmark)

    Fromont-Racine, M; Bucchini, D; Madsen, O

    1990-01-01

    Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific...... of the transgene was observed in cell types other than beta-islet cells....

  4. High glucose suppresses human islet insulin biosynthesis by inducing miR-133a leading to decreased polypyrimidine tract binding protein-expression.

    Directory of Open Access Journals (Sweden)

    Rikard G Fred

    Full Text Available BACKGROUND: Prolonged periods of high glucose exposure results in human islet dysfunction in vitro. The underlying mechanisms behind this effect of high glucose are, however, unknown. The polypyrimidine tract binding protein (PTB is required for stabilization of insulin mRNA and the PTB mRNA 3'-UTR contains binding sites for the microRNA molecules miR-133a, miR-124a and miR-146. The aim of this study was therefore to investigate whether high glucose increased the levels of these three miRNAs in association with lower PTB levels and lower insulin biosynthesis rates. METHODOLOGY/PRINCIPAL FINDINGS: Human islets were cultured for 24 hours in the presence of low (5.6 mM or high glucose (20 mM. Islets were also exposed to sodium palmitate or the proinflammatory cytokines IL-1beta and IFN-gamma, since saturated free fatty acids and cytokines also cause islet dysfunction. RNA was then isolated for real-time RT-PCR analysis of miR-133a, miR-124a, miR-146, insulin mRNA and PTB mRNA contents. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. Synthetic miR-133a precursor and inhibitor were delivered to dispersed islet cells by lipofection, and PTB was analyzed by immunoblotting following culture at low or high glucose. Culture in high glucose resulted in increased islet contents of miR-133a and reduced contents of miR-146. Cytokines increased the contents of miR-146. The insulin and PTB mRNA contents were unaffected by high glucose. However, both PTB protein levels and insulin biosynthesis rates were decreased in response to high glucose. The miR-133a inhibitor prevented the high glucose-induced decrease in PTB and insulin biosynthesis, and the miR-133a precursor decreased PTB levels and insulin biosynthesis similarly to high glucose. CONCLUSION: Prolonged high-glucose exposure down-regulates PTB levels and insulin biosynthesis rates in human islets by increasing miR-133a levels. We propose that this mechanism

  5. Free Radicals Formation of Irradiated Lyophilized Can-Cellous Human and Bovine Bone

    International Nuclear Information System (INIS)

    Abbas, Basril; Sudiro, Sutjipto; Hilmy, Nazly

    2000-01-01

    Radiation sterilization of lyophilized human and bovine bone as allograft and xenograft have been produced and used in orthopaedic practice in Indonesia routinely. It is well known from radio biologic studies that one of the most pronounce effects of ionizing radiation on biologic species produced the free radicals that influence the physico-chemical as well as the mechanical properties of irradiated bone. The aim of our study is to investigate the free radicals formation of irradiated lyophilized cancellous triple A bone (Autolyzed Antigen-Extracted Allograft) produced by Batan Research Tissue Bank in Jakarta. The cancellous triple A were prepared according to AATB (American Association of Tissue Bank) method. Gamma Irradiations was done at doses of 10, 20 and 30 kGy with a dose rate of 7,5 kGy/h at room temperature (30 o C± 2 o C). Measurements of free radicals was done at 24 o C ±1 o C within 30 minutes after irradiational and measurement were continued up to 9 months of storage using a JES-REIX ESR Spectrophotometer (JEOL) with Mn exp. ++ standard. Parameters measured, were the effects of mechanical grinding, water immersion and irradiation dose on free radicals formation in the bone. Results show that the signal area of ESR spectra from irradiated bovine bone of 30 kGy was higher than those of human bone I.e. 1,4 x 10 exp. 7 dan 6,4 x 10 exp. 6 Au (arbitrary unit)/g samples respectively. The signal of ESR spectra increased linearly with increasing dose in the range of 10-30 kGy and it will reduce about 30% caused by water immersion. The ESR signal reduced sharply after 2 days and gradually decreased up to 14 days and then became constant up to 9 months of storage at room temperature. A certain method of crushing can produce free radicals. Key Words: free radical, irradiation, allograft, xenograft, mechanical-grinding

  6. Insulin Resistance

    DEFF Research Database (Denmark)

    Jensen, Benjamin Anderschou Holbech

    Insulin resistance (IR) is escalating with alarming pace and is no longer restricted to westernized countries. As a forerunner for some of the most serious threats to human health including metabolic syndrome, cardiovascular diseases, and type 2-diabetes, the need for new treatment modalities...... interventions. We further show that improving the inflammatory toning, using fish oil as fat source, protects mice against diet induced obesity and -inflammation while preserving insulin sensitivity, even in the absence of free fatty acid receptor 4. Conversely, HFD-induced intestinal dysbiosis is associated...

  7. Insulin analogs with improved pharmacokinetic profiles.

    Science.gov (United States)

    Brange; Vølund

    1999-02-01

    The aim of insulin replacement therapy is to normalize blood glucose in order to reduce the complications of diabetes. The pharmacokinetics of the traditional insulin preparations, however, do not match the profiles of physiological insulin secretion. The introduction of the rDNA technology 20 years ago opened new ways to create insulin analogs with altered properties. Fast-acting analogs are based on the idea that an insulin with less tendency to self-association than human insulin would be more readily absorbed into the systemic circulation. Protracted-acting analogs have been created to mimic the slow, steady rate of insulin secretion in the fasting state. The present paper provides a historical review of the efforts to change the physicochemical and pharmacological properties of insulin in order to improve insulin therapy. The available clinical studies of the new insulins are surveyed and show, together with modeling results, that new strategies for optimal basal-bolus treatment are required for utilization of the new fast-acting analogs.

  8. Detection and characterisation of Complement protein activity in bovine milk by bactericidal sequestration assay.

    Science.gov (United States)

    Maye, Susan; Stanton, Catherine; Fitzgerald, Gerald F; Kelly, Philip M

    2015-08-01

    While the Complement protein system in human milk is well characterised, there is little information on its presence and activity in bovine milk. Complement forms part of the innate immune system, hence the importance of its contribution during milk ingestion to the overall defences of the neonate. A bactericidal sequestration assay, featuring a Complement sensitive strain, Escherichia coli 0111, originally used to characterise Complement activity in human milk was successfully applied to freshly drawn bovine milk samples, thus, providing an opportunity to compare Complement activities in both human and bovine milks. Although not identical in response, the levels of Complement activity in bovine milk were found to be closely comparable with that of human milk. Differential counts of Esch. coli 0111 after 2 h incubation were 6.20 and 6.06 log CFU/ml, for raw bovine and human milks, respectively - the lower value representing a stronger Complement response. Exposing bovine milk to a range of thermal treatments e.g. 42, 45, 65, 72, 85 or 95 °C for 10 min, progressively inhibited Complement activity by increasing temperature, thus confirming the heat labile nature of this immune protein system. Low level Complement activity was found, however, in 65 and 72 °C heat treated samples and in retailed pasteurised milk which highlights the outer limit to which high temperature, short time (HTST) industrial thermal processes should be applied if retention of activity is a priority. Concentration of Complement in the fat phase was evident following cream separation, and this was also reflected in the further loss of activity recorded in low fat variants of retailed pasteurised milk. Laboratory-based churning of the cream during simulated buttermaking generated an aqueous (buttermilk) phase with higher levels of Complement activity than the fat phase, thus pointing to a likely association with the milk fat globule membrane (MFGM) layer.

  9. Combining insulins for optimal blood glucose control in type 1 and 2 diabetes: focus on insulin glulisine

    Directory of Open Access Journals (Sweden)

    Heather Ulrich

    2007-07-01

    Full Text Available Heather Ulrich1,4, Benjamin Snyder1,Satish K Garg1,2,31Barbara Davis Center for Childhood Diabetes; 2Department of Medicine; 3Pediatrics; 4Department of Clinical Pharmacy, School of Pharmacy, University of Colorado at Denver and Health Sciences Center, Denver, CO, USAAbstract: Normalization of blood glucose is essential for the prevention of diabetes mellitus (DM-related microvascular and macrovascular complications. Despite substantial literature to support the benefits of glucose lowering and clear treatment targets, glycemic control remains suboptimal for most people with DM in the United States. Pharmacokinetic limitations of conventional insulins have been a barrier to achieving treatment targets secondary to adverse effects such as hypoglycemia and weight gain. Recombinant DNA technology has allowed modification of the insulin molecule to produce insulin analogues that overcome these pharmacokinetic limitations. With time action profiles that more closely mimic physiologic insulin secretion, rapid acting insulin analogues (RAAs reduce post-prandial glucose excursions and hypoglycemia when compared to regular human insulin (RHI. Insulin glulisine (Apidra® is a rapid-acting insulin analogue created by substituting lysine for asparagine at position B3 and glutamic acid for lysine at position B29 on the B chain of human insulin. The quick absorption of insulin glulisine more closely reproduces physiologic first-phase insulin secretion and its rapid acting profile is maintained across patient subtypes. Clinical trials have demonstrated comparable or greater efficacy of insulin glulisine versus insulin lispro or RHI, respectively. Efficacy is maintained even when insulin glulisine is administered post-meal. In addition, glulisine appears to have a more rapid time action profile compared with insulin lispro across various body mass indexes (BMIs. The safety and tolerability profile of insulin glulisine is also comparable to that of insulin

  10. Expression of microRNAs in bovine and human pre-implantation embryo culture media

    Science.gov (United States)

    Kropp, Jenna; Salih, Sana M.; Khatib, Hasan

    2014-01-01

    MicroRNAs (miRNA) are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, miR-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy. PMID:24795753

  11. Changing the insulin receptor to possess insulin-like growth factor I ligand specificity

    International Nuclear Information System (INIS)

    Andersen, A.S.; Kjeldsen, T.; Wiberg, F.C.; Christensen, P.M.; Rasmussen, J.S.; Norris, K.; Moeller, K.B.; Moeller, N.P.H.

    1990-01-01

    To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, the authors prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the α-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor

  12. Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells

    International Nuclear Information System (INIS)

    Timper, Katharina; Seboek, Dalma; Eberhardt, Michael; Linscheid, Philippe; Christ-Crain, Mirjam; Keller, Ulrich; Mueller, Beat; Zulewski, Henryk

    2006-01-01

    Mesenchymal stem cells (MSC) from mouse bone marrow were shown to adopt a pancreatic endocrine phenotype in vitro and to reverse diabetes in an animal model. MSC from human bone marrow and adipose tissue represent very similar cell populations with comparable phenotypes. Adipose tissue is abundant and easily accessible and could thus also harbor cells with the potential to differentiate in insulin producing cells. We isolated human adipose tissue-derived MSC from four healthy donors. During the proliferation period, the cells expressed the stem cell markers nestin, ABCG2, SCF, Thy-1 as well as the pancreatic endocrine transcription factor Isl-1. The cells were induced to differentiate into a pancreatic endocrine phenotype by defined culture conditions within 3 days. Using quantitative PCR a down-regulation of ABCG2 and up-regulation of pancreatic developmental transcription factors Isl-1, Ipf-1, and Ngn3 were observed together with induction of the islet hormones insulin, glucagon, and somatostatin

  13. Treatment of dwarfism with recombinant human insulin-like growth factor-1.

    Science.gov (United States)

    Ranke, Michael B; Wölfle, Joachim; Schnabel, Dirk; Bettendorf, Markus

    2009-10-01

    The growth hormone-IGF (insulin-like growth factor) system plays a central role in hormonal growth regulation. Recombinant human (rh) growth hormone (GH) has been available since the late 1980s for replacement therapy in GH-deficient patients and for the stimulation of growth in patients with short stature of various causes. Growth promotion by GH occurs in part indirectly through the induction of IGF-1 synthesis. In primary disturbances of IGF-1 production, short stature can only be treated with recombinant human IGF-1 (rhIGF-1). rhIGF-1 was recently approved for this indication but can also be used to treat other conditions. Selective review of the literature on IGF-1 therapy, based on a PubMed search. In children with severe primary IGF-1 deficiency (a rare condition whose prevalence is less than 1:10,000), the prognosis for final height is very poor (ca. 130 cm), and IGF-1 therapy is the appropriate form of pathophysiologically based treatment. There is no alternative treatment at present. The subcutaneous administration of IGF-1 twice daily in doses of 80 to 120 microg/kg accelerates growth and increases final height by 12 to 15 cm, according to current data. There is, however, a risk of hypoglycemia, as IGF-1 has an insulin-like effect. As treatment with IGF-1 is complex, this new medication should only be prescribed, for the time being, by experienced pediatric endocrinologists and diabetologists.

  14. Bovine rotavirus pentavalent vaccine development in India.

    Science.gov (United States)

    Zade, Jagdish K; Kulkarni, Prasad S; Desai, Sajjad A; Sabale, Rajendra N; Naik, Sameer P; Dhere, Rajeev M

    2014-08-11

    A bovine rotavirus pentavalent vaccine (BRV-PV) containing rotavirus human-bovine (UK) reassortant strains of serotype G1, G2, G3, G4 and G9 has been developed by the Serum Institute of India Ltd, in collaboration with the National Institute of Allergy and Infectious Diseases (NIAID), USA. The vaccine underwent animal toxicity studies and Phase I and II studies in adults, toddlers and infants. It has been found safe and immunogenic and will undergo a large Phase III study to assess efficacy against severe rotavirus gastroenteritis. Copyright © 2014. Published by Elsevier Ltd.

  15. Reduced insulin exocytosis in human pancreatic β-cells with gene variants linked to type 2 diabetes

    DEFF Research Database (Denmark)

    Rosengren, Anders H; Braun, Matthias; Mahdi, Taman

    2012-01-01

    The majority of genetic risk variants for type 2 diabetes (T2D) affect insulin secretion, but the mechanisms through which they influence pancreatic islet function remain largely unknown. We functionally characterized human islets to determine secretory, biophysical, and ultrastructural features ...

  16. A Branched-Chain Amino Acid-Related Metabolic Signature that Differentiates Obese and Lean Humans and Contributes to Insulin Resistance

    Science.gov (United States)

    Newgard, Christopher B; An, Jie; Bain, James R; Muehlbauer, Michael J; Stevens, Robert D; Lien, Lillian F; Haqq, Andrea M; Shah, Svati H.; Arlotto, Michelle; Slentz, Cris A; Rochon, James; Gallup, Dianne; Ilkayeva, Olga; Wenner, Brett R; Yancy, William E; Eisenson, Howard; Musante, Gerald; Surwit, Richard; Millington, David S; Butler, Mark D; Svetkey, Laura P

    2009-01-01

    Summary Metabolomic profiling of obese versus lean humans reveals a branched-chain amino acid (BCAA)-related metabolite signature that is suggestive of increased catabolism of BCAA and correlated with insulin resistance. To test its impact on metabolic homeostasis, we fed rats on high-fat (HF), HF with supplemented BCAA (HF/BCAA) or standard chow (SC) diets. Despite having reduced food intake and weight gain equivalent to the SC group, HF/BCAA rats were equally insulin resistant as HF rats. Pair-feeding of HF diet to match the HF/BCAA animals or BCAA addition to SC diet did not cause insulin resistance. Insulin resistance induced by HF/BCAA feeding was accompanied by chronic phosphorylation of mTOR, JNK, and IRS1(ser307), accumulation of multiple acylcarnitines in muscle, and was reversed by the mTOR inhibitor, rapamycin. Our findings show that in the context of a poor dietary pattern that includes high fat consumption, BCAA contributes to development of obesity-associated insulin resistance. PMID:19356713

  17. Insulin-Producing Cells Differentiated from Human Bone Marrow Mesenchymal Stem Cells In Vitro Ameliorate Streptozotocin-Induced Diabetic Hyperglycemia.

    Directory of Open Access Journals (Sweden)

    Ying Xin

    Full Text Available The two major obstacles in the successful transplantation of islets for diabetes treatment are inadequate supply of insulin-producing tissue and immune rejection. Induction of the differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs into insulin-producing cells (IPCs for autologous transplantation may alleviate those limitations.hMSCs were isolated and induced to differentiate into IPCs through a three-stage differentiation protocol in a defined media with high glucose, nicotinamide, and exendin-4. The physiological characteristics and functions of IPCs were then evaluated. Next, about 3 × 10(6 differentiated cells were transplanted into the renal sub-capsular space of streptozotocin (STZ-induced diabetic nude mice. Graft survival and function were assessed by immunohistochemistry, TUNEL staining and measurements of blood glucose levels in the mice.The differentiated IPCs were characterized by Dithizone (DTZ positive staining, expression of pancreatic β-cell markers, and human insulin secretion in response to glucose stimulation. Moreover, 43% of the IPCs showed L-type Ca2+ channel activity and similar changes in intracellular Ca2+ in response to glucose stimulation as that seen in pancreatic β-cells in the process of glucose-stimulated insulin secretion. Transplantation of functional IPCs into the renal subcapsular space of STZ-induced diabetic nude mice ameliorated the hyperglycemia. Immunofluorescence staining revealed that transplanted IPCs sustainably expressed insulin, c-peptide, and PDX-1 without apparent apoptosis in vivo.IPCs derived from hMSCs in vitro can ameliorate STZ-induced diabetic hyperglycemia, which indicates that these hMSCs may be a promising approach to overcome the limitations of islet transplantation.

  18. Sheep-passaged bovine spongiform encephalopathy agent exhibits altered pathobiological properties in bovine-PrP transgenic mice

    NARCIS (Netherlands)

    Espinosa, J.C.; Andreoletti, O.; Castilla, J.; Herva, M.E.; Morales, M.; Alamillo, E.; San-Segundo, F.D.; Lacroux, C.; Lugan, S.; Salguero, F.J.; Langeveld, J.P.M.; Torres, J.M.

    2007-01-01

    Sheep can be experimentally infected with bovine spongiform encephalopathy (BSE), and the ensuing disease is similar to scrapie in terms of pathogenesis and clinical signs. BSE infection in sheep is an animal and human health concern. In this study, the transmission in BoPrP-Tg110 mice of prions

  19. Differential insulin and steroidogenic signaling in insulin resistant and non-insulin resistant human luteinized granulosa cells-A study in PCOS patients.

    Science.gov (United States)

    Belani, Muskaan; Deo, Abhilash; Shah, Preeti; Banker, Manish; Singal, Pawan; Gupta, Sarita

    2018-04-01

    Insulin resistance (IR) is one of the significant aberrations in polycystic ovarian syndrome (PCOS), however is only observed in 70%-80% of obese PCOS and 20%-25% of lean PCOS. Hyperinsulinemia accompanies PCOS-IR along with hyperandrogenemia against normal insulin and androgen levels in PCOS-non insulin resistance (NIR). This could possibly be due to defects in the downstream signaling pathways. The study thus aims to unravel insulin and steroidogenic signaling pathways in luteinized granulosa cells isolated from PCOS-IR and NIR vs matched controls. Luteinized granulosa cells from 30 controls and 39 PCOS were classified for IR based on a novel method of down regulation of protein expression of insulin receptor-β (INSR- β) as shown in our previous paper. We evaluated expression of molecules involved in insulin, steroidogenic signaling and lipid metabolism in luteinized granulosa cells followed by analysis of estradiol, progesterone and testosterone in follicular fluid. Protein expression of INSR- β, pIRS (ser 307), PI(3)K, PKC-ζ, pAkt, ERK1/2, pP38MAPK and gene expression of IGF showed differential expression in the two groups. Increased protein expression of PPAR-γ was accompanied by up regulation in SREBP1c, FAS, CPT-1 and ACC-1 genes in PCOS-IR group. Expression of StAR, CYP19A1, 17 β- HSD and 3 β- HSD demonstrated significant decrease along with increase in CYP11A1, FSH-R and LH-R in both the groups. Follicular fluid testosterone increased and progesterone decreased in PCOS-IR group. This study shows how candidate molecules that were differentially expressed, aid in designing targeted therapy against the two phenotypes of PCOS. Copyright © 2018 Elsevier Ltd. All rights reserved.

  20. Treatment with insulin (analogues) and breast cancer risk in diabetics; a systematic review and meta-analysis of in vitro, animal and human evidence

    DEFF Research Database (Denmark)

    Bronsveld, Heleen K; ter Braak, Bas; Karlstad, Øystein

    2015-01-01

    INTRODUCTION: Several studies have suggested that anti-diabetic insulin analogue treatment might increase cancer risk. The aim of this study was to review the postulated association between insulin and insulin analogue treatment and breast cancer development, and plausible mechanisms. METHOD......: A systematic literature search was performed on breast cell-line, animal and human studies using the key words 'insulin analogue' and 'breast neoplasia' in MEDLINE at PubMed, EMBASE, and ISI Web of Science databases. A quantitative and qualitative review was performed on the epidemiological data; due...

  1. Effect of environmental particulates on cultured human and bovine endothelium. Cellular injury via an oxidant-dependent pathway

    International Nuclear Information System (INIS)

    Garcia, J.G.; Dodson, R.F.; Callahan, K.S.

    1989-01-01

    The effects of respirable environmental fibers on cultures of human umbilical vein and bovine pulmonary artery endothelial cell monolayers were studied. Interaction among endothelial cell monolayers and amosite and chrysotile asbestos, attapulgite, fiberglass, or latex beads resulted in rapid phagocytosis of the particulates. A gradient of time-dependent and concentration-dependent endothelial cell injury (measured by specific 51Cr release) was observed with amosite and attapulgite being markedly toxic. Chrysotile and fiberglass were much less toxic, and latex beads were not significantly injurious at any time or dose examined. Responses of bovine pulmonary artery and human endothelial vein endothelial cells to fiber phagocytosis and fiber-induced injury were similar. In human umbilical cell monolayers, fiber-mediated stimulation of the arachidonate metabolite prostacyclin paralleled endothelial cell injury; i.e. amosite and attapulgite were stimulatory, whereas fiberglass (0-500 micrograms/ml) and latex beads (10(9) beads/ml) did not significantly increase prostacyclin generation. Although chrysotile was only weakly cytotoxic, significant stimulation of prostacyclin was observed at the highest dose tested (500 micrograms/ml). To investigate whether toxic oxygen species may be involved in fiber-induced cytotoxicity, oxidant scavengers or inhibitors were used in injury studies. Both superoxide dismutase (a scavenger of O2-) and catalase (an inhibitor of H2O2) produced significant protection against fiber-mediated endothelial cell injury. In addition, chelation by deferoxamine of elemental Fe present in the fiber preparations was also protective, suggesting Fe, via the modified Haber-Weiss reaction, may promote hydroxyl radical formation and contribute to endothelial cell injury induced by these particulates

  2. Bovine colostrum improves neonatal growth, digestive function, and gut immunity relative to donor human milk and infant formula in preterm pigs

    DEFF Research Database (Denmark)

    Rasmussen, Stine Ostenfeldt; Martin, Lena; Østergaard, Mette Viberg

    2016-01-01

    Mother's own milk is the optimal first diet for preterm infants, but donor human milk (DM) or infant formula (IF) is used when supply is limited. We hypothesized that a gradual introduction of bovine colostrum (BC) or DM improves gut maturation, relative to IF during the first 11 days after preterm...

  3. [Targeting the brain through the nose. Effects of intranasally administered insulin].

    Science.gov (United States)

    Brünner, Y F; Benedict, C; Freiherr, J

    2013-08-01

    The assumption that the human brain is an insulin-independent organ was disproved with the discovery of insulin receptors in the central nervous system in the year 1978. Evidence has been provided for a high density of insulin receptors in brain regions responsible for cognitive memory processes (hippocampus) and for the regulation of appetite (hypothalamus). Accordingly, in animal studies an increased insulin level in the central nervous system leads to an improvement of hippocampal memory function and a decrease of food intake. Similar results were obtained in humans using the method of intranasal administration of insulin. Intranasal insulin reaches the brain and the cerebrospinal fluid via the olfactory epithelium and olfactory nerve fiber bundles leading through the lamina cribrosa to the olfactory bulb. Thus, this method renders the investigation of specific insulin effects in humans possible. The therapeutic potential of an intranasal insulin administration for the treatment of diseases for which an imbalance of the central nervous insulin metabolism is discussed (e.g. Alzheimer's disease, diabetes mellitus and obesity) can only be estimated with the help of further clinical studies.

  4. Insulin and the brain.

    Science.gov (United States)

    Derakhshan, Fatemeh; Toth, Cory

    2013-03-01

    Mainly known for its role in peripheral glucose homeostasis, insulin has also significant impact within the brain, functioning as a key neuromodulator in behavioral, cellular, biochemical and molecular studies. The brain is now regarded as an insulin-sensitive organ with widespread, yet selective, expression of the insulin receptor in the olfactory bulb, hypothalamus, hippocampus, cerebellum, amygdala and cerebral cortex. Insulin receptor signaling in the brain is important for neuronal development, glucoregulation, feeding behavior, body weight, and cognitive processes such as with attention, executive functioning, learning and memory. Emerging evidence has demonstrated insulin receptor signaling to be impaired in several neurological disorders. Moreover, insulin receptor signaling is recognized as important for dendritic outgrowth, neuronal survival, circuit development, synaptic plasticity and postsynaptic neurotransmitter receptor trafficking. We review the multiple roles of insulin in the brain, as well as its endogenous trafficking to the brain or its exogenous intervention. Although insulin can be directly targeted to the brain via intracerebroventricular (ICV) or intraparenchymal delivery, these invasive techniques are with significant risk, necessitating repeated surgical intervention and providing potential for systemic hypoglycemia. Another method, intranasal delivery, is a non-invasive, safe, and alternative approach which rapidly targets delivery of molecules to the brain while minimizing systemic exposure. Over the last decades, the delivery of intranasal insulin in animal models and human patients has evolved and expanded, permitting new hope for associated neurodegenerative and neurovascular disorders.

  5. Autophagy downregulation contributes to insulin resistance mediated injury in insulin receptor knockout podocytes in vitro

    Directory of Open Access Journals (Sweden)

    Ying Xu

    2016-04-01

    Full Text Available It is unknown whether autophagy activity is altered in insulin resistant podocytes and whether autophagy could be a therapeutic target for diabetic nephropathy (DN. Here we used shRNA transfection to knockdown the insulin receptor (IR gene in cultured human immortalized podocytes as an in vitro insulin resistant model. Autophagy related proteins LC3, Beclin, and p62 as well as nephrin, a podocyte injury marker, were assessed using western blot and immunofluorescence staining. Our results show that autophagy is suppressed when podocytes lose insulin sensitivity and that treatment of rapamycin, an mTOR specific inhibitor, could attenuate insulin resistance induced podocytes injury via autophagy activation. The present study deepens our understanding of the role of autophagy in the pathogenesis of DN.

  6. Dual effect of insulin resistance and cadmium on human granulosa cells - In vitro study

    Energy Technology Data Exchange (ETDEWEB)

    Belani, Muskaan, E-mail: muskaanbelani@gmail.com [Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390 002, Gujarat, India. (India); Shah, Preeti, E-mail: preeti.shah@novaivifertility.com [Nova IVI Fertility, Behind Xavier' s Ladies Hostel, 108, Swastik Society Rd., Navrangpura, Ahmedabad 390009, Gujarat, India. (India); Banker, Manish, E-mail: manish.banker@novaivifertility.com [Nova IVI Fertility, Behind Xavier' s Ladies Hostel, 108, Swastik Society Rd., Navrangpura, Ahmedabad 390009, Gujarat, India. (India); Gupta, Sarita, E-mail: saritagupta9@gmail.com [Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390 002, Gujarat, India. (India)

    2016-12-15

    Combined exposure of cadmium (Cd) and insulin resistance (IR) might be responsible for subfertility. In the present study, we investigated the effects of Cd in vitro in IR human granulosa cells. Isolated human granulosa cells from control and polycystic ovary syndrome (PCOS) follicular fluid samples were confirmed for IR by decrease in protein expression of insulin receptor-β. Control and IR human granulosa cells were then incubated with or without 32 μM Cd. The combined effect of IR with 32 μM Cd in granulosa cells demonstrated significant decrease in expression of StAR, CYP11A1, CYP19A1, 17β-HSD, 3β-HSD, FSH-R and LH-R. Decrease was also observed in progesterone and estradiol concentrations as compared to control. Additionally, increase in protein expression of cleaved PARP-F2, active caspase-3 and a positive staining for Annexin V and PI indicated apoptosis as the mode of increased cell death ultimately leading to decreased steroidogenesis, as observed through the combined exposure. Taken together the results suggest decrease in steroidogenesis ultimately leading to abnormal development of the follicle thus compromising fertility at the level of preconception. - Highlights: • Protein expression of INSR-β in granulosa cells to differentiate PCOS-IR and NIR • Cd and IR together decrease steroidogenesis in human granulosa cells in vitro. • Cd and IR increase human granulosa cell death by increase in apoptosis. • Environment and life style are set to hamper pregnancies at preconception level.

  7. Dual effect of insulin resistance and cadmium on human granulosa cells - In vitro study

    International Nuclear Information System (INIS)

    Belani, Muskaan; Shah, Preeti; Banker, Manish; Gupta, Sarita

    2016-01-01

    Combined exposure of cadmium (Cd) and insulin resistance (IR) might be responsible for subfertility. In the present study, we investigated the effects of Cd in vitro in IR human granulosa cells. Isolated human granulosa cells from control and polycystic ovary syndrome (PCOS) follicular fluid samples were confirmed for IR by decrease in protein expression of insulin receptor-β. Control and IR human granulosa cells were then incubated with or without 32 μM Cd. The combined effect of IR with 32 μM Cd in granulosa cells demonstrated significant decrease in expression of StAR, CYP11A1, CYP19A1, 17β-HSD, 3β-HSD, FSH-R and LH-R. Decrease was also observed in progesterone and estradiol concentrations as compared to control. Additionally, increase in protein expression of cleaved PARP-F2, active caspase-3 and a positive staining for Annexin V and PI indicated apoptosis as the mode of increased cell death ultimately leading to decreased steroidogenesis, as observed through the combined exposure. Taken together the results suggest decrease in steroidogenesis ultimately leading to abnormal development of the follicle thus compromising fertility at the level of preconception. - Highlights: • Protein expression of INSR-β in granulosa cells to differentiate PCOS-IR and NIR • Cd and IR together decrease steroidogenesis in human granulosa cells in vitro. • Cd and IR increase human granulosa cell death by increase in apoptosis. • Environment and life style are set to hamper pregnancies at preconception level.

  8. Selective insulin resistance in hepatocyte senescence

    International Nuclear Information System (INIS)

    Aravinthan, Aloysious; Challis, Benjamin; Shannon, Nicholas; Hoare, Matthew; Heaney, Judith; Alexander, Graeme J.M.

    2015-01-01

    Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence. - Highlights: • Senescent hepatocytes demonstrate selective insulin resistance. • GLUT changes act as markers of hepatocyte senescence and have prognostic value. • Study offers insight into long noticed intimacy of cirrhosis and insulin resistance

  9. Selective insulin resistance in hepatocyte senescence

    Energy Technology Data Exchange (ETDEWEB)

    Aravinthan, Aloysious [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Challis, Benjamin [Institute of Metabolic Sciences, University of Cambridge, Cambridge (United Kingdom); Shannon, Nicholas [Cancer Research UK Cambridge Institute, Cambridge (United Kingdom); Hoare, Matthew [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Cancer Research UK Cambridge Institute, Cambridge (United Kingdom); Heaney, Judith [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Foundation for Liver Research, Institute of Hepatology, London (United Kingdom); Alexander, Graeme J.M., E-mail: gja1000@doctors.org.uk [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom)

    2015-02-01

    Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence. - Highlights: • Senescent hepatocytes demonstrate selective insulin resistance. • GLUT changes act as markers of hepatocyte senescence and have prognostic value. • Study offers insight into long noticed intimacy of cirrhosis and insulin resistance.

  10. Activation of IGF-1 and insulin signaling pathways ameliorate mitochondrial function and energy metabolism in Huntington's Disease human lymphoblasts.

    Science.gov (United States)

    Naia, Luana; Ferreira, I Luísa; Cunha-Oliveira, Teresa; Duarte, Ana I; Ribeiro, Márcio; Rosenstock, Tatiana R; Laço, Mário N; Ribeiro, Maria J; Oliveira, Catarina R; Saudou, Frédéric; Humbert, Sandrine; Rego, A Cristina

    2015-02-01

    Huntington's disease (HD) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the huntingtin protein. Mitochondrial dysfunction associated with energy failure plays an important role in this untreated pathology. In the present work, we used lymphoblasts obtained from HD patients or unaffected parentally related individuals to study the protective role of insulin-like growth factor 1 (IGF-1) versus insulin (at low nM) on signaling and metabolic and mitochondrial functions. Deregulation of intracellular signaling pathways linked to activation of insulin and IGF-1 receptors (IR,IGF-1R), Akt, and ERK was largely restored by IGF-1 and, at a less extent, by insulin in HD human lymphoblasts. Importantly, both neurotrophic factors stimulated huntingtin phosphorylation at Ser421 in HD cells. IGF-1 and insulin also rescued energy levels in HD peripheral cells, as evaluated by increased ATP and phosphocreatine, and decreased lactate levels. Moreover, IGF-1 effectively ameliorated O2 consumption and mitochondrial membrane potential (Δψm) in HD lymphoblasts, which occurred concomitantly with increased levels of cytochrome c. Indeed, constitutive phosphorylation of huntingtin was able to restore the Δψm in lymphoblasts expressing an abnormal expansion of polyglutamines. HD lymphoblasts further exhibited increased intracellular Ca(2+) levels before and after exposure to hydrogen peroxide (H2O2), and decreased mitochondrial Ca(2+) accumulation, being the later recovered by IGF-1 and insulin in HD lymphoblasts pre-exposed to H2O2. In summary, the data support an important role for IR/IGF-1R mediated activation of signaling pathways and improved mitochondrial and metabolic function in HD human lymphoblasts.

  11. Transplanted human pancreatic islets after long-term insulin independence

    DEFF Research Database (Denmark)

    Muller, Y D; Gupta, Shashank; Morel, P

    2013-01-01

    Long-term insulin independence after islets of Langerhans transplantation is rarely achieved. The aims of this study were to identify the histological and immunological features of islets transplanted in a type 1 diabetic patient who died of a cerebral hemorrhage after >13 years insulin independe...

  12. Chondrogenic potential of physically treated bovine cartilage matrix derived porous scaffolds on human dermal fibroblast cells.

    Science.gov (United States)

    Moradi, Ali; Ataollahi, Forough; Sayar, Katayoun; Pramanik, Sumit; Chong, Pan-Pan; Khalil, Alizan Abdul; Kamarul, Tunku; Pingguan-Murphy, Belinda

    2016-01-01

    Extracellular matrices have drawn attention in tissue engineering as potential biomaterials for scaffold fabrication because of their bioactive components. Noninvasive techniques of scaffold fabrication and cross-linking treatments are believed to maintain the integrity of bioactive molecules while providing proper architectural and mechanical properties. Cartilage matrix derived scaffolds are designed to support the maintenance of chondrocytes and provide proper signals for differentiation of chondroinducible cells. Chondroinductive potential of bovine articular cartilage matrix derived porous scaffolds on human dermal fibroblasts and the effect of scaffold shrinkage on chondrogenesis were investigated. An increase in sulfated glycosaminoglycans production along with upregulation of chondrogenic genes confirmed that physically treated cartilage matrix derived scaffolds have chondrogenic potential on human dermal fibroblasts. © 2015 Wiley Periodicals, Inc.

  13. Novel and Reversible Mechanisms of Smoking-Induced Insulin Resistance in Humans

    OpenAIRE

    Bergman, Bryan C.; Perreault, Leigh; Hunerdosse, Devon; Kerege, Anna; Playdon, Mary; Samek, Ali M.; Eckel, Robert H.

    2012-01-01

    Smoking is the most common cause of preventable morbidity and mortality in the United States, in part because it is an independent risk factor for the development of insulin resistance and type 2 diabetes. However, mechanisms responsible for smoking-induced insulin resistance are unclear. In this study, we found smokers were less insulin sensitive compared with controls, which increased after either 1 or 2 weeks of smoking cessation. Improvements in insulin sensitivity after smoking cessation...

  14. Prevalence and characteristics of Listeria monocytogenes in bovine colostrum in Japan.

    Science.gov (United States)

    Hasegawa, Megumi; Iwabuchi, Eriko; Yamamoto, Shiori; Esaki, Hidetake; Kobayashi, Kazuhiko; Ito, Masahiko; Hirai, Katsuya

    2013-02-01

    This study was conducted to determine the prevalence and characteristics of Listeria monocytogenes in bovine colostrum in Japan. We collected bovine colostrum samples from 210 dams from 21 dairy farms in Hokkaido prefecture (Japan) between March and June 2009. L. monocytogenes was detected in samples from 6 (28.6%) of the 21 farms. Of the 210 samples, 16 (7.6%) were positive for L. monocytogenes. We recovered 80 L. monocytogenes isolates; 44 (55%) isolates were classified as serotype 1/2b and 36 (45%) were classified as serotype 4b. The isolates were susceptible to penicillin, ampicillin, amoxicillin, gentamicin, kanamycin, streptomycin, erythromycin, vancomycin, tetracycline, chloramphenicol, ciprofloxacin, and trimethoprim-sulfamethoxazole. Pulsed-field gel electrophoresis (PFGE) characterization of the 80 isolates revealed six PFGE types. Two PFGE types corresponded to human listeriosis cases. Most L. monocytogenes isolates possessed virulence-associated genes (actA, hly, iap, inlA, inlC, mpl, plcA, plcB, opuCA, prfA, and clpC). One PFGE type isolate possessed an epidemic clone II marker. Our findings suggest that isolates from bovine colostrum have the potential to cause human and animal listeriosis. This is the first study on the prevalence and characteristics of L. monocytogenes isolated from bovine colostrum obtained from dairy farms. Our results have important implications for improving public health and elucidating the epidemiology of L. monocytogenes in bovine colostrum.

  15. Insulin action in human thighs after one-legged immobilization

    DEFF Research Database (Denmark)

    Richter, Erik; Kiens, Bente; Mizuno, M.

    1989-01-01

    Insulin action was assessed in thighs of five healthy young males who had one knee immobilized for 7 days by a splint. The splint was not worn in bed. Subjects also used crutches to prevent weight bearing of the immobilized leg. Immobilization decreased the activity of citrate synthase and 3-OH......-acyl-CoA-dehydrogenase in the vastus lateralis muscle by 9 and 14%, respectively, and thigh volume by 5%. After 7 days of immobilization, a two-step euglycemic hyperinsulinemic clamp procedure combined with arterial and bilateral femoral venous catheterization was performed. Insulin action on glucose uptake and tyrosine release...... of the thighs at mean plasma insulin concentrations of 67 (clamp step I) and 447 microU/ml (clamp step II) was decreased by immobilization, whereas immobilization did not affect insulin action on thigh exchange of free fatty acids, glycerol, O2, or potassium. Before and during the clamp step I, lactate release...

  16. Insulin stimulates translocation of human GLUT4 to the membrane in fat bodies of transgenic Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Georgeta Crivat

    Full Text Available The fruit fly Drosophila melanogaster is an excellent model system for studies of genes controlling development and disease. However, its applicability to physiological systems is less clear because of metabolic differences between insects and mammals. Insulin signaling has been studied in mammals because of relevance to diabetes and other diseases but there are many parallels between mammalian and insect pathways. For example, deletion of Drosophila Insulin-Like Peptides resulted in 'diabetic' flies with elevated circulating sugar levels. Whether this situation reflects failure of sugar uptake into peripheral tissues as seen in mammals is unclear and depends upon whether flies harbor the machinery to mount mammalian-like insulin-dependent sugar uptake responses. Here we asked whether Drosophila fat cells are competent to respond to insulin with mammalian-like regulated trafficking of sugar transporters. Transgenic Drosophila expressing human glucose transporter-4 (GLUT4, the sugar transporter expressed primarily in insulin-responsive tissues, were generated. After expression in fat bodies, GLUT4 intracellular trafficking and localization were monitored by confocal and total internal reflection fluorescence microscopy (TIRFM. We found that fat body cells responded to insulin with increased GLUT4 trafficking and translocation to the plasma membrane. While the amplitude of these responses was relatively weak in animals reared on a standard diet, it was greatly enhanced in animals reared on sugar-restricted diets, suggesting that flies fed standard diets are insulin resistant. Our findings demonstrate that flies are competent to mobilize translocation of sugar transporters to the cell surface in response to insulin. They suggest that Drosophila fat cells are primed for a response to insulin and that these pathways are down-regulated when animals are exposed to constant, high levels of sugar. Finally, these studies are the first to use TIRFM to

  17. Insulin Stimulates Translocation of Human GLUT4 to the Membrane in Fat Bodies of Transgenic Drosophila melanogaster

    Science.gov (United States)

    Crivat, Georgeta; Lizunov, Vladimir A.; Li, Caroline R.; Stenkula, Karin G.; Zimmerberg, Joshua; Cushman, Samuel W.; Pick, Leslie

    2013-01-01

    The fruit fly Drosophila melanogaster is an excellent model system for studies of genes controlling development and disease. However, its applicability to physiological systems is less clear because of metabolic differences between insects and mammals. Insulin signaling has been studied in mammals because of relevance to diabetes and other diseases but there are many parallels between mammalian and insect pathways. For example, deletion of Drosophila Insulin-Like Peptides resulted in ‘diabetic’ flies with elevated circulating sugar levels. Whether this situation reflects failure of sugar uptake into peripheral tissues as seen in mammals is unclear and depends upon whether flies harbor the machinery to mount mammalian-like insulin-dependent sugar uptake responses. Here we asked whether Drosophila fat cells are competent to respond to insulin with mammalian-like regulated trafficking of sugar transporters. Transgenic Drosophila expressing human glucose transporter-4 (GLUT4), the sugar transporter expressed primarily in insulin-responsive tissues, were generated. After expression in fat bodies, GLUT4 intracellular trafficking and localization were monitored by confocal and total internal reflection fluorescence microscopy (TIRFM). We found that fat body cells responded to insulin with increased GLUT4 trafficking and translocation to the plasma membrane. While the amplitude of these responses was relatively weak in animals reared on a standard diet, it was greatly enhanced in animals reared on sugar-restricted diets, suggesting that flies fed standard diets are insulin resistant. Our findings demonstrate that flies are competent to mobilize translocation of sugar transporters to the cell surface in response to insulin. They suggest that Drosophila fat cells are primed for a response to insulin and that these pathways are down-regulated when animals are exposed to constant, high levels of sugar. Finally, these studies are the first to use TIRFM to monitor insulin

  18. Generation of insulin-producing cells from human bone marrow-derived mesenchymal stem cells: comparison of three differentiation protocols.

    Science.gov (United States)

    Gabr, Mahmoud M; Zakaria, Mahmoud M; Refaie, Ayman F; Khater, Sherry M; Ashamallah, Sylvia A; Ismail, Amani M; El-Badri, Nagwa; Ghoneim, Mohamed A

    2014-01-01

    Many protocols were utilized for directed differentiation of mesenchymal stem cells (MSCs) to form insulin-producing cells (IPCs). We compared the relative efficiency of three differentiation protocols. Human bone marrow-derived MSCs (HBM-MSCs) were obtained from three insulin-dependent type 2 diabetic patients. Differentiation into IPCs was carried out by three protocols: conophylline-based (one-step protocol), trichostatin-A-based (two-step protocol), and β -mercaptoethanol-based (three-step protocol). At the end of differentiation, cells were evaluated by immunolabeling for insulin production, expression of pancreatic endocrine genes, and release of insulin and c-peptide in response to increasing glucose concentrations. By immunolabeling, the proportion of generated IPCs was modest ( ≃ 3%) in all the three protocols. All relevant pancreatic endocrine genes, insulin, glucagon, and somatostatin, were expressed. There was a stepwise increase in insulin and c-peptide release in response to glucose challenge, but the released amounts were low when compared with those of pancreatic islets. The yield of functional IPCs following directed differentiation of HBM-MSCs was modest and was comparable among the three tested protocols. Protocols for directed differentiation of MSCs need further optimization in order to be clinically meaningful. To this end, addition of an extracellular matrix and/or a suitable template should be attempted.

  19. Insulin use and persistence in patients with type 2 diabetes adding mealtime insulin to a basal regimen: a retrospective database analysis

    Directory of Open Access Journals (Sweden)

    Torres Amelito M

    2011-01-01

    Full Text Available Abstract Background The objective of this study was to characterize insulin use and examine factors associated with persistence to mealtime insulin among patients with type 2 diabetes (T2D on stable basal insulin therapy initiating mealtime insulin therapy. Methods Insulin use among patients with T2D initiating mealtime insulin was investigated using Thomson Reuters MarketScan® research databases from July 2001 through September 2006. The first mealtime insulin claim preceded by 6 months with 2 claims for basal insulin was used as the index event. A total of 21 months of continuous health plan enrollment was required. Patients were required to have a second mealtime insulin claim during the 12-month follow-up period. Persistence measure 1 defined non-persistence as the presence of a 90-day gap in mealtime insulin claims, effective the date of the last claim prior to the gap. Persistence measure 2 required 1 claim per quarter to be persistent. Risk factors for non-persistence were assessed using logistic regression. Results Patients initiating mealtime insulin (n = 4752; 51% male, mean age = 60.3 years primarily used vial/syringe (87% and insulin analogs (60%. Patients filled a median of 2, 3, and 4 mealtime insulin claims at 3, 6, and 12 months, respectively, with a median time of 76 days between refills. According to measure 1, persistence to mealtime insulin was 40.7%, 30.2%, and 19.1% at 3, 6, and 12 months, respectively. Results for measure 2 were considerably higher: 74.3%, 55.3%, and 42.2% of patients were persistent at 3, 6, and 12 months, respectively. Initiating mealtime insulin with human insulin was a risk factor for non-persistence by both measures (OR Conclusions Mealtime insulin use and persistence were both considerably lower than expected, and were significantly lower for human insulin compared to analogs.

  20. Insulin-producing Cells from Adult Human Bone Marrow Mesenchymal Stromal Cells Could Control Chemically Induced Diabetes in Dogs: A Preliminary Study.

    Science.gov (United States)

    Gabr, Mahmoud M; Zakaria, Mahmoud M; Refaie, Ayman F; Ismail, Amani M; Khater, Sherry M; Ashamallah, Sylvia A; Azzam, Maha M; Ghoneim, Mohamed A

    2018-01-01

    Ten mongrel dogs were used in this study. Diabetes was chemically induced in 7 dogs, and 3 dogs served as normal controls. For each diabetic dog, 5 million human bone marrow-derived mesenchymal stem cells/kg were differentiated to form insulin-producing cells using a trichostatin-based protocol. Cells were then loaded in 2 TheraCyte capsules which were transplanted under the rectus sheath. One dog died 4 d postoperatively from pneumonia. Six dogs were followed up with for 6 to 18 mo. Euglycemia was achieved in 4 dogs. Their glucose tolerance curves exhibited a normal pattern demonstrating that the encapsulated cells were glucose sensitive and insulin responsive. In the remaining 2 dogs, the fasting blood sugar levels were reduced but did not reach normal values. The sera of all transplanted dogs contained human insulin and C-peptide with a negligible amount of canine insulin. Removal of the transplanted capsules was followed by prompt return of diabetes. Intracytoplasmic insulin granules were seen by immunofluorescence in cells from the harvested capsules. Furthermore, all pancreatic endocrine genes were expressed. This study demonstrated that the TheraCyte capsule or a similar device can provide adequate immunoisolation, an important issue when stem cells are considered for the treatment of type 1 diabetes mellitus.

  1. Nicotinic Acid Increases Adiponectin Secretion from Differentiated Bovine Preadipocytes through G-Protein Coupled Receptor Signaling

    Directory of Open Access Journals (Sweden)

    Christina Kopp

    2014-11-01

    Full Text Available The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p ≤ 0.001 and the mRNA abundances of GPR109A (p ≤ 0.05 and chemerin (p ≤ 0.01. Pre-incubation with PTX reduced the adiponectin response to NA (p ≤ 0.001. The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows.

  2. Grizzly bears exhibit augmented insulin sensitivity while obese prior to a reversible insulin resistance during hibernation.

    Science.gov (United States)

    Nelson, O Lynne; Jansen, Heiko T; Galbreath, Elizabeth; Morgenstern, Kurt; Gehring, Jamie Lauren; Rigano, Kimberly Scott; Lee, Jae; Gong, Jianhua; Shaywitz, Adam J; Vella, Chantal A; Robbins, Charles T; Corbit, Kevin C

    2014-08-05

    The confluence of obesity and diabetes as a worldwide epidemic necessitates the discovery of new therapies. Success in this endeavor requires translatable preclinical studies, which traditionally employ rodent models. As an alternative approach, we explored hibernation where obesity is a natural adaptation to survive months of fasting. Here we report that grizzly bears exhibit seasonal tripartite insulin responsiveness such that obese animals augment insulin sensitivity but only weeks later enter hibernation-specific insulin resistance (IR) and subsequently reinitiate responsiveness upon awakening. Preparation for hibernation is characterized by adiposity coupled to increased insulin sensitivity via modified PTEN/AKT signaling specifically in adipose tissue, suggesting a state of "healthy" obesity analogous to humans with PTEN haploinsufficiency. Collectively, we show that bears reversibly cope with homeostatic perturbations considered detrimental to humans and describe a mechanism whereby IR functions not as a late-stage metabolic adaptation to obesity, but rather a gatekeeper of the fed-fasting transition. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Metabolism and insulin signaling in common metabolic disorders and inherited insulin resistance.

    Science.gov (United States)

    Højlund, Kurt

    2014-07-01

    Type 2 diabetes, obesity and polycystic ovary syndrome (PCOS) are common metabolic disorders which are observed with increasing prevalences, and which are caused by a complex interplay between genetic and environmental factors, including increased calorie intake and physical inactivity. These metabolic disorders are all characterized by reduced plasma adiponectin and insulin resistance in peripheral tissues. Quantitatively skeletal muscle is the major site of insulin resistance. Both low plasma adiponectin and insulin resistance contribute to an increased risk of type 2 diabetes and cardiovascular disease. In several studies, we have investigated insulin action on glucose and lipid metabolism, and at the molecular level, insulin signaling to glucose transport and glycogen synthesis in skeletal muscle from healthy individuals and in obesity, PCOS and type 2 diabetes. Moreover, we have described a novel syndrome characterized by postprandial hyperinsulinemic hypoglycemia and insulin resistance. This syndrome is caused by a mutation in the tyrosine kinase domain of the insulin receptor gene (INSR). We have studied individuals with this mutation as a model of inherited insulin resistance. Type 2 diabetes, obesity and PCOS are characterized by pronounced defects in the insulin-stimulated glucose uptake, in particular glycogen synthesis and to a lesser extent glucose oxidation, and the ability of insulin to suppress lipid oxidation. In inherited insulin resistance, however, only insulin action on glucose uptake and glycogen synthesis is impaired. This suggests that the defects in glucose and lipid oxidation in the common metabolic disorders are secondary to other factors. In young women with PCOS, the degree of insulin resistance was similar to that seen in middle-aged patients with type 2 diabetes. This supports the hypothesis of an unique pathogenesis of insulin resistance in PCOS. Insulin in physiological concentrations stimulates glucose uptake in human skeletal

  4. Human umbilical cord-derived mesenchymal stem cells ameliorate insulin resistance by suppressing NLRP3 inflammasome-mediated inflammation in type 2 diabetes rats

    Directory of Open Access Journals (Sweden)

    Xiaoya Sun

    2017-11-01

    Full Text Available Abstract Background Insulin resistance is one of the most common and important pathological features of type 2 diabetes (T2D. Recently, insulin resistance is increasingly considered to be associated with systemic chronic inflammation. Elevated levels of tumor necrosis factor (TNF-α and interleukin (IL-1β in blood are predictive indicators of the development of T2D. Mesenchymal stem cell (MSC-based therapies have been proven to have potential immunomodulation and anti-inflammatory properties through their paracrine effects; however, the mechanism for the anti-inflammatory effect of MSCs in enhancing insulin sensitivity is still uncertain. Methods In the present experiment, we used HepG2 cells, a human hepatoma cell line, and a MSC-HepG2 transwell culturing system to investigate the anti-inflammatory mechanism of human umbilical cord-derived MSCs (UC-MSCs under palmitic acid (PA and lipopolysaccharide (LPS-induced insulin resistance in vitro. Insulin resistance was confirmed by glycogen assay kit and glucose assay kit. Inflammatory factor release was detected by ELISA, gene expression was tested by quantitative real-time PCR, and insulin signaling activation was determined by western blotting analysis. The changes of inflammatory factors and insulin signaling protein were also tested in T2D rats injected with UC-MSCs. Results Treating HepG2 cells with PA–LPS caused NLRP3 inflammation activation, including overexpression of NLRP3 and caspase-1, and overproduction of IL-1β and IL-18 as well as TNF-α from HepG2 cells. The elevated levels of these inflammatory cytokines impaired insulin receptor action and thereby prevented downstream signaling pathways, exacerbating insulin resistance in HepG2 cells. Importantly, UC-MSCs cocultured with HepG2 could effectively alleviate PA and LPS-induced insulin resistance by blocking the NLRP3 inflammasome activation and inflammatory agents. Furthermore, knockdown of NLRP3 or IL-1β partially improved PA and

  5. Comparative genomics and the role of lateral gene transfer in the evolution of bovine adapted Streptococcus agalactiae

    Science.gov (United States)

    Richards, Vincent P.; Lang, Ping; Pavinski Bitar, Paulina D.; Lefébure, Tristan; Schukken, Ynte H.; Zadoks, Ruth N.; Stanhope, Michael J.

    2011-01-01

    In addition to causing severe invasive infections in humans, Streptococcus agalactiae, or group B Streptococcus (GBS), is also a major cause of bovine mastitis. Here we provide the first genome sequence for S. agalactiae isolated from a cow diagnosed with clinical mastitis (strain FSL S3-026). Comparison to eight S. agalactiae genomes obtained from human disease isolates revealed 183 genes specific to the bovine strain. Subsequent polymerase chain reaction (PCR) screening for the presence/absence of a subset of these loci in additional bovine and human strains revealed strong differentiation between the two groups (Fisher exact test: p S. agalactiae with Streptococcus uberis (nisin U operon) and Streptococcus dysgalactiae subsp. dysgalactiae (lactose operon). We also found evidence for LGT, involving the salivaricin operon, between the bovine S. agalactiae strain and either Streptococcus pyogenes or Streptococcus salivarius. Our findings provide insight intomechanismsfacilitatingenvironmentaladaptationandacquisitionofpotential virulence factors, while highlighting both the key role LGT has played in the recent evolution of the bovine S. agalactiae strain, and the importance of LGT among pathogens within a shared environment. PMID:21536150

  6. Pregnancy, bovine somatotropin, and dietary n-3 fatty acids in lactating dairy cows: I. Ovarian, conceptus, and growth hormone-insulin-like growth factor system responses.

    Science.gov (United States)

    Bilby, T R; Sozzi, A; Lopez, M M; Silvestre, F T; Ealy, A D; Staples, C R; Thatcher, W W

    2006-09-01

    The objective was to examine effects of bovine somatotropin (bST), pregnancy, and dietary fatty acids on reproductive responses in lactating dairy cows. Beginning at approximately 17 d in milk (DIM), a comparison was made of isoenergetic diets comprising supplementary lipids of whole cottonseed vs. calcium salts of fish oil enriched lipid (FO). Ovulation was synchronized in cows with a presynchronization plus Ovsynch protocol, and cows were inseminated artificially by appointment or not inseminated (d 0 = time of synchronized ovulation; 77 +/- 12 DIM). On d 0 and 11, cows received bST (500 mg) or no bST. All cows were slaughtered on d 17. Number of cows in each group was as follows: control diet had 5 bST-treated cyclic (bST-C), 5 non-bST-treated cyclic (no bST-C), 4 bST-treated pregnant (bST-P), and 5 non-bST-treated pregnant (no bST-P) cows; and cyclic cows fed FO diet had 4 bST-treated (bST-FO) and 5 non-bST-treated cyclic (no bST-FO-C) cows. Feeding FO increased milk production, number of class 1 follicles (2 to 5 mm), and decreased insulin during the period before d 0 compared with control-fed cows. The bST increased milk production, pregnancy rate [83% (5/6) vs. 40% (4/10)], conceptus length (45 vs. 34 cm), and interferon-tau in the uterine luminal flushings (9.4 vs. 5.3 microg) with no effect on interferon-tau mRNA concentration in the conceptus. Treatment with bST increased plasma growth hormone (GH) and insulin-like growth factor (IGF)-I. Among control-fed cows (cyclic and pregnant), bST decreased progesterone concentration in plasma. Cows fed FO had less plasma insulin than control-fed cyclic cows, and FO altered the plasma GH (bST-FO > bST-C) and IGF-I (bST-C > bST-FO-C) responses to bST injections. Endometrial IGF-I mRNA was reduced in pregnant cows and tended to decrease in those fed FO. The IGF-II mRNA was increased in the endometrium of pregnant and bST-treated cows fed the control diet. Cows fed FO had increased concentrations of IGF-II mRNA, when b

  7. Protein kinase C is differentially regulated by thrombin, insulin, and epidermal growth factor in human mammary tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Gomez, M.L.; Tellez-Inon, M.T. (Instituto de Ingenieria Genetica y Biologia Molecular, Buenos Aires (Argentina)); Medrano, E.E.; Cafferatta, E.G.A. (Instituto de Investigaciones Bioquimicas Fundacion Campomar, Buenos Aires (Argentina))

    1988-03-01

    The exposure of serum-deprived mammary tumor cells MCF-7 and T-47D to insulin, thrombin, and epidermal growth factor (EGF) resulted in dramatic modifications in the activity and in the translocation capacity of protein kinase C from cytosol to membrane fractions. Insulin induces a 600% activation of the enzyme after 5 h of exposure to the hormone in MCF-7 cells; thrombin either activates (200% in MCF-7) or down-regulates (in T-47D), and EGF exerts only a moderate effect. Thus, the growth factors studied modulate differentially the protein kinase C activity in human mammary tumor cells. The physiological significance of the results obtained are discussed in terms of the growth response elicited by insulin, thrombin, and EGF.

  8. Insulin amyloid fibrillation studied by terahertz spectroscopy and other biophysical methods

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Rui [State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); He, Mingxia [College of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin 300072 (China); Su, Rongxin, E-mail: surx@tju.edu.cn [State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072 (China); Yu, Yanjun [State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Qi, Wei; He, Zhimin [State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072 (China)

    2010-01-01

    Assembly and fibrillation of amyloid proteins are believed to play a key role in the etiology of various human diseases, including Alzheimer's, Parkinson's, Huntington's and type II diabetes. Insights into conformational changes and formation processes during amyloid fibrillation are essential for the clinical diagnosis and drug discovery. To study the changes in secondary, tertiary, quaternary structures, and the alteration in the collective vibrational mode density of states during the amyloid fibrillation, bovine insulin in 20% acetic acid was incubated at 60 {sup o}C, and its multi-level structures were followed by various biophysical techniques, including circular dichroism (CD), thioflavin T fluorescence (ThT), dynamic light scattering (DLS), electron microscopy, and terahertz (THz) absorption spectroscopy. The experimental data demonstrated a transformation of {alpha}-helix into {beta}-sheet starting at 26 h. This was followed by the aggregation of insulin, as shown by ThT binding, with a transition midpoint at 41 h, and by the bulk formation of mature aggregates after about 71 h. THz is a quick and non-invasive technique, which has the advantage of allowing the study of the conformational state of biomolecules and tissues. We first applied THz spectroscopy to study the amyloid fibrillation. At the terahertz frequency range of 0.2-2.0 THz, there was an apparent increase in both the absorbance and refractive index in THz spectra. Thus, THz is expected to provide a new way of looking into amyloid fibrillation.

  9. Supplements in human islet culture: human serum albumin is inferior to fetal bovine serum.

    Science.gov (United States)

    Avgoustiniatos, Efstathios S; Scott, William E; Suszynski, Thomas M; Schuurman, Henk-Jan; Nelson, Rebecca A; Rozak, Phillip R; Mueller, Kate R; Balamurugan, A N; Ansite, Jeffrey D; Fraga, Daniel W; Friberg, Andrew S; Wildey, Gina M; Tanaka, Tomohiro; Lyons, Connor A; Sutherland, David E R; Hering, Bernhard J; Papas, Klearchos K

    2012-01-01

    Culture of human islets before clinical transplantation or distribution for research purposes is standard practice. At the time the Edmonton protocol was introduced, clinical islet manufacturing did not include culture, and human serum albumin (HSA), instead of fetal bovine serum (FBS), was used during other steps of the process to avoid the introduction of xenogeneic material. When culture was subsequently introduced, HSA was also used for medium supplementation instead of FBS, which was typically used for research islet culture. The use of HSA as culture supplement was not evaluated before this implementation. We performed a retrospective analysis of 103 high-purity islet preparations (76 research preparations, all with FBS culture supplementation, and 27 clinical preparations, all with HSA supplementation) for oxygen consumption rate per DNA content (OCR/DNA; a measure of viability) and diabetes reversal rate in diabetic nude mice (a measure of potency). After 2-day culture, research preparations exhibited an average OCR/DNA 51% higher (p < 0.001) and an average diabetes reversal rate 54% higher (p < 0.05) than clinical preparations, despite 87% of the research islet preparations having been derived from research-grade pancreata that are considered of lower quality. In a prospective paired study on islets from eight research preparations, OCR/DNA was, on average, 27% higher with FBS supplementation than that with HSA supplementation (p < 0.05). We conclude that the quality of clinical islet preparations can be improved when culture is performed in media supplemented with serum instead of albumin.

  10. The bovine kidney as an experimental model in urology: external gross anatomy.

    Science.gov (United States)

    Carvalho, Francismar S; Bagetti Filho, Hélio J S; Henry, Robert W; Pereira-Sampaio, Marco A

    2009-01-01

    The objective of this work was to obtain and record detailed and accurate measurements of the bovine kidney and to compare these new data with findings in humans. Thirty-eight bovine kidneys were used. The total number of lobes, along with the number of lobes located in the cranial polar, caudal polar and hilar regions, were recorded. Several measurements of the kidneys were made and evaluated. The hilar region presents the greatest length (mean of 76.87 mm) of the 3 renal regions of the kidney. The large area of the bovine renal hilus could make access to hilar structures easier than in the human kidney. The coefficient of variation for renal length was small (8.14%), while the coefficient of variation for the lobar number was high (26.82%). The number of renal lobes ranged from 13 to 35, with a mean of 20.62. The hilar region presents the highest number of lobes, while the cranial pole presents the lowest. The number of lobes in the cranial and caudal poles increases with the width of these regions. This is different from the hilar region, in which the lobar number increases with the length of the hilus. These data indicate that the adult bovine kidney can be used as a model for certain urologic procedures, but researchers must be aware that there are some major differences between the adult bovine kidney and the human kidney, as indicated by the data reported in this paper. (c) 2008 S. Karger AG, Basel.

  11. Insulin Resistance in Alzheimer's Disease

    Science.gov (United States)

    Dineley, Kelly T; Jahrling, Jordan B; Denner, Larry

    2014-01-01

    Insulin is a key hormone regulating metabolism. Insulin binding to cell surface insulin receptors engages many signaling intermediates operating in parallel and in series to control glucose, energy, and lipids while also regulating mitogenesis and development. Perturbations in the function of any of these intermediates, which occur in a variety of diseases, cause reduced sensitivity to insulin and insulin resistance with consequent metabolic dysfunction. Chronic inflammation ensues which exacerbates compromised metabolic homeostasis. Since insulin has a key role in learning and memory as well as directly regulating ERK, a kinase required for the type of learning and memory compromised in early Alzheimer's disease (AD), insulin resistance has been identified as a major risk factor for the onset of AD. Animal models of AD or insulin resistance or both demonstrate that AD pathology and impaired insulin signaling form a reciprocal relationship. Of note are human and animal model studies geared toward improving insulin resistance that have led to the identification of the nuclear receptor and transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ) as an intervention tool for early AD. Strategic targeting of alternate nodes within the insulin signaling network has revealed disease-stage therapeutic windows in animal models that coalesce with previous and ongoing clinical trial approaches. Thus, exploiting the connection between insulin resistance and AD provides powerful opportunities to delineate therapeutic interventions that slow or block the pathogenesis of AD. PMID:25237037

  12. Label-free detection of insulin and glucagon within human islets of Langerhans using Raman spectroscopy.

    Directory of Open Access Journals (Sweden)

    Janneke Hilderink

    Full Text Available Intrahepatic transplantation of donor islets of Langerhans is a promising therapy for patients with type 1 diabetes. It is of critical importance to accurately monitor islet quality before transplantation, which is currently done by standard histological methods that are performed off-line and require extensive sample preparation. As an alternative, we propose Raman spectroscopy which is a non-destructive and label-free technique that allows continuous real-time monitoring of the tissue to study biological changes as they occur. By performing Raman spectroscopic measurements on purified insulin and glucagon, we showed that the 520 cm(-1 band assigned to disulfide bridges in insulin, and the 1552 cm(-1 band assigned to tryptophan in glucagon are mutually exclusive and could therefore be used as indirect markers for the label-free distinction between both hormones. High-resolution hyperspectral Raman imaging for these bands showed the distribution of disulfide bridges and tryptophan at sub-micrometer scale, which correlated with the location of insulin and glucagon as revealed by conventional immunohistochemistry. As a measure for this correlation, quantitative analysis was performed comparing the Raman images with the fluorescence images, resulting in Dice coefficients (ranging between 0 and 1 of 0.36 for insulin and 0.19 for glucagon. Although the use of separate microscope systems with different spatial resolution and the use of indirect Raman markers cause some image mismatch, our findings indicate that Raman bands for disulfide bridges and tryptophan can be used as distinctive markers for the label-free detection of insulin and glucagon in human islets of Langerhans.

  13. Serum-converted platelet lysate can substitute for fetal bovine serum in human mesenchymal stromal cell cultures.

    Science.gov (United States)

    Mojica-Henshaw, Mariluz P; Jacobson, Pam; Morris, Julie; Kelley, Linda; Pierce, Jan; Boyer, Michael; Reems, Jo-Anna

    2013-12-01

    Fetal bovine serum (FBS) is commonly used as a serum supplement for culturing human mesenchymal stromal cells (hMSCs). However, human cells grown in FBS, especially for extended periods, risk potential exposure to bovine immunogenic proteins and infectious agents. To address this issue, we investigated the ability of a novel human platelet serum supplement to substitute for FBS in hMSC cultures. Platelet lysate-serum (PL-serum) was converted from platelet lysate-plasma (PL-plasma) that was manufactured from pooled platelet-rich plasma (PRP) apheresis units. Growth factor levels and the number of residual intact platelets in PL-serum and PL-plasma were compared with enzyme-linked immunosorbent assays and flow cytometry, respectively. Proliferation responses of hMSCs cultured in PL-serum, PL-plasma, or FBS were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the immunophenotype of harvested hMSCs was evaluated by flow cytometry and tri-lineage differentiation potential was evaluated by assessing adipogenic, osteogenic and chondrogenic development. Selected growth factor levels in PL-serum were not significantly different from PL-plasma (P > 0.05). hMSC cultures supplemented with PL-serum had comparable growth kinetics to PL-plasma, and hMSC yields were consistently greater than with FBS. hMSCs harvested from cultures supplemented with PL-serum, PL-plasma or FBS had similar cell surface phenotypes and maintained tri-lineage differentiation potential. PL-serum, similar to PL-plasma, can substitute for FBS in hMSC cultures. Use of PL-serum, in contrast to PL-plasma, has an added advantage of not requiring addition of a xenogeneic source of heparin, providing a completely xeno-free culture medium. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  14. Differentiation of Human Mesenchymal Stem Cells into Insulin Producing Cells by Using A Lentiviral Vector Carrying PDX1.

    Science.gov (United States)

    Allahverdi, Amir; Abroun, Saied; Jafarian, Arefeh; Soleimani, Masoud; Taghikhani, Mohammad; Eskandari, Fatemeh

    2015-01-01

    Type I diabetes is an immunologically-mediated devastation of insulin producing cells (IPCs) in the pancreatic islet. Stem cells that produce β-cells are a new promising tool. Adult stem cells such as mesenchymal stem cells (MSCs) are self renewing multi potent cells showing capabilities to differentiate into ectodermal, mesodermal and endodermal tissues. Pancreatic and duodenal homeobox factor 1 (PDX1) is a master regulator gene required for embryonic development of the pancreas and is crucial for normal pancreatic islets activities in adults. We induced the over-expression of the PDX1 gene in human bone marrow MSCs (BM-MSCs) by Lenti-PDX1 in order to generate IPCs. Next, we examine the ability of the cells by measuring insulin/c-peptide production and INSULIN and PDX1 gene expressions. After transduction, MSCs changed their morphology at day 5 and gradually differentiated into IPCs. INSULIN and PDX1 expressions were confirmed by real time polymerase chain reaction (RT-PCR) and immunostaining. IPC secreted insulin and C-peptide in the media that contained different glucose concentrations. MSCs differentiated into IPCs by genetic manipulation. Our result showed that lentiviral vectors could deliver PDX1 gene to MSCs and induce pancreatic differentiation.

  15. Microvascular Recruitment in Insulin Resistance

    DEFF Research Database (Denmark)

    Sjøberg, Kim Anker

    the resonating sound from the microbubbles in the systemic circulation were recorded for determination of microvascular recruitment in designated muscle segments. Results showed that microvascular recruitment increased with insulin stimulation by ~30% in rats and ~40% in humans (study I). Furthermore......, it was observed that muscle contractions increased muscle perfusion rapidly by 3-4 fold and by 1-2 fold compared to basal and insulin, respectively, in both rat and human skeletal muscle (study I). The real-time contrast-enhanced ultrasound method was applied to investigate the vaso-active effect of the incretin...... hormone glucagon-like-peptide-1 (GLP-1) in the microcirculation. Glucagon-like-peptide-1 analogs are drugs used for treatments of insulin resistance and type 2 diabetes but the vascular effects of GLP-1 in vivo are elusive. Here it was shown that GLP-1 rapidly increased the microvascular recruitment...

  16. Sulfated glycosaminoglycans in cultured endothelial cells from capillaries and large vessels of human and bovine origin

    International Nuclear Information System (INIS)

    Bar, R.S.; Dake, B.L.; Spanheimer, R.G.

    1985-01-01

    The ( 35 S)glycosaminoglycans (( 35 S)GAG) synthesized by capillary endothelial cells were analyzed and compared to GAG synthesized by endothelial cells cultured from 4 larger vessels. Two separate cultures of endothelial cells were established from bovine fat capillaries and from 4 larger vessels of human origin (umbilical vein) and bovine origin (pulmonary artery, pulmonary vein and aorta). After incubation with 35 SO 4 for 72 h, the ( 35 S)glycosaminoglycans (GAG) composition of the media, pericellular and cellular fractions of each culture were determined by selective degradation with nitrous acid, chondroitinase ABC and chondroitinase AC. All endothelial cells produced large amounts of ( 35 S)GAG with increased proportions of heparinoids (heparan sulfate and heparin) in the cellular and pericellular fractions. Each culture showed a distinct distribution of ( 35 S)GAG in the media, pericellular and cellular fractions with several specific differences found among the 5 cultures. The differences in GAG content were confirmed in a second group of separate cultures from each of the 5 vessels indicating that, although having several features of GAG metabolism in common, each endothelial cell culture demonstrated a characteristic complement of synthesized, secreted and cell surface-sulfated glycosaminoglycans. (author)

  17. Insulin resistance is associated with MCP1-mediated macrophage accumulation in skeletal muscle in mice and humans.

    Directory of Open Access Journals (Sweden)

    David Patsouris

    Full Text Available Inflammation is now recognized as a major factor contributing to type 2 diabetes (T2D. However, while the mechanisms and consequences associated with white adipose tissue inflammation are well described, very little is known concerning the situation in skeletal muscle. The aim of this study was to investigate, in vitro and in vivo, how skeletal muscle inflammation develops and how in turn it modulates local and systemic insulin sensitivity in different mice models of T2D and in humans, focusing on the role of the chemokine MCP1. Here, we found that skeletal muscle inflammation and macrophage markers are increased and associated with insulin resistance in mice models and humans. In addition, we demonstrated that intra-muscular TNFα expression is exclusively restricted to the population of intramuscular leukocytes and that the chemokine MCP1 was associated with skeletal muscle inflammatory markers in these models. Furthermore, we demonstrated that exposure of C2C12 myotubes to palmitate elevated the production of the chemokine MCP1 and that the muscle-specific overexpression of MCP1 in transgenic mice induced the local recruitment of macrophages and altered local insulin sensitivity. Overall our study demonstrates that skeletal muscle inflammation is clearly increased in the context of T2D in each one of the models we investigated, which is likely consecutive to the lipotoxic environment generated by peripheral insulin resistance, further increasing MCP1 expression in muscle. Consequently, our results suggest that MCP1-mediated skeletal muscle macrophages recruitment plays a role in the etiology of T2D.

  18. Proteomic analyses of host and pathogen responses during bovine mastitis.

    Science.gov (United States)

    Boehmer, Jamie L

    2011-12-01

    The pursuit of biomarkers for use as clinical screening tools, measures for early detection, disease monitoring, and as a means for assessing therapeutic responses has steadily evolved in human and veterinary medicine over the past two decades. Concurrently, advances in mass spectrometry have markedly expanded proteomic capabilities for biomarker discovery. While initial mass spectrometric biomarker discovery endeavors focused primarily on the detection of modulated proteins in human tissues and fluids, recent efforts have shifted to include proteomic analyses of biological samples from food animal species. Mastitis continues to garner attention in veterinary research due mainly to affiliated financial losses and food safety concerns over antimicrobial use, but also because there are only a limited number of efficacious mastitis treatment options. Accordingly, comparative proteomic analyses of bovine milk have emerged in recent years. Efforts to prevent agricultural-related food-borne illness have likewise fueled an interest in the proteomic evaluation of several prominent strains of bacteria, including common mastitis pathogens. The interest in establishing biomarkers of the host and pathogen responses during bovine mastitis stems largely from the need to better characterize mechanisms of the disease, to identify reliable biomarkers for use as measures of early detection and drug efficacy, and to uncover potentially novel targets for the development of alternative therapeutics. The following review focuses primarily on comparative proteomic analyses conducted on healthy versus mastitic bovine milk. However, a comparison of the host defense proteome of human and bovine milk and the proteomic analysis of common veterinary pathogens are likewise introduced.

  19. Blueberries? Impact on Insulin Resistance and Glucose Intolerance

    OpenAIRE

    Stull, April J.

    2016-01-01

    Blueberries are a rich source of polyphenols, which include anthocyanin bioactive compounds. Epidemiological evidence indicates that incorporating blueberries into the diet may lower the risk of developing type 2 diabetes (T2DM). These findings are supported by pre-clinical and clinical studies that have shown improvements in insulin resistance (i.e., increased insulin sensitivity) after obese and insulin-resistant rodents or humans consumed blueberries. Insulin resistance was assessed by hom...

  20. Increased bioactive lipids content in human subcutaneous and epicardial fat tissue correlates with insulin resistance.

    Science.gov (United States)

    Błachnio-Zabielska, Agnieszka U; Baranowski, Marcin; Hirnle, Tomasz; Zabielski, Piotr; Lewczuk, Anna; Dmitruk, Iwona; Górski, Jan

    2012-12-01

    Obesity is a risk factor for metabolic diseases. Intramuscular lipid accumulation of ceramides, diacylglycerols, and long chain acyl-CoA is responsible for the induction of insulin resistance. These lipids are probably implicated in obesity-associated insulin resistance not only in skeletal muscle but also in fat tissue. Only few data are available about ceramide content in human subcutaneous adipose tissue. However, there are no data on DAG and LCACoA content in adipose tissue. The aim of our study was to measure the lipids content in human SAT and epicardial adipose tissue we sought to determine the bioactive lipids content by LC/MS/MS in fat tissue from lean non-diabetic, obese non-diabetic, and obese diabetic subjects and test whether the lipids correlate with HOMA-IR. We found, that total content of measured lipids was markedly higher in OND and OD subjects in both types of fat tissue (for all p lipids content is greater in subcutaneous and epicardial fat tissue and the particular lipids content positively correlates with HOMA-IR.

  1. Development of an antibody to bovine IL-2 reveals multifunctional CD4 T(EM) cells in cattle naturally infected with bovine tuberculosis.

    Science.gov (United States)

    Whelan, Adam O; Villarreal-Ramos, Bernardo; Vordermeier, H Martin; Hogarth, Philip J

    2011-01-01

    Gaining a better understanding of the T cell mechanisms underlying natural immunity to bovine tuberculosis would help to identify immune correlates of disease progression and facilitate the rational design of improved vaccine and diagnostic strategies. CD4 T cells play an established central role in immunity to TB, and recent interest has focussed on the potential role of multifunctional CD4 T cells expressing IFN-γ, IL-2 and TNF-α. Until now, it has not been possible to assess the contribution of these multifunctional CD4 T cells in cattle due to the lack of reagents to detect bovine IL-2 (bIL-2). Using recombinant phage display technology, we have identified an antibody that recognises biologically active bIL-2. Using this antibody, we have developed a polychromatic flow cytometric staining panel that has allowed the investigation of multifunctional CD4 T-cells responses in cattle naturally infected with M. bovis. Assessment of the frequency of antigen specific CD4 T cell subsets reveals a dominant IFN-γ(+)IL-2(+)TNF-α(+) and IFN-γ(+) TNF-α(+) response in naturally infected cattle. These multifunctional CD4 T cells express a CD44(hi)CD45RO(+)CD62L(lo) T-effector memory (T(EM)) phenotype and display higher cytokine median fluorescence intensities than single cytokine producers, consistent with an enhanced 'quality of response' as reported for multifunctional cells in human and murine systems. Through our development of these novel immunological bovine tools, we provide the first description of multifunctional T(EM) cells in cattle. Application of these tools will improve our understanding of protective immunity in bovine TB and allow more direct comparisons of the complex T cell mediated immune responses between murine models, human clinical studies and bovine TB models in the future. © 2011 Whelan et al.

  2. Development of an antibody to bovine IL-2 reveals multifunctional CD4 T(EM cells in cattle naturally infected with bovine tuberculosis.

    Directory of Open Access Journals (Sweden)

    Adam O Whelan

    Full Text Available Gaining a better understanding of the T cell mechanisms underlying natural immunity to bovine tuberculosis would help to identify immune correlates of disease progression and facilitate the rational design of improved vaccine and diagnostic strategies. CD4 T cells play an established central role in immunity to TB, and recent interest has focussed on the potential role of multifunctional CD4 T cells expressing IFN-γ, IL-2 and TNF-α. Until now, it has not been possible to assess the contribution of these multifunctional CD4 T cells in cattle due to the lack of reagents to detect bovine IL-2 (bIL-2. Using recombinant phage display technology, we have identified an antibody that recognises biologically active bIL-2. Using this antibody, we have developed a polychromatic flow cytometric staining panel that has allowed the investigation of multifunctional CD4 T-cells responses in cattle naturally infected with M. bovis. Assessment of the frequency of antigen specific CD4 T cell subsets reveals a dominant IFN-γ(+IL-2(+TNF-α(+ and IFN-γ(+ TNF-α(+ response in naturally infected cattle. These multifunctional CD4 T cells express a CD44(hiCD45RO(+CD62L(lo T-effector memory (T(EM phenotype and display higher cytokine median fluorescence intensities than single cytokine producers, consistent with an enhanced 'quality of response' as reported for multifunctional cells in human and murine systems. Through our development of these novel immunological bovine tools, we provide the first description of multifunctional T(EM cells in cattle. Application of these tools will improve our understanding of protective immunity in bovine TB and allow more direct comparisons of the complex T cell mediated immune responses between murine models, human clinical studies and bovine TB models in the future.

  3. Different apoptotic responses of human and bovine pericytes to fluctuating glucose levels and protective role of thiamine.

    Science.gov (United States)

    Beltramo, Elena; Berrone, Elena; Tarallo, Sonia; Porta, Massimo

    2009-09-01

    Vascular cells in diabetes are subjected to daily fluctuations from high to low glucose. We aimed at investigating whether pulsed exposure to different glucose concentrations influences apoptosis in human retinal pericytes (HRP) versus bovine retinal pericytes (BRP), with consequences on the onset of diabetic retinopathy, and the possible protective role of thiamine. BRP and HRP (wild-type and immortalized) were grown in physiological/high glucose for 7 days, and then returned to physiological glucose for another 24, 48 or 72 h. Cells were also kept intermittently at 48-h intervals in high/normal glucose for 8 days, with/without thiamine/benfotiamine. Apoptosis was determined through ELISA, TUNEL, Bcl-2, Bax and p53 expression/concentration. Continuous exposure to high glucose increased apoptosis in BRP, but not HRP. BRP apoptosis normalized within 24 h of physiological glucose re-entry, while HRP apoptosis increased within 24-48 h of re-entry. Intermittent exposure to high glucose increased apoptosis in HRP and BRP. Bcl-2/Bax results were consistent with DNA fragmentation, while p53 was unchanged. Thiamine and benfotiamine countered intermittent high glucose-induced apoptosis. Human pericytes are less prone to apoptosis induced by persistently high glucose than bovine cells. However, while BRP recover after returning to physiological levels, HRP are more vulnerable to both downwardly fluctuating glucose levels and intermittent exposure. These findings reinforce the hypotheses that (1) glycaemic fluctuations play a role in the development of diabetic retinopathy and (2) species-specific models are needed. Thiamine and benfotiamine prevent human pericyte apoptosis, indicating this vitamin as an inexpensive approach to the prevention and/or treatment of diabetic complications.

  4. Bovine cysticercosis situation in Brazil

    Directory of Open Access Journals (Sweden)

    Gabriel Augusto Marques Rossi

    2014-02-01

    Full Text Available The taeniasis-cysticercosis complex is a long known zoonotic parasitosis characteristic of underdeveloped countries. In addition to its public health significance, this parasitosis is cause of economic losses to the beef production chain, and synonymous of technical inadequacy in relation to the adoption of Good Agricultural Practices. The occurrences of both human teniasis and bovine cysticercosis could and should be controlled with basic sanitary measures. However, there is much variation in the occurrence of the disease in cattle, characterizing a low rate of technical development as well as problems related to the adoption of basic sanitation measures. This review describes, in details, the causative agent and its epidemiological chain, besides raising current information about the occurrence of bovine cysticercosis in different regions of Brazil, aiming at the adoption of prophylactic measures by different segments responsible.

  5. LukMF′ is the major secreted leukocidin of bovine Staphylococcus aureus and is produced in vivo during bovine mastitis

    NARCIS (Netherlands)

    Vrieling, Manouk; Boerhout, Eveline M.; Van Wigcheren, Glenn F.; Koymans, Kirsten J.; Mols-Vorstermans, Tanja G.; de Haas, Carla J C; Aerts, Piet C.; Daemen, Ineke J J M; van Kessel, Kok P M; Koets, Ad P.; Rutten, Victor P M G; Nuijten, Piet J M; Van Strijp, Jos A G; Benedictus, Lindert

    2016-01-01

    Staphylococcus aureus is a major human and animal pathogen and a common cause of mastitis in cattle. S. aureus secretes several leukocidins that target bovine neutrophils, crucial effector cells in the defence against bacterial pathogens. In this study, we investigated the role of staphylococcal

  6. LukMF' is the major secreted leukocidin of bovine Staphylococcus aureus and is produced in vivo during bovine mastitis

    NARCIS (Netherlands)

    Vrieling, Manouk; Boerhout, Eveline M; van Wigcheren, Glenn F; Koymans, Kirsten J; Mols-Vorstermans, Tanja G; de Haas, Carla J C; Aerts, Piet C; Daemen, Ineke J J M; van Kessel, Kok P M; Koets, Ad P|info:eu-repo/dai/nl/194306992; Rutten, Victor P M G|info:eu-repo/dai/nl/092848028; Nuijten, Piet J M; van Strijp, Jos A G; Benedictus, Lindert|info:eu-repo/dai/nl/37157742X

    2016-01-01

    Staphylococcus aureus is a major human and animal pathogen and a common cause of mastitis in cattle. S. aureus secretes several leukocidins that target bovine neutrophils, crucial effector cells in the defence against bacterial pathogens. In this study, we investigated the role of staphylococcal

  7. LukMF′ is the major secreted leukocidin of bovine Staphylococcus aureus and is produced in vivo during bovine mastitis

    NARCIS (Netherlands)

    Vrieling, Manouk; Boerhout, Eveline M.; Wigcheren, Van Glenn F.; Koymans, Kirsten J.; Mols-Vorstermans, Tanja G.; Haas, de Carla J.C.; Aerts, Piet C.; Daemen, Ineke J.J.M.; Kessel, Van Kok P.M.; Koets, Ad P.; Rutten, Victor P.M.G.; Nuijten, Piet J.M.; Strijp, van Jos A.G.; Benedictus, Lindert

    2016-01-01

    Staphylococcus aureus is a major human and animal pathogen and a common cause of mastitis in cattle. S. aureus secretes several leukocidins that target bovine neutrophils, crucial effector cells in the defence against bacterial pathogens. In this study, we investigated the role of staphylococcal

  8. Ex vivo permeation of carprofen from nanoparticles: A comprehensive study through human, porcine and bovine skin as anti-inflammatory agent.

    Science.gov (United States)

    Parra, Alexander; Clares, Beatriz; Rosselló, Ana; Garduño-Ramírez, María L; Abrego, Guadalupe; García, María L; Calpena, Ana C

    2016-03-30

    The purpose of this study was the development of poly(d,l-lactide-co-glycolide) acid (PLGA) nanoparticles (NPs) for the dermal delivery of carprofen (CP). The developed nanovehicle was then lyophilized using hydroxypropyl-β-cyclodextrin (HPβCD) as cryoprotectant. The ex vivo permeation profiles were evaluated using Franz diffusion cells using three different types of skin membranes: human, porcine and bovine. Furthermore, biomechanical properties of skin (trans-epidermal water loss and skin hydration) were tested. Finally, the in vivo skin irritation and the anti-inflammatory efficacy were also assayed. Results demonstrated the achievement of NPs 187.32 nm sized with homogeneous distribution, negatively charged surface (-23.39 mV) and high CP entrapment efficiency (75.38%). Permeation studies showed similar diffusion values between human and porcine skins and higher for bovine. No signs of skin irritation were observed in rabbits. Topically applied NPs significantly decreased in vivo inflammation compared to the reference drug in a TPA-induced mouse ear edema model. Thus, it was concluded that NPs containing CP may be a useful tool for the dermal treatment of local inflammation. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. High Prevalence of Schistosoma japonicum and Fasciola gigantica in Bovines from Northern Samar, the Philippines

    Science.gov (United States)

    Gordon, Catherine A.; Acosta, Luz P.; Gobert, Geoffrey N.; Jiz, Mario; Olveda, Remigio M.; Ross, Allen G.; Gray, Darren J.; Williams, Gail M.; Harn, Donald; Li, Yuesheng; McManus, Donald P.

    2015-01-01

    The cause of zoonotic schistosomiasis in the Philippines is Schistosoma japonicum, which infects up to 46 mammalian hosts, including humans and bovines. In China, water buffaloes have been identified as major reservoir hosts for schistosomiasis japonica, contributing up to 75% of human transmission. In the Philippines, water buffaloes (carabao; Bubalus bubalis carabanesis) have, historically, been considered unimportant reservoirs. We therefore revisited the possible role of bovines in schistosome transmission in the Philippines, using the recently described formalin-ethyl acetate sedimentation (FEA-SD) technique and a qPCR assay to examine fecal samples from 153 bovines (both carabao and cattle) from six barangays in Northern Samar. A high prevalence of S. japonicum was found using qPCR and FEA-SD in both cattle (87.50% and 77.08%, respectively) and carabao (80.00% and 55.24%, respectively). The average daily egg output for each bovine was calculated at 195,000. High prevalence and infection intensity of F. gigantica was also found in the bovines by qPCR and FEA-SD (95.33% and 96.00%, respectively). The identification of bovines as major reservoir hosts for S. japonicum transmission suggests that bovine treatment and/or vaccination, as one becomes available, should be included in any future control program that aims to reduce the disease burden due to schistosomiasis in the Philippines. PMID:25643317

  10. High prevalence of Schistosoma japonicum and Fasciola gigantica in bovines from Northern Samar, the Philippines.

    Directory of Open Access Journals (Sweden)

    Catherine A Gordon

    2015-02-01

    Full Text Available The cause of zoonotic schistosomiasis in the Philippines is Schistosoma japonicum, which infects up to 46 mammalian hosts, including humans and bovines. In China, water buffaloes have been identified as major reservoir hosts for schistosomiasis japonica, contributing up to 75% of human transmission. In the Philippines, water buffaloes (carabao; Bubalus bubalis carabanesis have, historically, been considered unimportant reservoirs. We therefore revisited the possible role of bovines in schistosome transmission in the Philippines, using the recently described formalin-ethyl acetate sedimentation (FEA-SD technique and a qPCR assay to examine fecal samples from 153 bovines (both carabao and cattle from six barangays in Northern Samar. A high prevalence of S. japonicum was found using qPCR and FEA-SD in both cattle (87.50% and 77.08%, respectively and carabao (80.00% and 55.24%, respectively. The average daily egg output for each bovine was calculated at 195,000. High prevalence and infection intensity of F. gigantica was also found in the bovines by qPCR and FEA-SD (95.33% and 96.00%, respectively. The identification of bovines as major reservoir hosts for S. japonicum transmission suggests that bovine treatment and/or vaccination, as one becomes available, should be included in any future control program that aims to reduce the disease burden due to schistosomiasis in the Philippines.

  11. Racl Signaling Is Required for Insulin-Stimulated Glucose Uptake and Is Dysregulated in Insulin-Resistant Murine and Human Skeletal Muscle

    DEFF Research Database (Denmark)

    Sylow, L.; Jensen, T. E.; Kleinert, M.

    2013-01-01

    The actin cytoskeleton-regulating GTPase Racl is required for insulin-stimulated GLUT4 translocation in cultured muscle cells. However, involvement of Racl and its downstream signaling in glucose transport in insulin-sensitive and insulin-resistant mature skeletal muscle has not previously been i...

  12. High oxygen condition facilitates the differentiation of mouse and human pluripotent stem cells into pancreatic progenitors and insulin-producing cells.

    Science.gov (United States)

    Hakim, Farzana; Kaitsuka, Taku; Raeed, Jamiruddin Mohd; Wei, Fan-Yan; Shiraki, Nobuaki; Akagi, Tadayuki; Yokota, Takashi; Kume, Shoen; Tomizawa, Kazuhito

    2014-04-04

    Pluripotent stem cells have potential applications in regenerative medicine for diabetes. Differentiation of stem cells into insulin-producing cells has been achieved using various protocols. However, both the efficiency of the method and potency of differentiated cells are insufficient. Oxygen tension, the partial pressure of oxygen, has been shown to regulate the embryonic development of several organs, including pancreatic β-cells. In this study, we tried to establish an effective method for the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells by culturing under high oxygen (O2) conditions. Treatment with a high O2 condition in the early stage of differentiation increased insulin-positive cells at the terminus of differentiation. We found that a high O2 condition repressed Notch-dependent gene Hes1 expression and increased Ngn3 expression at the stage of pancreatic progenitors. This effect was caused by inhibition of hypoxia-inducible factor-1α protein level. Moreover, a high O2 condition activated Wnt signaling. Optimal stage-specific treatment with a high O2 condition resulted in a significant increase in insulin production in both mouse embryonic stem cells and human iPSCs and yielded populations containing up to 10% C-peptide-positive cells in human iPSCs. These results suggest that culturing in a high O2 condition at a specific stage is useful for the efficient generation of insulin-producing cells.

  13. Human ketone body production and utilization studied using tracer techniques: Regulation by free fatty acids, insulin, catecholamines, and thyroid hormones

    Energy Technology Data Exchange (ETDEWEB)

    Keller, U.; Lustenberger, M.; Mueller-Brand, J.G.; Gerber, P.P.; Stauffacher, W.

    1989-05-01

    Ketone body concentrations fluctuate markedly during physiological and pathological conditions. Tracer techniques have been developed in recent years to study production, utilization, and the metabolic clearance rate of ketone bodies. This review describes data on the roles of insulin, catecholamines, and thyroid hormones in the regulation of ketone body kinetics. The data indicate that insulin lowers ketone body concentrations by three independent mechanisms: first, it inhibits lipolysis, and thus lowers free fatty acid availability for ketogenesis; second, it restrains ketone body production within the liver; third, it enhances peripheral ketone body utilization. To assess these effects in humans in vivo, experimental models were developed to study insulin effects with controlled concentrations of free fatty acids, insulin, glucagon, and ketone bodies. Presently available data also support an important role of catecholamines in increasing ketone body concentrations. Evidence was presented that norepinephrine increases ketogenesis not only by stimulating lipolysis, and thus releasing free fatty acids, but also by increasing intrahepatic ketogenesis. Thyroid hormone availability was associated with lipolysis and ketogenesis. Ketone body concentrations after an overnight fast were only modestly elevated in hyperthyroidism resulting from increased peripheral ketone body clearance. There was a significant correlation between serum triiodothyronine levels and the ketone body metabolic clearance rate. Thus, ketone body homeostasis in human subjects resulted from the interaction of hormones such as insulin, catecholamines, and thyroid hormones regulating lipolysis, intrahepatic ketogenesis, and peripheral ketone body utilization. 58 references.

  14. Human ketone body production and utilization studied using tracer techniques: Regulation by free fatty acids, insulin, catecholamines, and thyroid hormones

    International Nuclear Information System (INIS)

    Keller, U.; Lustenberger, M.; Mueller-Brand, J.G.; Gerber, P.P.; Stauffacher, W.

    1989-01-01

    Ketone body concentrations fluctuate markedly during physiological and pathological conditions. Tracer techniques have been developed in recent years to study production, utilization, and the metabolic clearance rate of ketone bodies. This review describes data on the roles of insulin, catecholamines, and thyroid hormones in the regulation of ketone body kinetics. The data indicate that insulin lowers ketone body concentrations by three independent mechanisms: first, it inhibits lipolysis, and thus lowers free fatty acid availability for ketogenesis; second, it restrains ketone body production within the liver; third, it enhances peripheral ketone body utilization. To assess these effects in humans in vivo, experimental models were developed to study insulin effects with controlled concentrations of free fatty acids, insulin, glucagon, and ketone bodies. Presently available data also support an important role of catecholamines in increasing ketone body concentrations. Evidence was presented that norepinephrine increases ketogenesis not only by stimulating lipolysis, and thus releasing free fatty acids, but also by increasing intrahepatic ketogenesis. Thyroid hormone availability was associated with lipolysis and ketogenesis. Ketone body concentrations after an overnight fast were only modestly elevated in hyperthyroidism resulting from increased peripheral ketone body clearance. There was a significant correlation between serum triiodothyronine levels and the ketone body metabolic clearance rate. Thus, ketone body homeostasis in human subjects resulted from the interaction of hormones such as insulin, catecholamines, and thyroid hormones regulating lipolysis, intrahepatic ketogenesis, and peripheral ketone body utilization. 58 references

  15. Generation of Insulin-Producing Cells from Human Bone Marrow-Derived Mesenchymal Stem Cells: Comparison of Three Differentiation Protocols

    Directory of Open Access Journals (Sweden)

    Mahmoud M. Gabr

    2014-01-01

    Full Text Available Introduction. Many protocols were utilized for directed differentiation of mesenchymal stem cells (MSCs to form insulin-producing cells (IPCs. We compared the relative efficiency of three differentiation protocols. Methods. Human bone marrow-derived MSCs (HBM-MSCs were obtained from three insulin-dependent type 2 diabetic patients. Differentiation into IPCs was carried out by three protocols: conophylline-based (one-step protocol, trichostatin-A-based (two-step protocol, and β-mercaptoethanol-based (three-step protocol. At the end of differentiation, cells were evaluated by immunolabeling for insulin production, expression of pancreatic endocrine genes, and release of insulin and c-peptide in response to increasing glucose concentrations. Results. By immunolabeling, the proportion of generated IPCs was modest (≃3% in all the three protocols. All relevant pancreatic endocrine genes, insulin, glucagon, and somatostatin, were expressed. There was a stepwise increase in insulin and c-peptide release in response to glucose challenge, but the released amounts were low when compared with those of pancreatic islets. Conclusion. The yield of functional IPCs following directed differentiation of HBM-MSCs was modest and was comparable among the three tested protocols. Protocols for directed differentiation of MSCs need further optimization in order to be clinically meaningful. To this end, addition of an extracellular matrix and/or a suitable template should be attempted.

  16. Challenges constraining access to insulin in the private-sector market of Delhi, India.

    Science.gov (United States)

    Sharma, Abhishek; Kaplan, Warren A

    2016-01-01

    India's majority of patients-including those living with diabetes-seek healthcare in the private sector through out-of-pocket (OOP) payments. We studied access to insulin in the private-sector market of Delhi state, India. A modified World Health Organization/Health Action International (WHO/HAI) standard survey to assess insulin availability and prices, and qualitative interviews with insulin retailers (pharmacists) and wholesalers to understand insulin market dynamics. In 40 pharmacy outlets analysed, mean availability of the human and analogue insulins on the 2013 Delhi essential medicine list was 44.4% and 13.1%, respectively. 82% of pharmacies had domestically manufactured human insulin phials, primarily was made in India under licence to overseas pharmaceutical companies. Analogue insulin was only in cartridge and pen forms that were 4.42 and 5.81 times, respectively, the price of human insulin phials. Domestically manufactured human phial and cartridge insulin (produced for foreign and Indian companies) was less expensive than their imported counterparts. The lowest paid unskilled government worker in Delhi would work about 1.5 and 8.6 days, respectively, to be able to pay OOP for a monthly supply of human phial and analogue cartridge insulin. Interviews suggest that the Delhi insulin market is dominated by a few multinational companies that import and/or license in-country production. Several factors influence insulin uptake by patients, including doctor's prescribing preference. Wholesalers have negative perceptions about domestic insulin manufacturing. The Delhi insulin market is an oligopoly with limited market competition. Increasing competition from Indian companies is going to require some additional policies, not presently in place. As more Indian companies produce biosimilars, brand substitution policies are needed to be able to benefit from market competition.

  17. Challenges constraining access to insulin in the private-sector market of Delhi, India

    Science.gov (United States)

    Kaplan, Warren A

    2016-01-01

    Objective India's majority of patients—including those living with diabetes—seek healthcare in the private sector through out-of-pocket (OOP) payments. We studied access to insulin in the private-sector market of Delhi state, India. Methods A modified World Health Organization/Health Action International (WHO/HAI) standard survey to assess insulin availability and prices, and qualitative interviews with insulin retailers (pharmacists) and wholesalers to understand insulin market dynamics. Results In 40 pharmacy outlets analysed, mean availability of the human and analogue insulins on the 2013 Delhi essential medicine list was 44.4% and 13.1%, respectively. 82% of pharmacies had domestically manufactured human insulin phials, primarily was made in India under licence to overseas pharmaceutical companies. Analogue insulin was only in cartridge and pen forms that were 4.42 and 5.81 times, respectively, the price of human insulin phials. Domestically manufactured human phial and cartridge insulin (produced for foreign and Indian companies) was less expensive than their imported counterparts. The lowest paid unskilled government worker in Delhi would work about 1.5 and 8.6 days, respectively, to be able to pay OOP for a monthly supply of human phial and analogue cartridge insulin. Interviews suggest that the Delhi insulin market is dominated by a few multinational companies that import and/or license in-country production. Several factors influence insulin uptake by patients, including doctor's prescribing preference. Wholesalers have negative perceptions about domestic insulin manufacturing. Conclusions The Delhi insulin market is an oligopoly with limited market competition. Increasing competition from Indian companies is going to require some additional policies, not presently in place. As more Indian companies produce biosimilars, brand substitution policies are needed to be able to benefit from market competition. PMID:28588966

  18. Insulin analogues and severe hypoglycaemia in type 1 diabetes

    DEFF Research Database (Denmark)

    Kristensen, P L; Hansen, L S; Jespersen, M J

    2012-01-01

    The effect of insulin analogues on glycaemic control is well-documented, whereas the effect on avoidance of severe hypoglycaemia remains tentative. We studied the frequency of severe hypoglycaemia in unselected patients with type 1 diabetes treated with insulin analogues, human insulin, or mixed...

  19. Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI)

    DEFF Research Database (Denmark)

    Valnickova, Zuzana; Thaysen-Andersen, Morten; Højrup, Peter

    2009-01-01

    -activatable fibrinolysis inhibitor (TAFI), and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI. RESULTS: The four N-linked glycosylation sequons within the activation peptide were all occupied...

  20. Surface characterization of insulin-coated Ti6Al4V medical implants conditioned in cell culture medium: An XPS study

    Energy Technology Data Exchange (ETDEWEB)

    Shchukarev, Andrey, E-mail: andrey.shchukarev@umu.se [Department of Chemistry, Umeå University, Umeå SE-90187 (Sweden); Malekzadeh, Behnosh Öhrnell [Department of Orthodontics, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, SE-40530 (Sweden); Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, SE-40530 (Sweden); Ransjö, Maria [Department of Orthodontics, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, SE-40530 (Sweden); Tengvall, Pentti [Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, SE-40530 (Sweden); Westerlund, Anna [Department of Orthodontics, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, SE-40530 (Sweden)

    2017-04-15

    Highlights: • In the absence of FBS, chemically immobilized insulin layer remains intact; • The immobilized insulin expose hydrophobic domains outward the implant; • In the presence of FBS, a partial replacement of insulin occurs; • The immobilized insulin stabilizes the secondary structure of adsorbed proteins. - Abstract: Surface characterization of insulin-coated Ti6Al4V medical implants, after incubation in α-minimum essential medium (α-MEM), was done by X-ray photoelectron spectroscopy (XPS), in order to analyze the insulin behavior at the implant – α-MEM interface. In the absence of serum proteins in cell culture medium, the coated insulin layer remained intact, but experienced a time-dependent structural transformation exposing hydrophobic parts of the protein toward the solution. The presence of fetal bovine serum (FBS) in the medium resulted in partial substitution of insulin by serum proteins. In spite of some insulin release, the remaining coated layer demonstrated a direct surface effect by stabilizing the structure of protein competitors, and by supporting the accumulation of calcium and phosphate ions at the interface. A structurally stable protein layer with incorporated calcium and phosphate ions at the implant–tissue interface could be an important prerequisite for enhanced bone formation.

  1. Surface characterization of insulin-coated Ti6Al4V medical implants conditioned in cell culture medium: An XPS study

    International Nuclear Information System (INIS)

    Shchukarev, Andrey; Malekzadeh, Behnosh Öhrnell; Ransjö, Maria; Tengvall, Pentti; Westerlund, Anna

    2017-01-01

    Highlights: • In the absence of FBS, chemically immobilized insulin layer remains intact; • The immobilized insulin expose hydrophobic domains outward the implant; • In the presence of FBS, a partial replacement of insulin occurs; • The immobilized insulin stabilizes the secondary structure of adsorbed proteins. - Abstract: Surface characterization of insulin-coated Ti6Al4V medical implants, after incubation in α-minimum essential medium (α-MEM), was done by X-ray photoelectron spectroscopy (XPS), in order to analyze the insulin behavior at the implant – α-MEM interface. In the absence of serum proteins in cell culture medium, the coated insulin layer remained intact, but experienced a time-dependent structural transformation exposing hydrophobic parts of the protein toward the solution. The presence of fetal bovine serum (FBS) in the medium resulted in partial substitution of insulin by serum proteins. In spite of some insulin release, the remaining coated layer demonstrated a direct surface effect by stabilizing the structure of protein competitors, and by supporting the accumulation of calcium and phosphate ions at the interface. A structurally stable protein layer with incorporated calcium and phosphate ions at the implant–tissue interface could be an important prerequisite for enhanced bone formation.

  2. In vitro generation of functional insulin-producing cells from lipoaspirated human adipose tissue-derived stem cells.

    Science.gov (United States)

    Mohamad Buang, Mohamad Lizan; Seng, Heng Kien; Chung, Lee Han; Saim, Aminuddin Bin; Idrus, Ruszymah Bt Hj

    2012-01-01

    Tissue engineering strategy has been considered as an alternative treatment for diabetes mellitus due to lack of permanent pharmaceutical treatment and islet donors for transplantation. Various cell lines have been used to generate functional insulin-producing cells (IPCs) including progenitor pancreatic cell lines, embryonic stem cells (ESCs), umbilical cord blood stem cells (UCB-SCs), adult bone marrow stem cells (BMSCs), and adipose tissue-derived stem cells (ADSCs). Human ADSCs from lipoaspirated abdominal fat tissue was differentiated into IPCs following a two-step induction protocol based on a combination of alternating high and low glucose, nicotinamide, activin A and glucagon-like peptide 1 (GLP-1) for a duration of 3 weeks. During differentiation, histomorphological changes of the stem cells towards pancreatic β-islet characteristics were observed via light microscope and transmission electron microscope (TEM). Dithizone (DTZ) staining, which is selective towards IPCs, was used to stain the new islet-like cells. Production of insulin hormone by the cells was analyzed via enzyme-linked immunosorbent assay (ELISA), whereas its hormonal regulation was tested via a glucose challenge test. Histomorphological changes of the differentiated cells were noted to resemble pancreatic β-cells, whereas DTZ staining positively stained the cells. The differentiated cells significantly produced human insulin as compared to the undifferentiated ADSCs, and its production was increased with an increase of glucose concentration in the culture medium. These initial data indicate that human lipoaspirated ADSCs have the potential to differentiate into functional IPCs, and could be used as a therapy to treat diabetes mellitus in the future. Copyright © 2012 IMSS. Published by Elsevier Inc. All rights reserved.

  3. Human-Based Human Milk Fortifier as Rescue Therapy in Very Low Birth Weight Infants Demonstrating Intolerance to Bovine-Based Human Milk Fortifier.

    Science.gov (United States)

    Sandhu, Amanjot; Fast, Sharla; Bonnar, Kari; Baier, Ronald John; Narvey, Michael

    2017-11-01

    To describe the results of utilizing a human milk-based human milk fortifier (HMHMF) as rescue therapy to meet nutritional requirements in very low birth weight and preterm infants demonstrating feeding intolerance to bovine-based human milk fortifier (BHMF) in the Canadian Neonatal Intensive Care Unit (NICU) setting. At two Level III NICUs in Winnipeg, MB, Canada, a rescue protocol was implemented to provide HMHMF for infants demonstrating intolerance to BHMF. To qualify for rescue, infants were required to experience two episodes of significant gastrointestinal (GI) symptoms associated with fortification with BHMF. A case series report was conducted retrospectively examining the success of rescue therapy, growth rates, protein, and calorie intakes before and after initiation of HMHMF in seven infants. Seven infants (birth weight 723 ± 247 g, gestation 25.3 ± 3.4 weeks) were treated with rescue fortification with HMHMF. All infants were transitioned off parenteral nutrition (PN) without relapse of GI symptoms. Growth rate, protein, and calorie intakes improved with the use of HMHMF. Very low birth weight and preterm infants with GI intolerance to BHMF were successfully rescued with use of HMHMF. Improvements in growth were achieved without need for supplementation with PN through achievement of sufficient enteral calorie and protein intakes.

  4. Virulence Gene Pool Detected in Bovine Group C Streptococcus dysgalactiae subsp. dysgalactiae Isolates by Use of a Group A S. pyogenes Virulence Microarray ▿

    Science.gov (United States)

    Rato, Márcia G.; Nerlich, Andreas; Bergmann, René; Bexiga, Ricardo; Nunes, Sandro F.; Vilela, Cristina L.; Santos-Sanches, Ilda; Chhatwal, Gursharan S.

    2011-01-01

    A custom-designed microarray containing 220 virulence genes of Streptococcus pyogenes (group A Streptococcus [GAS]) was used to test group C Streptococcus dysgalactiae subsp. dysgalactiae (GCS) field strains causing bovine mastitis and group C or group G Streptococcus dysgalactiae subsp. equisimilis (GCS/GGS) isolates from human infections, with the latter being used for comparative purposes, for the presence of virulence genes. All bovine and all human isolates carried a fraction of the 220 genes (23% and 39%, respectively). The virulence genes encoding streptolysin S, glyceraldehyde-3-phosphate dehydrogenase, the plasminogen-binding M-like protein PAM, and the collagen-like protein SclB were detected in the majority of both bovine and human isolates (94 to 100%). Virulence factors, usually carried by human beta-hemolytic streptococcal pathogens, such as streptokinase, laminin-binding protein, and the C5a peptidase precursor, were detected in all human isolates but not in bovine isolates. Additionally, GAS bacteriophage-associated virulence genes encoding superantigens, DNase, and/or streptodornase were detected in bovine isolates (72%) but not in the human isolates. Determinants located in non-bacteriophage-related mobile elements, such as the gene encoding R28, were detected in all bovine and human isolates. Several virulence genes, including genes of bacteriophage origin, were shown to be expressed by reverse transcriptase PCR (RT-PCR). Phylogenetic analysis of superantigen gene sequences revealed a high level (>98%) of identity among genes of bovine GCS, of the horse pathogen Streptococcus equi subsp. equi, and of the human pathogen GAS. Our findings indicate that alpha-hemolytic bovine GCS, an important mastitis pathogen and considered to be a nonhuman pathogen, carries important virulence factors responsible for virulence and pathogenesis in humans. PMID:21525223

  5. Sequence of a New World primate insulin having low biological potency and immunoreactivity

    Energy Technology Data Exchange (ETDEWEB)

    Seino, S.; Steiner, D.F.; Bell, G.I.

    1987-11-01

    The organization of the insulin gene of the owl or night monkey (Aotus trivirgatus), a New World primate, is similar to that of the human gene. The sequences of these two genes and flanking regions possess 84.3% homology. An unusual feature of the owl monkey gene is the partial duplication and insertion of a portion of the A-chain coding sequence into the 3' untranslated region. The insulin gene of this primate also lacks a region of tandem repeats that is present in the 5' flanking region of the human and chimpanzee genes. Owl monkey preproinsulin has 85.5% identity with the human insulin precursor and is the most divergent of the primate insulins/preproinsulins yet described. The differences between owl monkey and human preproinsulin include three substitutions in the signal peptide, two in the B chain, seven in the C peptide, and three in the A chain. One of these replacements is the conservative substitution of valine for isoleucine a position A2, an invariant site in all other vertebrate insulins and insulin-like growth factors. The substitutions in owl monkey insulin at B9, B27, A2, A4, and A17 alter its structure so that it has only 20% of the receptor-binding activity and 1% of the affinity with guinea pig anti-porcine insulin antibodies as compared to human insulin.

  6. Sequence of a New World primate insulin having low biological potency and immunoreactivity

    International Nuclear Information System (INIS)

    Seino, S.; Steiner, D.F.; Bell, G.I.

    1987-01-01

    The organization of the insulin gene of the owl or night monkey (Aotus trivirgatus), a New World primate, is similar to that of the human gene. The sequences of these two genes and flanking regions possess 84.3% homology. An unusual feature of the owl monkey gene is the partial duplication and insertion of a portion of the A-chain coding sequence into the 3' untranslated region. The insulin gene of this primate also lacks a region of tandem repeats that is present in the 5' flanking region of the human and chimpanzee genes. Owl monkey preproinsulin has 85.5% identity with the human insulin precursor and is the most divergent of the primate insulins/preproinsulins yet described. The differences between owl monkey and human preproinsulin include three substitutions in the signal peptide, two in the B chain, seven in the C peptide, and three in the A chain. One of these replacements is the conservative substitution of valine for isoleucine a position A2, an invariant site in all other vertebrate insulins and insulin-like growth factors. The substitutions in owl monkey insulin at B9, B27, A2, A4, and A17 alter its structure so that it has only 20% of the receptor-binding activity and 1% of the affinity with guinea pig anti-porcine insulin antibodies as compared to human insulin

  7. Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats.

    Science.gov (United States)

    Matveyenko, Aleksey V; Georgia, Senta; Bhushan, Anil; Butler, Peter C

    2010-11-01

    Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in development of viable glucose-responsive pancreatic endocrine tissue. Athymic nude rats were implanted with PE derived from hESC either via implantation into the epididymal fat pads or by subcutaneous implantation into TheraCyte encapsulation devices for 20 wk. Blood glucose, weight, and human insulin/C-peptide secretion were monitored by weekly blood draws. Graft β-cell function was assessed by a glucose tolerance test, and graft morphology was assessed by immunohistochemistry and immunofluorescence. At 20 wk postimplantation, epididymal fat-implanted PE progressed to develop islet-like structures in 50% of implants, with a mean β-cell fractional area of 0.8 ± 0.3%. Human C-peptide and insulin were detectable, but at very low levels (C-peptide = 50 ± 26 pmol/l and insulin = 15 ± 7 pmol/l); however, there was no increase in human C-peptide/insulin levels after glucose challenge. There was no development of viable pancreatic tissue or meaningful secretory function when human PE was implanted in the TheraCyte encapsulation devices. These data confirm that islet-like structures develop from hESC differentiated to PE by the protocol developed by NovoCell. However, the extent of endocrine cell formation and secretory function is not yet sufficient to be clinically relevant.

  8. Interfacial adsorption of insulin - Conformational changes and reversibility of adsorption

    NARCIS (Netherlands)

    Mollmann, SH; Jorgensen, L; Bukrinsky, JT; Elofsson, U; Norde, W; Frokjaer, S

    The adsorption of human insulin to Teflon particles was studied with respect to conformational changes and the reversibility of adsorption was examined by total internal reflection fluorescence (TIRF). Adsorption isotherms for the adsorption of human insulin indicated high affinity adsorption, even

  9. Sulfate Anion Delays the Self-Assembly of Human Insulin by Modifying the Aggregation Pathway

    OpenAIRE

    Owczarz, Marta; Arosio, Paolo

    2014-01-01

    The understanding of the molecular mechanisms underlying protein self-assembly and of their dependence on solvent composition has implications in a large number of biological and biotechnological systems. In this work, we characterize the aggregation process of human insulin at acidic pH in the presence of sulfate ions using a combination of Thioflavin T fluorescence, dynamic light scattering, size exclusion chromatography, Fourier transform infrared spectroscopy, and transmission electron mi...

  10. Biological and Clinical Study of 6-Deoxy-6-Iodo-D-Glucose: a iodinated tracer of glucose transport and of insulin-resistance in human

    International Nuclear Information System (INIS)

    Barone-Rochette, Gilles

    2013-01-01

    Insulin resistance (IR), characterized by a depressed cellular sensitivity to insulin in insulin-sensitive organs, is a central feature to obesity, the metabolic syndrome, and diabetes mellitus and leads to increase cardiovascular diseases, particularly heart failure. All these events are today serious public health problems. But actually, there is no simple tool to measure insulin resistance. The gold standard technique remains the hyperinsulinemic euglycemic clamp. However, the complexity and length of this technique render it unsuitable for routine clinical use. Many methods or index have been proposed to assess insulin resistance in human, but none have shown enough relevance to be used in clinical use. The U1039 INSERM unit previously has validated a new tracer of glucose transport, radiolabelled with 123 iodine and has developed a fast and simple imaging protocol with a small animal gamma camera, which allows the obtaining of an IR index for each organ, showing more discriminating for the heart. The project of my thesis was the human transfer of this measurement technique, perfectly validated in animal. The first part of this thesis evaluated to tolerance, in vivo kinetics, distribution and dosimetry of novel tracer of glucose transport, the [ 123 I]-6DIG. The safeties of new tracer and measurement technique were adequate. There were no adverse effects with excellent tolerance of the whole protocol. 6DIG eliminating was fast, primarily in the urine and complete within 72 h. The effective whole-body absorbed dose for a complete scan with injection of 92.5 * 2 MBq was between 3 to 4 mSv. The second part of this thesis evaluated in human feasibility and reproducibility of the measurement technique validated in animal. The third part showed techniques used to allow human transfer of this method. The study protocol was applied on 12 subjects (healthy volunteers (n=6) and type 2 diabetic patients (n=6)). With a method adapted to measure in humans, we determined an

  11. Palmitate-induced inflammatory pathways in human adipose microvascular endothelial cells promote monocyte adhesion and impair insulin transcytosis.

    Science.gov (United States)

    Pillon, Nicolas J; Azizi, Paymon M; Li, Yujin E; Liu, Jun; Wang, Changsen; Chan, Kenny L; Hopperton, Kathryn E; Bazinet, Richard P; Heit, Bryan; Bilan, Philip J; Lee, Warren L; Klip, Amira

    2015-07-01

    Obesity is associated with inflammation and immune cell recruitment to adipose tissue, muscle and intima of atherosclerotic blood vessels. Obesity and hyperlipidemia are also associated with tissue insulin resistance and can compromise insulin delivery to muscle. The muscle/fat microvascular endothelium mediates insulin delivery and facilitates monocyte transmigration, yet its contribution to the consequences of hyperlipidemia is poorly understood. Using primary endothelial cells from human adipose tissue microvasculature (HAMEC), we investigated the effects of physiological levels of fatty acids on endothelial inflammation and function. Expression of cytokines and adhesion molecules was measured by RT-qPCR. Signaling pathways were evaluated by pharmacological manipulation and immunoblotting. Surface expression of adhesion molecules was determined by immunohistochemistry. THP1 monocyte interaction with HAMEC was measured by cell adhesion and migration across transwells. Insulin transcytosis was measured by total internal reflection fluorescence microscopy. Palmitate, but not palmitoleate, elevated the expression of IL-6, IL-8, TLR2 (Toll-like receptor 2), and intercellular adhesion molecule 1 (ICAM-1). HAMEC had markedly low fatty acid uptake and oxidation, and CD36 inhibition did not reverse the palmitate-induced expression of adhesion molecules, suggesting that inflammation did not arise from palmitate uptake/metabolism. Instead, inhibition of TLR4 to NF-κB signaling blunted palmitate-induced ICAM-1 expression. Importantly, palmitate-induced surface expression of ICAM-1 promoted monocyte binding and transmigration. Conversely, palmitate reduced insulin transcytosis, an effect reversed by TLR4 inhibition. In summary, palmitate activates inflammatory pathways in primary microvascular endothelial cells, impairing insulin transport and increasing monocyte transmigration. This behavior may contribute in vivo to reduced tissue insulin action and enhanced tissue

  12. Insulin and GH signaling in human skeletal muscle in vivo following exogenous GH exposure: impact of an oral glucose load.

    Directory of Open Access Journals (Sweden)

    Thomas Krusenstjerna-Hafstrøm

    2011-05-01

    Full Text Available GH induces acute insulin resistance in skeletal muscle in vivo, which in rodent models has been attributed to crosstalk between GH and insulin signaling pathways. Our objective was to characterize time course changes in signaling pathways for GH and insulin in human skeletal muscle in vivo following GH exposure in the presence and absence of an oral glucose load.Eight young men were studied in a single-blinded randomized crossover design on 3 occasions: 1 after an intravenous GH bolus 2 after an intravenous GH bolus plus an oral glucose load (OGTT, and 3 after intravenous saline plus OGTT. Muscle biopsies were taken at t = 0, 30, 60, and 120. Blood was sampled at frequent intervals for assessment of GH, insulin, glucose, and free fatty acids (FFA.GH increased AUC(glucose after an OGTT (p<0.05 without significant changes in serum insulin levels. GH induced phosphorylation of STAT5 independently of the OGTT. Conversely, the OGTT induced acute phosphorylation of the insulin signaling proteins Akt (ser(473 and thr(308, and AS160.The combination of OGTT and GH suppressed Akt activation, whereas the downstream expression of AS160 was amplified by GH. WE CONCLUDED THE FOLLOWING: 1 A physiological GH bolus activates STAT5 signaling pathways in skeletal muscle irrespective of ambient glucose and insulin levels 2 Insulin resistance induced by GH occurs without a distinct suppression of insulin signaling proteins 3 The accentuation of the glucose-stimulated activation of AS 160 by GH does however indicate a potential crosstalk between insulin and GH.ClinicalTrials.gov NCT00477997.

  13. Bovine Staphylococcus aureus secretes the leukocidin LukMF′ to kill migrating neutrophils through CCR1

    NARCIS (Netherlands)

    Vrieling, M.; Koymans, K.J.; Heesterbeek, D.A.C.; Aerts, P.C.; Rutten, V.P.M.G.; de Haas, C.J.C.; van Kessel, K.P.M.; Koets, A.P.; Nijland, R; van Strijp, J.A.G.

    2015-01-01

    Although Staphylococcus aureus is best known for infecting humans, bovine-specific strains are a major cause of mastitis in dairy cattle. The bicomponent leukocidin LukMF′, exclusively harbored by S. aureus of ruminant origin, is a virulence factor associated with bovine infections. In this study,

  14. Bovine Staphylococcus aureus secretes the leukocidin LukMF' to kill migrating neutrophils through CCR1

    NARCIS (Netherlands)

    Vrieling, M.; Koymans, K.J.; Heesterbeek, D.A.C.; Aerts, P.C.; Rutten, V.P.M.G.; Haas, de C.J.C.; Kessel, van K.P.M.; Koets, A.P.; Nijland, R.; Strijp, van J.A.G.

    2015-01-01

    Although Staphylococcus aureus is best known for infecting humans, bovine-specific strains are a major cause of mastitis in dairy cattle. The bicomponent leukocidin LukMF′, exclusively harbored by S. aureus of ruminant origin, is a virulence factor associated with bovine infections. In this study,

  15. A high resolution radiation hybrid map of bovine chromosome 14 identifies scaffold rearrangement in the latest bovine assembly

    Directory of Open Access Journals (Sweden)

    Wang Zhiquan

    2007-07-01

    Full Text Available Abstract Background Radiation hybrid (RH maps are considered to be a tool of choice for fine mapping closely linked loci, considering that the resolution of linkage maps is determined by the number of informative meiosis and recombination events which may require very large mapping populations. Accurately defining the marker order on chromosomes is crucial for correct identification of quantitative trait loci (QTL, haplotype map construction and refinement of candidate gene searches. Results A 12 k Radiation hybrid map of bovine chromosome 14 was constructed using 843 single nucleotide polymorphism markers. The resulting map was aligned with the latest version of the bovine assembly (Btau_3.1 as well as other previously published RH maps. The resulting map identified distinct regions on Bovine chromosome 14 where discrepancies between this RH map and the bovine assembly occur. A major region of discrepancy was found near the centromere involving the arrangement and order of the scaffolds from the assembly. The map further confirms previously published conserved synteny blocks with human chromosome 8. As well, it identifies an extra breakpoint and conserved synteny block previously undetected due to lower marker density. This conserved synteny block is in a region where markers between the RH map presented here and the latest sequence assembly are in very good agreement. Conclusion The increase of publicly available markers shifts the rate limiting step from marker discovery to the correct identification of their order for further use by the research community. This high resolution map of bovine chromosome 14 will facilitate identification of regions in the sequence assembly where additional information is required to resolve marker ordering.

  16. Aβ-Induced Insulin Resistance and the Effects of Insulin on the Cholesterol Synthesis Pathway and Aβ Secretion in Neural Cells.

    Science.gov (United States)

    Najem, Dema; Bamji-Mirza, Michelle; Yang, Ze; Zhang, Wandong

    2016-06-01

    Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) toxicity, tau pathology, insulin resistance, neuroinflammation, and dysregulation of cholesterol homeostasis, all of which play roles in neurodegeneration. Insulin has polytrophic effects on neurons and may be at the center of these pathophysiological changes. In this study, we investigated possible relationships among insulin signaling and cholesterol biosynthesis, along with the effects of Aβ42 on these pathways in vitro. We found that neuroblastoma 2a (N2a) cells transfected with the human gene encoding amyloid-β protein precursor (AβPP) (N2a-AβPP) produced Aβ and exhibited insulin resistance by reduced p-Akt and a suppressed cholesterol-synthesis pathway following insulin treatment, and by increased phosphorylation of insulin receptor subunit-1 at serine 612 (p-IRS-S612) as compared to parental N2a cells. Treatment of human neuroblastoma SH-SY5Y cells with Aβ42 also increased p-IRS-S612, suggesting that Aβ42 is responsible for insulin resistance. The insulin resistance was alleviated when N2a-AβPP cells were treated with higher insulin concentrations. Insulin increased Aβ release from N2a-AβPP cells, by which it may promote Aβ clearance. Insulin increased cholesterol-synthesis gene expression in SH-SY5Y and N2a cells, including 24-dehydrocholesterol reductase (DHCR24) and 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) through sterol-regulatory element-binding protein-2 (SREBP2). While Aβ42-treated SH-SY5Y cells exhibited increased HMGCR expression and c-Jun phosphorylation as pro-inflammatory responses, they also showed down-regulation of neuro-protective/anti-inflammatory DHCR24. These results suggest that Aβ42 may cause insulin resistance, activate JNK for c-Jun phosphorylation, and lead to dysregulation of cholesterol homeostasis, and that enhancing insulin signaling may relieve the insulin-resistant phenotype and the dysregulated cholesterol-synthesis pathway to promote A

  17. A model to estimate insulin sensitivity in dairy cows

    Directory of Open Access Journals (Sweden)

    Holtenius Kjell

    2007-10-01

    Full Text Available Abstract Impairment of the insulin regulation of energy metabolism is considered to be an etiologic key component for metabolic disturbances. Methods for studies of insulin sensitivity thus are highly topical. There are clear indications that reduced insulin sensitivity contributes to the metabolic disturbances that occurs especially among obese lactating cows. Direct measurements of insulin sensitivity are laborious and not suitable for epidemiological studies. We have therefore adopted an indirect method originally developed for humans to estimate insulin sensitivity in dairy cows. The method, "Revised Quantitative Insulin Sensitivity Check Index" (RQUICKI is based on plasma concentrations of glucose, insulin and free fatty acids (FFA and it generates good and linear correlations with different estimates of insulin sensitivity in human populations. We hypothesized that the RQUICKI method could be used as an index of insulin function in lactating dairy cows. We calculated RQUICKI in 237 apparently healthy dairy cows from 20 commercial herds. All cows included were in their first 15 weeks of lactation. RQUICKI was not affected by the homeorhetic adaptations in energy metabolism that occurred during the first 15 weeks of lactation. In a cohort of 24 experimental cows fed in order to obtain different body condition at parturition RQUICKI was lower in early lactation in cows with a high body condition score suggesting disturbed insulin function in obese cows. The results indicate that RQUICKI might be used to identify lactating cows with disturbed insulin function.

  18. Receptor-isoform-selective insulin analogues give tissue-preferential effects

    DEFF Research Database (Denmark)

    Vienberg, Sara Gry; Bouman, Stephan D; Sørensen, Heidi

    2011-01-01

    The relative expression patterns of the two IR (insulin receptor) isoforms, +/- exon 11 (IR-B/IR-A respectively), are tissue-dependent. Therefore we have developed insulin analogues with different binding affinities for the two isoforms to test whether tissue-preferential biological effects can...... be attained. In rats and mice, IR-B is the most prominent isoform in the liver (> 95%) and fat (> 90%), whereas in muscles IR-A is the dominant isoform (> 95%). As a consequence, the insulin analogue INS-A, which has a higher relative affinity for human IR-A, had a higher relative potency [compared with HI...... (human insulin)] for glycogen synthesis in rat muscle strips (26%) than for glycogen accumulation in rat hepatocytes (5%) and for lipogenesis in rat adipocytes (4%). In contrast, the INS-B analogue, which has an increased affinity for human IR-B, had higher relative potencies (compared with HI...

  19. Loss of ABHD15 Impairs the Anti-lipolytic Action of Insulin by Altering PDE3B Stability and Contributes to Insulin Resistance.

    Science.gov (United States)

    Xia, Wenmin; Pessentheiner, Ariane R; Hofer, Dina C; Amor, Melina; Schreiber, Renate; Schoiswohl, Gabriele; Eichmann, Thomas O; Walenta, Evelyn; Itariu, Bianca; Prager, Gerhard; Hackl, Hubert; Stulnig, Thomas; Kratky, Dagmar; Rülicke, Thomas; Bogner-Strauss, Juliane G

    2018-05-15

    Elevated circulating fatty acids (FAs) contribute to obesity-associated metabolic complications, but the mechanisms by which insulin suppresses lipolysis are poorly understood. We show that α/β-hydrolase domain-containing 15 (ABHD15) is required for the anti-lipolytic action of insulin in white adipose tissue (WAT). Neither insulin nor glucose treatments can suppress FA mobilization in global and conditional Abhd15-knockout (KO) mice. Accordingly, insulin signaling is impaired in Abhd15-KO adipocytes, as indicated by reduced AKT phosphorylation, glucose uptake, and de novo lipogenesis. In vitro data reveal that ABHD15 associates with and stabilizes phosphodiesterase 3B (PDE3B). Accordingly, PDE3B expression is decreased in the WAT of Abhd15-KO mice, mechanistically explaining increased protein kinase A (PKA) activity, hormone-sensitive lipase (HSL) phosphorylation, and undiminished FA release upon insulin signaling. Ultimately, Abhd15-KO mice develop insulin resistance. Notably, ABHD15 expression is decreased in humans with obesity and diabetes compared to humans with obesity and normal glucose tolerance, identifying ABHD15 as a potential therapeutic target to mitigate insulin resistance. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Liraglutide, a once-daily human GLP-1 analogue, improves pancreatic B-cell function and arginine-stimulated insulin secretion during hyperglycaemia in patients with Type 2 diabetes mellitus

    DEFF Research Database (Denmark)

    Vilsbøll, Tina; Brock, Birgitte; Perrild, Hans

    2008-01-01

    To assess the effect of liraglutide, a once-daily human glucagon-like peptide-1 analogue on pancreatic B-cell function. methods: Patients with Type 2 diabetes (n = 39) were randomized to treatment with 0.65, 1.25 or 1.9 mg/day liraglutide or placebo for 14 weeks. First- and second-phase insulin...... release were measured by means of the insulin-modified frequently sampled intravenous glucose tolerance test. Arginine-stimulated insulin secretion was measured during a hyperglycaemic clamp (20 mmol/l). Glucose effectiveness and insulin sensitivity were estimated by means of the insulin...

  1. A Novel Genetic Group of Bovine Hepacivirus in Archival Serum Samples from Brazilian Cattle

    Directory of Open Access Journals (Sweden)

    Cláudio W. Canal

    2017-01-01

    Full Text Available Hepatitis C virus (HCV (genus Hepacivirus; family Flaviviridae is a major human pathogen causing persistent infection and hepatic injury. Recently, emerging HCV-like viruses were described infecting wild animals, such as bats and rodents, and domestic animals, including dogs, horses, and cattle. Using degenerate primers for detecting bovine pestiviruses in a 1996 survey three bovine serum samples showed a low identity with the genus Pestivirus of the Flaviviridae family. A virus could not be isolated in cell culture. The description of bovine hepaciviruses (BovHepV in 2015 allowed us to retrospectively identify the sequences as BovHepV, with a 88.9% nucleotide identity. In a reconstructed phylogenetic tree, the Brazilian BovHepV samples grouped within the bovine HCV-like cluster in a separated terminal node that was more closely related to the putative bovine Hepacivirus common ancestor than to bovine hepaciviruses detected in Europe and Africa.

  2. Binding and Endocytosis of Bovine Hololactoferrin by the Parasite Entamoeba histolytica

    Directory of Open Access Journals (Sweden)

    Guillermo Ortíz-Estrada

    2015-01-01

    Full Text Available Entamoeba histolytica is a human parasite that requires iron (Fe for its metabolic function and virulence. Bovine lactoferrin (B-Lf and its peptides can be found in the digestive tract after dairy products are ingested. The aim of this study was to compare virulent trophozoites recently isolated from hamster liver abscesses with nonvirulent trophozoites maintained for more than 30 years in cultures in vitro regarding their interaction with iron-charged B-Lf (B-holo-Lf. We performed growth kinetics analyses of trophozoites in B-holo-Lf and throughout several consecutive transfers. The virulent parasites showed higher growth and tolerance to iron than nonvirulent parasites. Both amoeba variants specifically bound B-holo-Lf with a similar Kd. However, averages of 9.45 × 105 and 6.65 × 106 binding sites/cell were found for B-holo-Lf in nonvirulent and virulent amoebae, respectively. Virulent amoebae bound more efficiently to human and bovine holo-Lf, human holo-transferrin, and human and bovine hemoglobin than nonvirulent amoebae. Virulent amoebae showed two types of B-holo-Lf binding proteins. Although both amoebae endocytosed this glycoprotein through clathrin-coated vesicles, the virulent amoebae also endocytosed B-holo-Lf through a cholesterol-dependent mechanism. Both amoeba variants secreted cysteine proteases cleaving B-holo-Lf. These data demonstrate that the B-Lf endocytosis is more efficient in virulent amoebae.

  3. Binding and Endocytosis of Bovine Hololactoferrin by the Parasite Entamoeba histolytica.

    Science.gov (United States)

    Ortíz-Estrada, Guillermo; Calderón-Salinas, Víctor; Shibayama-Salas, Mineko; León-Sicairos, Nidia; de la Garza, Mireya

    2015-01-01

    Entamoeba histolytica is a human parasite that requires iron (Fe) for its metabolic function and virulence. Bovine lactoferrin (B-Lf) and its peptides can be found in the digestive tract after dairy products are ingested. The aim of this study was to compare virulent trophozoites recently isolated from hamster liver abscesses with nonvirulent trophozoites maintained for more than 30 years in cultures in vitro regarding their interaction with iron-charged B-Lf (B-holo-Lf). We performed growth kinetics analyses of trophozoites in B-holo-Lf and throughout several consecutive transfers. The virulent parasites showed higher growth and tolerance to iron than nonvirulent parasites. Both amoeba variants specifically bound B-holo-Lf with a similar K d . However, averages of 9.45 × 10(5) and 6.65 × 10(6) binding sites/cell were found for B-holo-Lf in nonvirulent and virulent amoebae, respectively. Virulent amoebae bound more efficiently to human and bovine holo-Lf, human holo-transferrin, and human and bovine hemoglobin than nonvirulent amoebae. Virulent amoebae showed two types of B-holo-Lf binding proteins. Although both amoebae endocytosed this glycoprotein through clathrin-coated vesicles, the virulent amoebae also endocytosed B-holo-Lf through a cholesterol-dependent mechanism. Both amoeba variants secreted cysteine proteases cleaving B-holo-Lf. These data demonstrate that the B-Lf endocytosis is more efficient in virulent amoebae.

  4. Attempts to grow human noroviruses, a sapovirus, and a bovine norovirus in vitro.

    Directory of Open Access Journals (Sweden)

    Tomoichiro Oka

    Full Text Available Noroviruses (NoVs and Sapoviruses (SaVs are enteric caliciviruses that have been detected in multiple mammalian species, including humans. Currently, efficient cell culture systems have been established only for murine NoVs and porcine SaV Cowden strain. Establishment of an efficient in vitro cell culture system for other NoVs and SaVs remains challenging; however, human NoV (HuNoV replication in 3D cultured Caco-2 cells and a clone of Caco-2 cells, C2BBe1, human enteroids and in human B cells has been reported. In this study, we tested various cells and culture conditions to grow HuNoVs and a human SaV (HuSaV to test the possibility of the propagation in different cells and culture conditions. We also attempted to grow a bovine NoV (BoNoV in ex vivo organ cultures. We did not observe significant RNA level increases for HuSaV and BoNoV under our test conditions. HuNoV RNA levels increased to a maximum of ~600-fold in long-term Caco-2 cells that were cultured for 1-2 months in multi-well plates and inoculated with HuNoV-positive and bacteria-free human stool suspensions using serum-free medium supplemented with the bile acid, GCDCA. However, this positive result was inconsistent. Our results demonstrated that HuNoVs, BoNoV and HuSaV largely failed to grow in vitro under our test conditions. Our purpose is to share our findings with other researchers with the goal to develop efficient, reproducible simplified and cost-effective culture systems for human and animal NoVs and SaVs in the future.

  5. Sulfated glycosaminoglycans in cultured endothelial cells from capillaries and large vessels of human and bovine origin

    Energy Technology Data Exchange (ETDEWEB)

    Bar, R.S.; Dake, B.L.; Spanheimer, R.G.

    1985-07-01

    The (/sup 35/S)glycosaminoglycans ((/sup 35/S)GAG) synthesized by capillary endothelial cells were analyzed and compared to GAG synthesized by endothelial cells cultured from 4 larger vessels. Two separate cultures of endothelial cells were established from bovine fat capillaries and from 4 larger vessels of human origin (umbilical vein) and bovine origin (pulmonary artery, pulmonary vein and aorta). After incubation with /sup 35/SO/sub 4/ for 72 h, the (/sup 35/S)glycosaminoglycans (GAG) composition of the media, pericellular and cellular fractions of each culture were determined by selective degradation with nitrous acid, chondroitinase ABC and chondroitinase AC. All endothelial cells produced large amounts of (/sup 35/S)GAG with increased proportions of heparinoids (heparan sulfate and heparin) in the cellular and pericellular fractions. Each culture showed a distinct distribution of (/sup 35/S)GAG in the media, pericellular and cellular fractions with several specific differences found among the 5 cultures. The differences in GAG content were confirmed in a second group of separate cultures from each of the 5 vessels indicating that, although having several features of GAG metabolism in common, each endothelial cell culture demonstrated a characteristic complement of synthesized, secreted and cell surface-sulfated glycosaminoglycans. (author). 16 refs.

  6. Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination.

    Science.gov (United States)

    Jeong, Young-Hee; Kim, Yeong Ji; Kim, Eun Young; Kim, Se Eun; Kim, Jiwoo; Park, Min Jee; Lee, Hong-Gu; Park, Se Pill; Kang, Man-Jong

    2016-06-01

    Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine β-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the β-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine β-casein gene.

  7. Repopulation of the atrophied thymus in diabetic rats by insulin-like growth factor I

    International Nuclear Information System (INIS)

    Binz, K.; Joller, P.; Froesch, P.; Binz, H.; Zapf, J.; Froesch, E.R.

    1990-01-01

    Atrophy of the thymus is one of the consequences of severe insulin deficiency. The authors describe here that the weight and the architecture of the thymus of diabetic rats is restored towards normal not only by insulin but also by insulin-like growth factor I (IGF-I) treatment. In contrast to insulin, this effect of IGF-I occurs despite persisting hyperglycemia and adrenal hyperplasia. They also investigated the in vivo effect of IGF-I on replication and differentiation of thymocytes from streptozotocin-induced diabetic rats. Thymocytes from diabetic rats incorporated less [ 3 H]thymidine than did thymocytes from healthy rats. Insulin, as well as IGF-I treatment of diabetic rats increased [ 3 H]thymidine incorporation by thymocytes. Flow cytometry of thymocytes labeled with monoclonal antibodies revealed a decreased expression of the Thy-1 antigen in diabetic rats compared with control rats. In addition, a major deficiency of thymocytes expressing simultaneously the W3/25 and the Ox8 antigens was observed. These changes were restored towards normal by insulin as well as by IGF-I treatment. The antibody response to a T cell-dependent antigen (bovine serum albumin) was comparable in normal and diabetic rats. They conclude that IGF-I has important effects on the thymocyte number and the presence of CD4 + /CD8 + immature cells in the thymus of diabetic rats despite persisting hyperglycemia. However, helper T-cell function for antibody production appears to be preserved even in the severely diabetic state

  8. Effects of exogenous human insulin dose adjustment on body mass ...

    African Journals Online (AJOL)

    glycaemic control by frequent exogenous insulin injections. To maintain fasting ... mass index in adult patients with type 1 diabetes mellitus at Kalafong Hospital ..... The Diabetes Control and Complications Trial cited in the review by Kaufman[2] also .... in obese insulin-resistant children: A randomized clinical trial. Diabetes ...

  9. Immunoassay of serum polypeptide hormones by using 125I-labelled anti(-immunoglobulin G) antibodies.

    Science.gov (United States)

    Beck, P; Nicholas, H

    1975-03-01

    1. A technique for indirectly labelling antibodies to polypeptide hormones, by combining them with radioactively labelled anti-(immunoglobulin G) is described. (a) 125I-labelled anti-(rabbit immunoglobulin G) and anti-(guinea-pig immunoglobulin G) antibodies with high specific radioactivity were prepared after purification of the antibodies on immunoadsorbents containing the respective antigens. (b) Rabbit immunoglobulin G antibodies to human growth hormone, porcine glucagon and guinea-pig immunoglobulin G antibodies to bovine insulin and bovine parathyroid hormone were combined with immunoadsorbents containing the respective polypeptide hormone antigen. (c) The immunoglobulin G antibodies to the polypeptide hormones were reacted with 125-I-labelled anti-(immunoglobulin G) antibodies directed against the appropriate species of immunoglobulin G,and the anti-hormone antibodies were combined with the hormone-containing immunoadsorbent. (d) 125I-labelled anti-(immunoglobulin G) antibodies and anti-hormone antibodies were simultaneously eluted from the hormone-containing immunoadsorbent by dilute HCl, pH 2.0. After elution the anti-(immunoglobulin G) antibodies and antihormone antibodies were allowed to recombine at pH 8.0 and 4 degrees C. 2. The resultant immunoglobulin G-anti-immunoglobulin G complex was used in immunoradiometric (labelled antibody) and two-site assays of the respective polypeptide hormone. 3. By using these immunoassays, concentrations down to 90pg of human growth hormone/ml, 100 pg of bovine insulin/ml, 80 pg of bovine parathyroid hormone/ml and 150 pg of glucagon/ml were readily detected. Assays of human plasma for growth hormone and insulin by these methods showed good agreement with results obtained by using a directly 125I-labelled anti-hormone antibody in an immunoradiometric assay of human growth hormone or by radioimmunoassay of human insulin. 4. The method described allows immunoradiometric or two-site assays to be performed starting with as

  10. Biopharmaceutical characterisation of insulin and recombinant human growth hormone loaded lipid submicron particles produced by supercritical gas micro-atomisation.

    Science.gov (United States)

    Salmaso, Stefano; Bersani, Sara; Elvassore, Nicola; Bertucco, Alberto; Caliceti, Paolo

    2009-09-08

    Homogeneous dispersions of insulin and recombinant human growth hormone (rh-GH) in tristearin/phosphatidylcholine/PEG mixtures (1.3:1.3:0.25:0.15 w/w ratio) were processed by supercritical carbon dioxide gas micro-atomisation to produce protein-loaded lipid particles. The process yielded spherical particles, with a 197+/-94 nm mean diameter, and the insulin and rh-GH recovery in the final product was 57+/-8% and 48+/-5%, respectively. In vitro, the proteins were slowly released for about 70-80 h according to a diffusive mechanism. In vivo, the insulin and glucose profiles in plasma obtained by subcutaneous administration of a dose of particles containing 2 microg insulin to diabetic mice overlapped that obtained with 2 microg of insulin in solution. Administration of a dose of particles containing 5 microg insulin resulted in faster and longer glycaemia reduction. Oral administration of 20 and 50 microg insulin equivalent particles produced a significant hypoglycaemic effect. The glucose levels decreased since 2h after administration, reaching about 50% and 70% glucose reduction in 1-2h with the lower and higher dose, respectively. As compared to subcutaneous administration, the relative pharmacological bioavailability obtained with 20 and 50 microg equivalent insulin particles was 7.7% and 6.7%, respectively. Daily subcutaneous administration of 40 microg of rh-GH-loaded particles to hypophysectomised rats induced similar body weight increase as 40 microg rh-GH in solution. The daily oral administration of 400 microg rh-GH equivalent particles elicited a slight body weight increase, which corresponded to a relative pharmacological bioavailability of 3.4% compared to subcutaneous administration.

  11. Lipid droplets hypertrophy: a crucial determining factor in insulin regulation by adipocytes

    Science.gov (United States)

    Sanjabi, Bahram; Dashty, Monireh; Özcan, Behiye; Akbarkhanzadeh, Vishtaseb; Rahimi, Mehran; Vinciguerra, Manlio; van Rooij, Felix; Al-Lahham, Saad; Sheedfar, Fareeba; van Kooten, Theo G.; Spek, C. Arnold; Rowshani, Ajda T.; van der Want, Johannes; Klaassen, Rene; Sijbrands, Eric; Peppelenbosch, Maikel P.; Rezaee, Farhad

    2015-03-01

    Lipid droplets (LDs) hypertrophy in adipocytes is the main cause of energy metabolic system dysfunction, obesity and its afflictions such as T2D. However, the role of adipocytes in linking energy metabolic disorders with insulin regulation is unknown in humans. Human adipocytes constitutively synthesize and secrete insulin, which is biologically functional. Insulin concentrations and release are fat mass- and LDs-dependent respectively. Fat reduction mediated by bariatric surgery repairs obesity-associated T2D. The expression of genes, like PCSK1 (proinsulin conversion enzyme), GCG (Glucagon), GPLD1, CD38 and NNAT, involved in insulin regulation/release were differentially expressed in pancreas and adipose tissue (AT). INS (insulin) and GCG expression reduced in human AT-T2D as compared to AT-control, but remained unchanged in pancreas in either state. Insulin levels (mRNA/protein) were higher in AT derived from prediabetes BB rats with destructed pancreatic β-cells and controls than pancreas derived from the same rats respectively. Insulin expression in 10 human primary cell types including adipocytes and macrophages is an evidence for extrapancreatic insulin-producing cells. The data suggest a crosstalk between AT and pancreas to fine-tune energy metabolic system or may minimize the metabolic damage during diabetes. This study opens new avenues towards T2D therapy with a great impact on public health.

  12. A Review of Basal-Bolus Therapy Using Insulin Glargine and Insulin Lispro in the Management of Diabetes Mellitus.

    Science.gov (United States)

    Candido, Riccardo; Wyne, Kathleen; Romoli, Ester

    2018-04-13

    Basal-bolus therapy (BBT) refers to the combination of a long-acting basal insulin with a rapid-acting insulin at mealtimes. Basal insulin glargine 100 U/mL and prandial insulin lispro have been available for many years and there is a substantial evidence base to support the efficacy and safety of these agents when they are used in BBT or basal-plus therapy for patients with type 1 or type 2 diabetes mellitus (T1DM, T2DM). With the growing availability of alternative insulins for use in such regimens, it seems timely to review the data regarding BBT with insulin glargine 100 U/mL and insulin lispro. In patients with T1DM, BBT with insulin glargine plus insulin lispro provides similar or better glycemic control and leads to less nocturnal hypoglycemia compared to BBT using human insulin as the basal and/or prandial component, and generally provides similar glycemic control and rates of severe hypoglycemia to those achieved with insulin lispro administered by continuous subcutaneous insulin infusion (CSII). Studies evaluating BBT with insulin glargine plus insulin lispro in patients with T2DM also demonstrate the efficacy and safety of these insulins. Available data suggest that BBT with insulin glargine and insulin lispro provides similar levels of efficacy and safety in pediatric and adult populations with T1DM and in adult patients and those aged more than 65 years with T2DM. These insulin preparations also appear to be safe and effective for controlling T2DM in people of different ethnicities and in patients with T1DM or T2DM and comorbidities. Eli Lilly and Company.

  13. The effects of injection of bovine vaccine into a human digit: a case report

    Directory of Open Access Journals (Sweden)

    Ricketts David M

    2005-10-01

    Full Text Available Abstract Background The incidence of needlestick injuries in farmers and veterinary surgeons is significant and the consequences of such an injection can be serious. Case presentation We report accidental injection of bovine vaccine into the base of the little finger. This resulted in increased pressure in the flexor sheath causing signs and symptoms of ischemia. Amputation of the digit was required despite repeated surgical debridement and decompression. Conclusion There have been previous reports of injection of oil-based vaccines into the human hand resulting in granulomatous inflammation or sterile abscess and causing morbidity and tissue loss. Self-injection with veterinary vaccines is an occupational hazard for farmers and veterinary surgeons. Injection of vaccine into a closed compartment such as the human finger can have serious sequelae including loss of the injected digit. These injuries are not to be underestimated. Early debridement and irrigation of the injected area with decompression is likely to give the best outcome. Frequent review is necessary after the first procedure because repeat operations may be required.

  14. Human conglutinin-like protein

    DEFF Research Database (Denmark)

    Jensenius, J C; Thiel, S; Baatrup, G

    1985-01-01

    The presence in human plasma of a molecule homologous to bovine conglutinin is indicated by the results of biological and immunochemical analysis. The human conglutinin-like protein shows calcium-dependent binding to complement-treated solid phase IgG and immunological cross-reaction with chicken...... anti-bovine conglutinin. The binding of the human protein to complement-treated IgG was inhibited by N-acetyl-D-glucosamine but not by other sugars. Analysis by SDS-PAGE and Western blotting showed reaction of anti-conglutinin with molecules of similar mobility to the monomer and hexamer of bovine...

  15. Dimethylarginine Dimethylaminohydrolase Overexpression enhances Insulin Sensitivity

    Science.gov (United States)

    Sydow, Karsten; Mondon, Carl E.; Schrader, Joerg; Konishi, Hakuoh; Cooke, John P.

    2011-01-01

    Objective Previous studies suggest that nitric oxide (NO) may modulate insulin-induced uptake of glucose in insulin-sensitive tissues. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase (NOS). We hypothesized that a reduction in endogenous ADMA would increase NO synthesis and thereby enhance insulin sensitivity. Methods and Results To test this hypothesis we employed a transgenic mouse in which we overexpressed human dimethylarginine dimethylaminohydrolase (DDAH-I). The DDAH-I mice had lower plasma ADMA at all ages (22–70 weeks) by comparison to wild-type (WT) littermates. With a glucose challenge, WT mice showed a prompt increase in ADMA, whereas DDAH-I mice had a blunted response. Furthermore, DDAH-I mice had a blunted increase in plasma insulin and glucose levels after glucose challenge, with a 50% reduction in the insulin resistence index, consistent with enhanced sensitivity to insulin. In liver, we observed an increased Akt phosphorylation in the DDAH-I mice after i.p. glucose challenge. Incubation of skeletal muscle from WT mice ex vivo with ADMA (2μM) markedly suppressed insulin-induced glycogen synthesis in fast-twitch but not slow-twitch muscle. Conclusions These findings suggest that the endogenous NOS inhibitor ADMA reduces insulin sensitivity, consistent with previous observations that NO plays a role in insulin sensitivity. PMID:18239148

  16. Anti-Bovine Programmed Death-1 Rat–Bovine Chimeric Antibody for Immunotherapy of Bovine Leukemia Virus Infection in Cattle

    Directory of Open Access Journals (Sweden)

    Tomohiro Okagawa

    2017-06-01

    Full Text Available Blockade of immunoinhibitory molecules, such as programmed death-1 (PD-1/PD-ligand 1 (PD-L1, is a promising strategy for reinvigorating exhausted T cells and preventing disease progression in a variety of chronic infections. Application of this therapeutic strategy to cattle requires bovinized chimeric antibody targeting immunoinhibitory molecules. In this study, anti-bovine PD-1 rat–bovine chimeric monoclonal antibody 5D2 (Boch5D2 was constructed with mammalian expression systems, and its biochemical function and antiviral effect were characterized in vitro and in vivo using cattle infected with bovine leukemia virus (BLV. Purified Boch5D2 was capable of detecting bovine PD-1 molecules expressed on cell membranes in flow cytometric analysis. In particular, Biacore analysis determined that the binding affinity of Boch5D2 to bovine PD-1 protein was similar to that of the original anti-bovine PD-1 rat monoclonal antibody 5D2. Boch5D2 was also capable of blocking PD-1/PD-L1 binding at the same level as 5D2. The immunomodulatory and therapeutic effects of Boch5D2 were evaluated by in vivo administration of the antibody to a BLV-infected calf. Inoculated Boch5D2 was sustained in the serum for a longer period. Boch5D2 inoculation resulted in activation of the proliferation of BLV-specific CD4+ T cells and decrease in the proviral load of BLV in the peripheral blood. This study demonstrates that Boch5D2 retains an equivalent biochemical function to that of the original antibody 5D2 and is a candidate therapeutic agent for regulating antiviral immune response in vivo. Clinical efficacy of PD-1/PD-L1 blockade awaits further experimentation with a large number of animals.

  17. Pooled human platelet lysate versus fetal bovine serum—investigating the proliferation rate, chromosome stability and angiogenic potential of human adipose tissue-derived stem cells intended for clinical use

    DEFF Research Database (Denmark)

    Trojahn Kølle, Stig-Frederik; Oliveri, Roberto S; Glovinski, Peter V

    2013-01-01

    Because of an increasing focus on the use of adipose-derived stem cells (ASCs) in clinical trials, the culture conditions for these cells are being optimized. We compared the proliferation rates and chromosomal stability of ASCs that had been cultured in Dulbecco's modified Eagle's Medium (DMEM) ......) supplemented with either pooled human platelet lysate (pHPL) or clinical-grade fetal bovine serum (FBS) (DMEM(pHPL) versus DMEM(FBS))....

  18. The interplay between noncoding RNAs and insulin in diabetes.

    Science.gov (United States)

    Tian, Yan; Xu, Jia; Du, Xiao; Fu, Xianghui

    2018-04-10

    Noncoding RNAs (ncRNAs), including microRNAs, long noncoding RNAs and circular RNAs, regulate various biological processes and are involved in the initiation and progression of human diseases. Insulin, a predominant hormone secreted from pancreatic β cells, is an essential factor in regulation of systemic metabolism through multifunctional insulin signaling. Insulin production and action are tightly controlled. Dysregulations of insulin production and action can impair metabolic homeostasis, and eventually lead to the development of multiple metabolic diseases, especially diabetes. Accumulating data indicates that ncRNAs modulate β cell mass, insulin synthesis, secretion and signaling, and their role in diabetes is dramatically emerging. This review summarizes our current knowledge of ncRNAs as regulators of insulin, with particular emphasis on the implications of this interplay in the development of diabetes. We outline the role of ncRNAs in pancreatic β cell mass and function, which is critical for insulin production and secretion. We also highlight the involvement of ncRNAs in insulin signaling in peripheral tissues including liver, muscle and adipose, and discuss ncRNA-mediated inter-organ crosstalk under diabetic conditions. A more in-depth understanding of the interplay between ncRNAs and insulin may afford valuable insights and novel therapeutic strategies for treatment of diabetes, as well as other human diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Processed bovine cartilage: an improved biosynthetic implant for contour defects

    International Nuclear Information System (INIS)

    Ersek, R.A.; Hart, W.G. Jr.; Greer, D.; Beisang, A.A.; Flynn, P.J.; Denton, D.R.

    1984-01-01

    Irradiated human cartilage has been found to be a superior implant material for correction of contour defects; however, availability problems have prevented this material from gaining wide acceptance. Implantation of processed irradiated bovine cartilage in primates and rabbits, as described here, provides strong evidence that this material performs like irradiated allograft cartilage antigenically and has certain cosmetic advantages over allograft cartilage. Our studies in primates have shown that there is no systemically measurable antibody-antigen reaction, either cellular or noncellular, to irradiated processed bovine cartilage. Neither primary nor second-set provocative implantations produced any measurable rejection. In rabbits, composite grafts of two pieces of irradiated bovine cartilage adjacent to each other were also well tolerated, with no measurable absorption and with capsule formation typical of a foreign body reaction to an inert object

  20. SP-A binding sites on bovine alveolar macrophages.

    Science.gov (United States)

    Plaga, S; Plattner, H; Schlepper-Schaefer, J

    1998-11-25

    Surfactant protein A (SP-A) binding to bovine alveolar macrophages was examined in order to characterize SP-A binding proteins on the cell surface and to isolate putative receptors from these cells that could be obtained in large amounts. Human SP-A, unlabeled or labeled with gold particles, was bound to freshly isolated macrophages and analyzed with ELISA or the transmission electron microscope. Binding of SP-A was inhibited by Ca2+ chelation, by an excess of unlabeled SP-A, or by the presence of 20 mg/ml mannan. We conclude that bovine alveolar macrophages expose binding sites for SP-A that are specific and that depend on Ca2+ and on mannose residues. For isolation of SP-A receptors with homologous SP-A as ligand we isolated SP-A from bovine lung lavage. SDS-PAGE analysis of the purified SP-A showed a protein of 32-36 kDa. Functional integrity of the protein was demonstrated. Bovine SP-A bound to Dynabeads was used to isolate SP-A binding proteins. From the fractionated and blotted proteins of the receptor preparation two proteins bound SP-A in a Ca2+-dependent manner, a 40-kDa protein showing mannose dependency and a 210-kDa protein, showing no mannose sensitivity. Copyright 1998 Academic Press.

  1. Thermal dissociation and unfolding of insulin

    DEFF Research Database (Denmark)

    Huus, Kasper; Havelund, Svend; Olsen, Helle B

    2005-01-01

    The thermal stability of human insulin was studied by differential scanning microcalorimetry and near-UV circular dichroism as a function of zinc/protein ratio, to elucidate the dissociation and unfolding processes of insulin in different association states. Zinc-free insulin, which is primarily...... dimeric at room temperature, unfolded at approximately 70 degrees C. The two monomeric insulin mutants Asp(B28) and Asp(B9),Glu(B27) unfolded at higher temperatures, but with enthalpies of unfolding that were approximately 30% smaller. Small amounts of zinc caused a biphasic thermal denaturation pattern...... of insulin. The biphasic denaturation is caused by a redistribution of zinc ions during the heating process and results in two distinct transitions with T(m)'s of approximately 70 and approximately 87 degrees C corresponding to monomer/dimer and hexamer, respectively. At high zinc concentrations (>or=5 Zn(2...

  2. Ultra-high resolution X-ray structures of two forms of human recombinant insulin at 100 K.

    Science.gov (United States)

    Lisgarten, David R; Palmer, Rex A; Lobley, Carina M C; Naylor, Claire E; Chowdhry, Babur Z; Al-Kurdi, Zakieh I; Badwan, Adnan A; Howlin, Brendan J; Gibbons, Nicholas C J; Saldanha, José W; Lisgarten, John N; Basak, Ajit K

    2017-08-01

    The crystal structure of a commercially available form of human recombinant (HR) insulin, Insugen (I), used in the treatment of diabetes has been determined to 0.92 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16,000 keV (λ = 0.77 Å). Refinement carried out with anisotropic displacement parameters, removal of main-chain stereochemical restraints, inclusion of H atoms in calculated positions, and 220 water molecules, converged to a final value of R = 0.1112 and R free  = 0.1466. The structure includes what is thought to be an ordered propanol molecule (POL) only in chain D(4) and a solvated acetate molecule (ACT) coordinated to the Zn atom only in chain B(2). Possible origins and consequences of the propanol and acetate molecules are discussed. Three types of amino acid representation in the electron density are examined in detail: (i) sharp with very clearly resolved features; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry; (iii) poor density and difficult or impossible to model. An example of type (ii) is observed for the intra-chain disulphide bridge in chain C(3) between Sγ6-Sγ11 which has two clear conformations with relative refined occupancies of 0.8 and 0.2, respectively. In contrast the corresponding S-S bridge in chain A(1) shows one clearly defined conformation. A molecular dynamics study has provided a rational explanation of this difference between chains A and C. More generally, differences in the electron density features between corresponding residues in chains A and C and chains B and D is a common observation in the Insugen (I) structure and these effects are discussed in detail. The crystal structure, also at 0.92 Å and 100 K, of a second commercially available form of human recombinant insulin, Intergen (II), deposited in the Protein Data Bank as 3W7Y which remains otherwise unpublished is compared here

  3. Insulin Resistance Induced by Short term Fructose Feeding may not ...

    African Journals Online (AJOL)

    Fructose feeding causes insulin resistance and invariably Non-Insulin Dependent Diabetes Mellitus (NIDDM) in rats and genetically predisposed humans. The effect of insulin resistance induced by short term fructose feeding on fertility in female rats was investigated using the following parameters: oestrous phase and ...

  4. The relationship between bone turnover and insulin sensitivity and secretion

    DEFF Research Database (Denmark)

    Frost, Morten; Balkau, Beverley; Hatunic, Mensud

    2018-01-01

    Bone metabolism appears to influence insulin secretion and sensitivity, and insulin promotes bone formation in animals, but similar evidence in humans is limited. The objectives of this study are to explore if bone turnover markers were associated with insulin secretion and sensitivity and to det...

  5. Eccentric exercise decreases maximal insulin action in humans

    DEFF Research Database (Denmark)

    Asp, Svend; Daugaard, J R; Kristiansen, S

    1996-01-01

    subjects participated in two euglycaemic clamps, performed in random order. One clamp was preceded 2 days earlier by one-legged eccentric exercise (post-eccentric exercise clamp (PEC)) and one was without the prior exercise (control clamp (CC)). 2. During PEC the maximal insulin-stimulated glucose uptake...... for all three clamp steps used (P maximal activity of glycogen synthase was identical in the two thighs for all clamp steps. 3. The glucose infusion rate (GIR......) necessary to maintain euglycaemia during maximal insulin stimulation was lower during PEC compared with CC (15.7%, 81.3 +/- 3.2 vs. 96.4 +/- 8.8 mumol kg-1 min-1, P maximal...

  6. Design and in vivo evaluation of a patch delivery system for insulin based on thiolated polymers.

    Science.gov (United States)

    Grabovac, Vjera; Föger, Florian; Bernkop-Schnürch, Andreas

    2008-02-04

    The aim of this study was to develop and evaluate a novel three-layered oral delivery system for insulin in vivo. The patch system consisted of a mucoadhesive layer, a water insoluble backing layer made of ethylcellulose and an enteric coating made of Eudragit. Drug release studies were performed in media mimicking stomach and intestinal fluids. For in vivo studies patch systems were administered orally to conscious non-diabetic rats. Orally administered insulin in aqueous solution was used as control. After the oral administration of the patch systems a decrease of glucose and increase of insulin blood levels were measured. The mucoadhesive layer, exhibiting a diameter of 2.5mm and a weight of 5mg, comprised polycarbophil-cysteine conjugate (49%), bovine insulin (26%), gluthatione (5%) and mannitol (20%). 74.8+/-4.8% of insulin was released from the delivery system over 6h. Six hours after administration of the patch system mean maximum decrease of blood glucose level of 31.6% of the initial value could be observed. Maximum insulin concentration in blood was 11.3+/-6.2ng/ml and was reached 6h after administration. The relative bioavailability of orally administered patch system versus subcutaneous injection was 2.2%. The results indicate that the patch system provides enhancement of intestinal absorption and thereby offers a promising strategy for peroral peptide delivery.

  7. Acclimation temperature affects the metabolic response of amphibian skeletal muscle to insulin.

    Science.gov (United States)

    Petersen, Ann M; Gleeson, Todd T

    2011-09-01

    Frog skeletal muscle mainly utilizes the substrates glucose and lactate for energy metabolism. The goal of this study was to determine the effect of insulin on the uptake and metabolic fate of lactate and glucose at rest in skeletal muscle of the American bullfrog, Lithobates catesbeiana, under varying temperature regimens. We hypothesize that lactate and glucose metabolic pathways will respond differently to the presence of insulin in cold versus warm acclimated frog tissues, suggesting an interaction between temperature and metabolism under varying environmental conditions. We employed radiolabeled tracer techniques to measure in vitro uptake, oxidation, and incorporation of glucose and lactate into glycogen by isolated muscles from bullfrogs acclimated to 5 °C (cold) or 25 °C (warm). Isolated bundles from Sartorius muscles were incubated at 5 °C, 15 °C, or 25 °C, and in the presence and absence of 0.05 IU/mL bovine insulin. Insulin treatment in the warm acclimated and incubated frogs resulted in an increase in glucose incorporation into glycogen, and an increase in intracellular [glucose] of 0.5 μmol/g (Pmuscle. When compared to the warm treatment group, cold acclimation and incubation resulted in increased rates of glucose oxidation and glycogen synthesis, and a reduction in free intracellular glucose levels (Pmuscles from either acclimation group were incubated at an intermediate temperature of 15 °C, insulin's effect on substrate metabolism was attenuated or even reversed. Therefore, a significant interaction between insulin and acclimation condition in controlling skeletal muscle metabolism appears to exist. Our findings further suggest that one of insulin's actions in frog muscle is to increase glucose incorporation into glycogen, and to reduce reliance on lactate as the primary metabolic fuel. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Hypoglycemia in type 1 diabetic pregnancy: role of preconception insulin aspart treatment in a randomized study

    DEFF Research Database (Denmark)

    Heller, Simon; Damm, Peter; Mersebach, Henriette

    2010-01-01

    OBJECTIVE A recent randomized trial compared prandial insulin aspart (IAsp) with human insulin in type 1 diabetic pregnancy. The aim of this exploratory analysis was to investigate the incidence of severe hypoglycemia during pregnancy and compare women enrolled preconception with women enrolled...... during early pregnancy. RESEARCH DESIGN AND METHODS IAsp administered immediately before each meal was compared with human insulin administered 30 min before each meal in 99 subjects (44 to IAsp and 55 to human insulin) randomly assigned preconception and in 223 subjects (113 for IAsp and 110 for human...... insulin) randomly assigned in early pregnancy (...

  9. Human circulating monocytes internalize 125I-insulin in a similar fashion to rat hepatocytes: relevance to receptor regulation in target and nontarget tissues

    International Nuclear Information System (INIS)

    Grunberger, G.; Robert, A.; Carpentier, J.L.; Dayer, J.M.; Roth, A.; Stevenson, H.C.; Orci, L.; Gorden, P.

    1985-01-01

    Circulating monocytes bind 125 I-insulin in a specific fashion and have been used to analyze the ambient receptor status in humans. When freshly isolated circulating monocytes are incubated with 125 I-insulin and examined by electron microscopic autoradiography, approximately 18% of the labeled material is internalized after 15 minutes at 37 degrees C. By 2 hours at 37 degrees C, approximately one half of the 125 I-insulin is internalized. Internalization occurs also at 15 degrees C but at a slower rate. Furthermore, the monocytes bind and internalize 125 I-insulin in a manner that mirrors that of major target tissues, such as rat hepatocytes. These data suggest that the insulin receptor of the circulating monocyte might be regulated by adsorptive endocytosis in a manner analogous to that of target tissue, such as the liver

  10. Nutritional Modulation of Insulin Resistance

    Directory of Open Access Journals (Sweden)

    Martin O. Weickert

    2012-01-01

    Full Text Available Insulin resistance has been proposed as the strongest single predictor for the development of Type 2 Diabetes (T2DM. Chronic oversupply of energy from food, together with inadequate physical activity, have been recognized as the most relevant factors leading to overweight, abdominal adiposity, insulin resistance, and finally T2DM. Conversely, energy reduced diets almost invariably to facilitate weight loss and reduce abdominal fat mass and insulin resistance. However, sustained weight loss is generally difficult to achieve, and distinct metabolic characteristics in patients with T2DM further compromise success. Therefore, investigating the effects of modulating the macronutrient composition of isoenergetic diets is an interesting concept that may lead to additional important insights. Metabolic effects of various different dietary concepts and strategies have been claimed, but results from randomized controlled studies and particularly from longer-term-controlled interventions in humans are often lacking. However, some of these concepts are supported by recent research, at least in animal models and short-term studies in humans. This paper provides an update of the current literature regarding the role of nutrition in the modulation of insulin resistance, which includes the discussion of weight-loss-independent metabolic effects of commonly used dietary concepts.

  11. Human placental growth hormone, insulin-like growth factor I and -II, and insulin requirements during pregnancy in type 1 diabetes

    DEFF Research Database (Denmark)

    Fuglsang, Jens; Lauszus, Finn; Flyvbjerg, Allan

    2003-01-01

    between hPGH and IGF-I in type 1 diabetes mellitus has not been investigated thoroughly. Furthermore, hPGH may be involved in the development of insulin resistance during pregnancy. In this prospective, longitudinal study, 51 type 1 diabetic subjects were followed with repeated blood sampling during...... pregnancy in type 1 diabetic subjects could not be related to hPGH levels.......Human placental GH (hPGH) replaces pituitary GH during pregnancy. hPGH is correlated to serum IGF-I in normal pregnancies and in pregnancies complicated by fetoplacental disorders. In gestational diabetes and type 2 diabetes no correlation between hPGH and IGF-I has been found. The relationship...

  12. Neurotrophin Signaling Is Required for Glucose-Induced Insulin Secretion.

    Science.gov (United States)

    Houtz, Jessica; Borden, Philip; Ceasrine, Alexis; Minichiello, Liliana; Kuruvilla, Rejji

    2016-11-07

    Insulin secretion by pancreatic islet β cells is critical for glucose homeostasis, and a blunted β cell secretory response is an early deficit in type 2 diabetes. Here, we uncover a regulatory mechanism by which glucose recruits vascular-derived neurotrophins to control insulin secretion. Nerve growth factor (NGF), a classical trophic factor for nerve cells, is expressed in pancreatic vasculature while its TrkA receptor is localized to islet β cells. High glucose rapidly enhances NGF secretion and increases TrkA phosphorylation in mouse and human islets. Tissue-specific deletion of NGF or TrkA, or acute disruption of TrkA signaling, impairs glucose tolerance and insulin secretion in mice. We show that internalized TrkA receptors promote insulin granule exocytosis via F-actin reorganization. Furthermore, NGF treatment augments glucose-induced insulin secretion in human islets. These findings reveal a non-neuronal role for neurotrophins and identify a new regulatory pathway in insulin secretion that can be targeted to ameliorate β cell dysfunction. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. From Human Mesenchymal Stem Cells to Insulin-Producing Cells: Comparison between Bone Marrow- and Adipose Tissue-Derived Cells.

    Science.gov (United States)

    Gabr, Mahmoud M; Zakaria, Mahmoud M; Refaie, Ayman F; Abdel-Rahman, Engy A; Reda, Asmaa M; Ali, Sameh S; Khater, Sherry M; Ashamallah, Sylvia A; Ismail, Amani M; Ismail, Hossam El-Din A; El-Badri, Nagwa; Ghoneim, Mohamed A

    2017-01-01

    The aim of this study is to compare human bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs), for their differentiation potentials to form insulin-producing cells. BM-MSCs were obtained during elective orthotopic surgery and AT-MSCs from fatty aspirates during elective cosmetics procedures. Following their expansion, cells were characterized by phenotyping, trilineage differentiation ability, and basal gene expression of pluripotency genes and for their metabolic characteristics. Cells were differentiated according to a Trichostatin-A based protocol. The differentiated cells were evaluated by immunocytochemistry staining for insulin and c-peptide. In addition the expression of relevant pancreatic endocrine genes was determined. The release of insulin and c-peptide in response to a glucose challenge was also quantitated. There were some differences in basal gene expression and metabolic characteristics. After differentiation the proportion of the resulting insulin-producing cells (IPCs), was comparable among both cell sources. Again, there were no differences neither in the levels of gene expression nor in the amounts of insulin and c-peptide release as a function of glucose challenge. The properties, availability, and abundance of AT-MSCs render them well-suited for applications in regenerative medicine. Conclusion . BM-MSCs and AT-MSCs are comparable regarding their differential potential to form IPCs. The availability and properties of AT-MSCs render them well-suited for applications in regenerative medicine.

  14. Concentrated insulins: the new basal insulins

    Directory of Open Access Journals (Sweden)

    Lamos EM

    2016-03-01

    Full Text Available Elizabeth M Lamos,1 Lisa M Younk,2 Stephen N Davis3 1Division of Endocrinology, Diabetes and Nutrition, 2Department of Medicine, University of Maryland School of Medicine, 3Department of Medicine, University of Maryland Medical Center, Baltimore, MD, USA Introduction: Insulin therapy plays a critical role in the treatment of type 1 and type 2 diabetes mellitus. However, there is still a need to find basal insulins with 24-hour coverage and reduced risk of hypoglycemia. Additionally, with increasing obesity and insulin resistance, the ability to provide clinically necessary high doses of insulin at low volume is also needed. Areas covered: This review highlights the published reports of the pharmacokinetic (PK and glucodynamic properties of concentrated insulins: Humulin-R U500, insulin degludec U200, and insulin glargine U300, describes the clinical efficacy, risk of hypoglycemic, and metabolic changes observed, and finally, discusses observations about the complexity of introducing a new generation of concentrated insulins to the therapeutic market. Conclusion: Humulin-R U500 has a similar onset but longer duration of action compared with U100 regular insulin. Insulin glargine U300 has differential PK/pharmacodynamic effects when compared with insulin glargine U100. In noninferiority studies, glycemic control with degludec U200 and glargine U300 is similar to insulin glargine U100 and nocturnal hypoglycemia is reduced. Concentrated formulations appear to behave as separate molecular entities when compared with earlier U100 insulin analog compounds. In the review of available published data, newer concentrated basal insulins may offer an advantage in terms of reduced intraindividual variability as well as reducing the injection burden in individuals requiring high-dose and large volume insulin therapy. Understanding the PK and pharmacodynamic properties of this new generation of insulins is critical to safe dosing, dispensing, and administration

  15. Insulin Receptor Substrate 2 Is a Negative Regulator of Memory Formation

    Science.gov (United States)

    Irvine, Elaine E.; Drinkwater, Laura; Radwanska, Kasia; Al-Qassab, Hind; Smith, Mark A.; O'Brien, Melissa; Kielar, Catherine; Choudhury, Agharul I.; Krauss, Stefan; Cooper, Jonathan D.; Withers, Dominic J.; Giese, Karl Peter

    2011-01-01

    Insulin has been shown to impact on learning and memory in both humans and animals, but the downstream signaling mechanisms involved are poorly characterized. Insulin receptor substrate-2 (Irs2) is an adaptor protein that couples activation of insulin- and insulin-like growth factor-1 receptors to downstream signaling pathways. Here, we have…

  16. Expression and functional assessment of candidate type 2 diabetes susceptibility genes identify four new genes contributing to human insulin secretion

    Directory of Open Access Journals (Sweden)

    Fatou K. Ndiaye

    2017-06-01

    Full Text Available Objectives: Genome-wide association studies (GWAS have identified >100 loci independently contributing to type 2 diabetes (T2D risk. However, translational implications for precision medicine and for the development of novel treatments have been disappointing, due to poor knowledge of how these loci impact T2D pathophysiology. Here, we aimed to measure the expression of genes located nearby T2D associated signals and to assess their effect on insulin secretion from pancreatic beta cells. Methods: The expression of 104 candidate T2D susceptibility genes was measured in a human multi-tissue panel, through PCR-free expression assay. The effects of the knockdown of beta-cell enriched genes were next investigated on insulin secretion from the human EndoC-βH1 beta-cell line. Finally, we performed RNA-sequencing (RNA-seq so as to assess the pathways affected by the knockdown of the new genes impacting insulin secretion from EndoC-βH1, and we analyzed the expression of the new genes in mouse models with altered pancreatic beta-cell function. Results: We found that the candidate T2D susceptibility genes' expression is significantly enriched in pancreatic beta cells obtained by laser capture microdissection or sorted by flow cytometry and in EndoC-βH1 cells, but not in insulin sensitive tissues. Furthermore, the knockdown of seven T2D-susceptibility genes (CDKN2A, GCK, HNF4A, KCNK16, SLC30A8, TBC1D4, and TCF19 with already known expression and/or function in beta cells changed insulin secretion, supporting our functional approach. We showed first evidence for a role in insulin secretion of four candidate T2D-susceptibility genes (PRC1, SRR, ZFAND3, and ZFAND6 with no previous knowledge of presence and function in beta cells. RNA-seq in EndoC-βH1 cells with decreased expression of PRC1, SRR, ZFAND6, or ZFAND3 identified specific gene networks related to T2D pathophysiology. Finally, a positive correlation between the expression of Ins2 and the

  17. Bovine TLR2 and TLR4 mediate Cryptosporidium parvum recognition in bovine intestinal epithelial cells.

    Science.gov (United States)

    Yang, Zhengtao; Fu, Yunhe; Gong, Pengtao; Zheng, Jingtong; Liu, Li; Yu, Yuqiang; Li, Jianhua; Li, He; Yang, Ju; Zhang, Xichen

    2015-08-01

    Cryptosporidium parvum (C. parvum) is an intestinal parasite that causes diarrhea in neonatal calves. It results in significant morbidity of neonatal calves and economic losses for producers worldwide. Innate resistance against C. parvum is thought to depend on engagement of pattern recognition receptors. However, the role of innate responses to C. parvum has not been elucidated in bovine. The aim of this study was to evaluate the role of TLRs in host-cell responses during C. parvum infection of cultured bovine intestinal epithelial cells. The expressions of TLRs in bovine intestinal epithelial cells were detected by qRT-PCR. To determine which, if any, TLRs may play a role in the response of bovine intestinal epithelial cells to C. parvum, the cells were stimulated with C. parvum and the expression of TLRs were tested by qRT-PCR. The expression of NF-κB was detected by western blotting. Further analyses were carried out in bovine TLRs transfected HEK293 cells and by TLRs-DN transfected bovine intestinal epithelial cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs. The expression of TLR2 and TLR4 were up-regulated when bovine intestinal epithelial cells were treated with C. parvum. Meanwhile, C. parvum induced IL-8 production in TLR2 or TLR4/MD-2 transfected HEK293 cells. Moreover, C. parvum induced NF-κB activation and cytokine expression in bovine intestinal epithelial cells. The induction of NF-κB activation and cytokine expression by C. parvum were reduced in TLR2-DN and TLR4-DN transfected cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs, and bovine intestinal epithelial cells recognized and responded to C. parvum via TLR2 and TLR4. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Differential effects of prednisone and growth hormone on fuel metabolism and insulin antagonism in humans

    International Nuclear Information System (INIS)

    Horber, F.F.; Marsh, H.M.; Haymond, M.W.

    1991-01-01

    Human growth hormone (hGH) and prednisone cause insulin resistance and glucose intolerance. However, it is unknown whether hGH and prednisone antagonize insulin action on protein, fat, and carbohydrate metabolism by a common or independent mechanism. Therefore, protein, fat, and carbohydrate metabolism was assessed simultaneously in four groups of eight subjects each after 7 days of placebo, recombinant DNA hGH (rhGH; 0.1 mg.kg-1.day-1), prednisone (0.8 mg.kg-1.day-1), or rhGH and prednisone administration after an 18-h fast and during gut infusion of glucose and amino acids (fed state). Fasting plasma glucose concentrations were similar during placebo and rhGH but elevated (P less than 0.001) during combined treatment, whereas plasma insulin concentrations were higher (237 +/- 57 pmol/ml, P less than 0.001) during combined than during placebo, rhGH, or prednisone treatment (34, 52, and 91 pM, respectively). In the fed state, plasma glucose concentrations were elevated only during combined treatment (11.3 +/- 2.1 mM, P less than 0.001). Plasma insulin concentrations were elevated during therapy with prednisone alone and rhGH alone (667 +/- 72 and 564 +/- 65 pmol/ml, respectively, P less than 0.001) compared with placebo (226 +/- 44 pmol/ml) but lower than with the combined rhGH and prednisone treatment (1249 +/- 54 pmol/ml, P less than 0.01). Protein oxidation 14 C leucine increased (P less than 0.001) with prednisone therapy, decreased (P less than 0.001) with rhGH treatment, and was normal during the combined treatment

  19. Cancer risk among insulin users

    DEFF Research Database (Denmark)

    But, Anna; De Bruin, Marie L.; Bazelier, Marloes T.

    2017-01-01

    Aims/hypothesis: The aim of this work was to investigate the relationship between use of certain insulins and risk for cancer, when addressing the limitations and biases involved in previous studies. Methods: National Health Registries from Denmark (1996–2010), Finland (1996–2011), Norway (2005......–2010) and Sweden (2007–2012) and the UK Clinical Practice Research Datalink database (1987–2013) were used to conduct a cohort study on new insulin users (N = 327,112). By using a common data model and semi-aggregate approach, we pooled individual-level records from five cohorts and applied Poisson regression...... models. For each of ten cancer sites studied, we estimated the rate ratios (RRs) by duration (≤0.5, 0.5–1, 1–2, 2–3, 3–4, 4–5, 5–6 and >6 years) of cumulative exposure to insulin glargine or insulin detemir relative to that of human insulin. Results: A total of 21,390 cancer cases occurred during a mean...

  20. Effects of heparin on insulin binding and biological activity

    International Nuclear Information System (INIS)

    Kriauciunas, K.M.; Grigorescu, F.; Kahn, C.R.

    1987-01-01

    The effect of heparin, a polyanionic glycosaminoglycan known to alter the function of many proteins, on insulin binding and bioactivity was studied. Cultured human lymphocytes (IM-9) were incubated with varying concentrations of heparin, then extensively washed, and 125 I-labeled insulin binding was measured. Heparin at concentrations used clinically for anticoagulation (1-50 U/ml) inhibited binding in a dose-dependent manner; 50% inhibition of binding occurred with 5-10 U/ml. Scatchard analysis indicated that the decrease in binding was due to a decrease in both the affinity and the apparent number of available insulin receptors. The effect occurred within 10 min at 22 degrees C and persisted even after the cells were extensively washed. Inhibition of insulin binding also occurred when cells were preincubated with heparinized plasma or heparinized serum but not when cells were incubated with normal serum or plasma from blood anticoagulated with EDTA. By contrast, other polyanions and polycations, e.g., poly-L-glutamic acid, poly-L-lysine, succinylated poly-L-lysine, and histone, did not inhibit binding. Heparin also inhibited insulin binding in Epstein-Barr (EB) virus-transformed lymphocytes but had no effect on insulin binding to isolated adipocytes, human erythrocytes, or intact hepatoma cells. When isolated adipocytes were incubated with heparin, there was a dose-dependent inhibition of insulin-stimulated glucose oxidation and, to a lesser extent, of basal glucose oxidation. Although heparin has no effect on insulin binding to intact hepatoma cells, heparin inhibited both insulin binding and insulin-stimulated autophosphorylation in receptors solubilized from these cells