Role of transglutaminase in insulin release. Study with glycine and sarcosine methylesters

International Nuclear Information System (INIS)

Sener, A.; Dunlop, M.E.; Gomis, R.; Mathias, P.C.; Malaisse-Lagae, F.; Malaisse, W.J.

1985-01-01

The Ca2+-responsive enzyme transglutaminase, which catalyzes the cross-bridging of proteins, is present in pancreatic islet cells, but its participation in the process of insulin release remains to be documented. Glycine methylester (1.0-10.0 mM) inhibited, in a dose-related manner, transglutaminase activity in rat pancreatic islet homogenates, decreased [ 14 C]methylamine incorporation into endogenous proteins of intact islets, and caused a rapid and reversible inhibition of insulin release evoked by D-glucose, while failing to affect D-[U- 14 C]glucose oxidation. Glycine methylester also inhibited insulin release induced by other nutrient or nonnutrient secretagogues. Sarcosine methylester failed to affect transglutaminase activity, [ 14 C]methylamine incorporation, and insulin release. Both methylesters mobilized 45 Ca from prelabeled intact islets, from membranes of islet cells, liver or brain, and from artificial lipid multilayers, this Ca mobilization being apparently unrelated to changes in transglutaminase activity. It is proposed that, in the pancreatic B cell, transglutaminase participates in the machinery controlling the access of secretory granules to the exocytotic sites

The application and evaluation of insulin release test and quantitative parameter in diabetic type II

International Nuclear Information System (INIS)

Huang Chenggang; Chen Xiaoyan; Guan Xiaofeng

2002-01-01

Objective: To analyse the curve of Insulin Release Test (IRT) about the patients whit type II diabetes, to evaluate β-cell function and the sensitivity of body to Insulin using Insulin Release Index (IRI) and Insulin Sensitivity Index (ISI), and to probe the value for clinical therapy. Methods: 1) Have a IRT of 396 cases with type II diabetes and 17 normal bodies and acquire the IRT curve, 2) Design the count methods about IRI and ISI, IRI = Ins max/Ins FBI x Δ Ins max/T max (minute), ISI=(Ins max-Ins FBI)/(Ins 180'-Ins FBI), 3) Compare IRI Changes of before and after treatment for 12 cases with no insulin release and 9 cases with less insulin release. Results: IRT curve type (No release type 21.0%, less release type 33.3%, peak delay type 36.9%, high insulin type 6.0%, release delay type 2.8%); respective IRI, ISI compared to normal, P<0.01; IRI of before and after treatment with insulin P<0.01. Conclusions: IRT Curve combining IRI and ISI can reflect accurately β-cell function with type II diabetes and the sensitivity of body to insulin, Also it has some reference value for clinical therapy

Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors.

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Paola Llanos

Full Text Available Glucose-stimulated insulin secretion (GSIS from pancreatic β-cells requires an increase in intracellular free Ca2+ concentration ([Ca2+]. Glucose uptake into β-cells promotes Ca2+ influx and reactive oxygen species (ROS generation. In other cell types, Ca2+ and ROS jointly induce Ca2+ release mediated by ryanodine receptor (RyR channels. Therefore, we explored here if RyR-mediated Ca2+ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC, which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H2O2 in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H2O2-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca2+] produced by incubation of dissociated β-cells with H2O2. Addition of stimulatory glucose or H2O2 (in basal glucose to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca2+ release, induced by the concomitant increases in [Ca2+] and ROS produced by stimulatory glucose, is an essential step in GSIS.

Insulin activates single amiloride-blockable Na channels in a distal nephron cell line (A6).

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Marunaka, Y; Hagiwara, N; Tohda, H

1992-09-01

Using the patch-clamp technique, we studied the effect of insulin on an amiloride-blockable Na channel in the apical membrane of a distal nephron cell line (A6) cultured on permeable collagen films for 10-14 days. NPo (N, number of channels per patch membrane; Po, average value of open probability of individual channels in the patch) under baseline conditions was 0.88 +/- 0.12 (SE)(n = 17). After making cell-attached patches on the apical membrane which contained Na channels, insulin (1 mU/ml) was applied to the serosal bath. While maintaining the cell-attached patch, NPo significantly increased to 1.48 +/- 0.19 (n = 17; P less than 0.001) after 5-10 min of insulin application. The open probability of Na channels was 0.39 +/- 0.01 (n = 38) under baseline condition, and increased to 0.66 +/- 0.03 (n = 38, P less than 0.001) after addition of insulin. The baseline single-channel conductance was 4pS, and neither the single-channel conductance nor the current-voltage relationship was significantly changed by insulin. These results indicate that insulin increases Na absorption in the distal nephron by increasing the open probability of the amiloride-blockable Na channel.

Rosiglitazone stimulates the release and synthesis of insulin by enhancing GLUT-2, glucokinase and BETA2/NeuroD expression

International Nuclear Information System (INIS)

Kim, Hyo-Sup; Noh, Jung-Hyun; Hong, Seung-Hyun; Hwang, You-Cheol; Yang, Tae-Young; Lee, Myung-Shik; Kim, Kwang-Won; Lee, Moon-Kyu

2008-01-01

Peroxisome proliferator-activated receptor (PPAR)-γ is a member of the nuclear receptor superfamily, and its ligands, the thiazolidinediones, might directly stimulate insulin release and insulin synthesis in pancreatic β-cells. In the present study, we examined the effects of rosiglitazone (RGZ) on insulin release and synthesis in pancreatic β-cell (INS-1). Insulin release and synthesis were stimulated by treatment with RGZ for 24 h. RGZ upregulated the expressions of GLUT-2 and glucokinase (GCK). Moreover, it was found that RGZ increased the expression of BETA2/NeuroD gene which could regulate insulin gene expression. These results suggest that RGZ could stimulate the release and synthesis of insulin through the upregulation of GLUT-2, GCK, and BETA2/NeuroD gene expression

High performance liquid chromatographic analysis of insulin degradation products from a cultured kidney cell line

International Nuclear Information System (INIS)

Duckworth, W.C.; Hamel, F.G.; Liepnieks, J.; Frank, B.H.; Yagil, C.; Rabkin, R.

1988-01-01

The kidney is a major site for insulin removal and degradation, but the subcellular processes and enzymes involved have not been established. We have examined this process by analyzing insulin degradation products by HPLC. Monoiodoinsulin specifically labeled on either the A14 or B26 tyrosine residue was incubated with a cultured kidney epithelial cell line, and both intracellular and extracellular products were examined on HPLC. The products were then compared with products of known structure generated by hepatocytes and the enzyme insulin protease. Intracellular and extracellular products were different, suggesting two different degradative pathways, as previously shown in liver. The extracellular degradation products eluted from HPLC both before and after sulfitolysis similarly with hepatocyte products and products generated by insulin protease. The intracellular products also eluted identically with hepatocyte products. Based on comparisons with identified products, the kidney cell generates two fragments from the A chain of intact insulin, one with a cleavage at A13-A14 and the other at A14-A15. The B chain of intact insulin is cleaved in a number of different sites, resulting in peptides that elute identically with B chain peptides cleaved at B9-B10, B13-B14, B16-B17, B24-B25, and B25-B26. These similarities with hepatocytes and insulin protease suggest that liver and kidney have similar mechanisms for insulin degradation and that insulin protease or a very similar enzyme is involved in both tissues

Subcellular localization, mobility, and kinetic activity of glucokinase in glucose-responsive insulin-secreting cells.

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Stubbs, M; Aiston, S; Agius, L

2000-12-01

We investigated the subcellular localization, mobility, and activity of glucokinase in MIN6 cells, a glucose-responsive insulin-secreting beta-cell line. Glucokinase is present in the cytoplasm and a vesicular/granule compartment that is partially colocalized with insulin granules. The granular staining of glucokinase is preserved after permeabilization of the cells with digitonin. There was no evidence for changes in distribution of glucokinase between the cytoplasm and the granule compartment during incubation of the cells with glucose. The rate of release of glucokinase and of phosphoglucoisomerase from digitonin-permeabilized cells was slower when cells were incubated at an elevated glucose concentration (S0.5 approximately 15 mmol/l). This effect of glucose was counteracted by competitive inhibitors of glucokinase (5-thioglucose and mannoheptulose) but was unaffected by fructose analogs and may be due to changes in cell shape or conformation of the cytoskeleton that are secondary to glucose metabolism. Based on the similar release of glucokinase and phosphoglucoisomerase, we found no evidence for specific binding of cytoplasmic digitonin-extractable glucokinase. The affinity of beta-cells for glucose is slightly lower than that in cell extracts and, unlike that in hepatocytes, is unaffected by fructose, tagatose, or a high-K+ medium, which is consistent with the lack of change in glucokinase distribution or release. We conclude that glucokinase is present in two locations, cytoplasm and the granular compartment, and that it does not translocate between them. This conclusion is consistent with the lack of adaptive changes in the glucose phosphorylation affinity. The glucokinase activity associated with the insulin granules may have a role in either direct or indirect coupling between glucose phosphorylation and insulin secretion.

Hyperinsulinemia is associated with increased soluble insulin receptors release from hepatocytes

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Marcia eHiriart

2014-06-01

Full Text Available It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l-1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia the amount of this soluble receptor increases, this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance.

Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor.

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Delanoue, Renald; Meschi, Eleonora; Agrawal, Neha; Mauri, Alessandra; Tsatskis, Yonit; McNeill, Helen; Léopold, Pierre

2016-09-30

Animals adapt their growth rate and body size to available nutrients by a general modulation of insulin-insulin-like growth factor signaling. In Drosophila, dietary amino acids promote the release in the hemolymph of brain insulin-like peptides (Dilps), which in turn activate systemic organ growth. Dilp secretion by insulin-producing cells involves a relay through unknown cytokines produced by fat cells. Here, we identify Methuselah (Mth) as a secretin-incretin receptor subfamily member required in the insulin-producing cells for proper nutrient coupling. We further show, using genetic and ex vivo organ culture experiments, that the Mth ligand Stunted (Sun) is a circulating insulinotropic peptide produced by fat cells. Therefore, Sun and Mth define a new cross-organ circuitry that modulates physiological insulin levels in response to nutrients. Copyright © 2016, American Association for the Advancement of Science.

Effect of the hexane extract of Piper auritum on insulin release from β-cell and oxidative stress in streptozotocin-induced diabetic rat.

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Gutierrez, Rosa Martha Perez

2012-10-01

The large-leafed perennial plant Piper auritum known as Hoja Santa, is used for its leaves that because of their spicy aromatic scent and flavor have an important presence in Mexican cuisine, and in many regions, this plant is known for its therapeutic properties. In the present study, we investigated the effect of hexane, chloroform and methanol extracts from Piper auritum on cell culture system and the effect in streptozotocin-induced type 1 diabetic rats treated by 28 days on the physiological, metabolic parameters and oxidative stress. The hexane extract of P. auritum (HS) treatment significantly reduced the intake of both food, water and body weight loss as well as levels of blood glucose, serum cholesterol, triglycerides and increase HDL-cholesterol. After 4-week administration of HS antioxidant enzyme as SOD, CAT, GSH, GPx in pancreas were determined. These enzyme increased significantly compared with those of the diabetic rats control and normal animals. For all estimated, the results of HS treated groups leading to a restoration of the defense mechanism. The treatment also improves pancreatic TBARS-reactive substance level and serum NO and iNOS. To determine the insulin releasing activity, after extract treatment the serum and pancreatic sections were processed for examination of insulin-releasing activity using an immunocytochemistry kit. The results showed that administration of the hexane extract (200 and 400 mg/kg) exhibited a significant increase in serum and pancreas tissue insulin. Administration of streptozotocin decreased the insulin secretory activity in comparison with intact rats, but treatment with the HS extract increased significantly the activity of the beta cells in comparison with the diabetic control rats. The extract decreased serum glucose in streptozotocin-induced diabetic rats and increased insulin release from the beta cells of the pancreas. In cultured RIN-5F cells, we examined whether hexane extract of P. auritum would protect the

Apoptosis may determine the release of skeletal alkaline phosphatase activity from human osteoblast-line cells.

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Farley, J R; Stilt-Coffing, B

2001-01-01

Although quantitative measurement of skeletal alkaline phosphatase (sALP) activity in serum can provide an index of the rate of bone formation, the metabolic process that determines the release of sALP - from the surface of osteoblasts, into circulation-is unknown. The current studies were intended to examine the hypothesis that the release of sALP from human osteoblasts is a consequence of apoptotic cell death. We measured the release of sALP activity from human osteosarcoma (SaOS-2) cells and normal human bone cells, under basal conditions and in response to agents that increased apoptosis (TNF-a, okadiac acid) and agents that inhibit apoptosis (IGF-I, calpain, and caspase inhibitors). Apoptosis was determined by the presence of nucleosomes (histone-associated DNA) in the cytoplasm of the cells by using a commercial kit. The results of these studies showed that TNF-a and okadiac acid caused dose- and time-dependent increases in apoptosis in the SaOS-2 cells (r = 0.78 for doses of TNF-a and r = 0.93 for doses of okadiac acid, P sALP activity (e.g., r = 0.89 for TNF-a and r = 0.75 for okadiac acid, P sALP activity (P sALP activity (P sALP release. The associations between apoptosis and sALP release were not unique to osteosarcoma (i.e., SaOS-2) cells, but also seen with osteoblast-line cells derived from normal human bone. Together, these data demonstrate that the release of sALP activity from human osteoblast-line cells in vitro is associated with, and may be a consequence of, apoptotic cell death. These findings are consistent with the general hypothesis that the appearance of sALP activity in serum may reflect the turnover of osteoblast-line cells.

Intra-peritoneal administration of interleukin-1 beta induces impaired insulin release from the perfused rat pancreas

DEFF Research Database (Denmark)

Wogensen, L; Helqvist, S; Pociot, F

1990-01-01

Previous studies have demonstrated a stimulatory effect of interleukin-1 beta (IL-1 beta) on insulin and glucagon release from the perfused rat pancreas, accompanied by selective lysis of 20% of beta-cells as assessed by electronmicroscopy. However, we have not observed an inhibitory action of IL-1...... beta on insulin release from the perfused pancreas as shown for isolated islets. To test whether periodical exposure of the endocrine pancreas to circulating IL-1 beta in vivo affects insulin release from the intact perfused pancreas, rats were treated with daily intraperitoneal injections of 4...

Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells

International Nuclear Information System (INIS)

Vaca, Pilar; Berna, Genoveva; Araujo, Raquel; Carneiro, Everardo M.; Bedoya, Francisco J.; Soria, Bernat; Martin, Franz

2008-01-01

The poly(ADP-ribose) polymerase (PARP) inhibitor, nicotinamide, induces differentiation and maturation of fetal pancreatic cells. In addition, we have previously reported evidence that nicotinamide increases the insulin content of cells differentiated from embryonic stem (ES) cells, but the possibility of nicotinamide acting as a differentiating agent on its own has never been completely explored. Islet cell differentiation was studied by: (i) X-gal staining after neomycin selection; (ii) BrdU studies; (iii) single and double immunohistochemistry for insulin, C-peptide and Glut-2; (iv) insulin and C-peptide content and secretion assays; and (v) transplantation of differentiated cells, under the kidney capsule, into streptozotocin (STZ)-diabetic mice. Here we show that undifferentiated mouse ES cells treated with nicotinamide: (i) showed an 80% decrease in cell proliferation; (ii) co-expressed insulin, C-peptide and Glut-2; (iii) had values of insulin and C-peptide corresponding to 10% of normal mouse islets; (iv) released insulin and C-peptide in response to stimulatory glucose concentrations; and (v) after transplantation into diabetic mice, normalized blood glucose levels over 7 weeks. Our data indicate that nicotinamide decreases ES cell proliferation and induces differentiation into insulin-secreting cells. Both aspects are very important when thinking about cell therapy for the treatment of diabetes based on ES cells

Transfer plate radioassay using adsorbed anti-insulin antibody to detect insulin secreted by islet cell cultures

International Nuclear Information System (INIS)

Scearce, R.M.; Oie, H.K.; Gazdar, A.F.; Chick, W.L.; Eisenbarth, G.S.

1981-01-01

A solid-phase radioimmunoassay for detection of insulin synthesized by islet cell clones is described. This assay employs anti-insulin antibody adsorbed onto fenestrated polyvinyl chloride 96-well plates ('transfer plates'). The calibrated aperture in the bottom of each transfer plate well permits fluid to enter the wells when transfer plates are lowered into microculture wells containing insulin. With this assay it is possible to rapidly screen hundreds of islet cell cultures for insulin production. The authors have used this assay to facilitate cloning of the RIN rat insulinoma cell line. The assay readily detects insulin synthesis by RIN cells and [ 125 I]insulin is not displaced by culture medium from cells which do not produce insulin. The transfer plate format should be applicable to semiautomate other radioimmunoassays. (Auth.)

Methylmercury inhibits prolactin release in a cell line of pituitary origin

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L.A.L. Maués

2015-08-01

Full Text Available Heavy metals, such as methylmercury, are key environmental pollutants that easily reach human beings by bioaccumulation through the food chain. Several reports have demonstrated that endocrine organs, and especially the pituitary gland, are potential targets for mercury accumulation; however, the effects on the regulation of hormonal release are unclear. It has been suggested that serum prolactin could represent a biomarker of heavy metal exposure. The aim of this study was to evaluate the effect of methylmercury on prolactin release and the role of the nitrergic system using prolactin secretory cells (the mammosomatotroph cell line, GH3B6. Exposure to methylmercury (0-100 μM was cytotoxic in a time- and concentration-dependent manner, with an LC50 higher than described for cells of neuronal origin, suggesting GH3B6 cells have a relative resistance. Methylmercury (at exposures as low as 1 μM for 2 h also decreased prolactin release. Interestingly, inhibition of nitric oxide synthase by N-nitro-L-arginine completely prevented the decrease in prolactin release without acute neurotoxic effects of methylmercury. These data indicate that the decrease in prolactin production occurs via activation of the nitrergic system and is an early effect of methylmercury in cells of pituitary origin.

IRS-1: essential for insulin- and IL-4-stimulated mitogenesis in hematopoietic cells.

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Wang, L M; Myers, M G; Sun, X J; Aaronson, S A; White, M; Pierce, J H

1993-09-17

Although several interleukin-3 (IL-3)-dependent cell lines proliferate in response to IL-4 or insulin, the 32D line does not. Insulin and IL-4 sensitivity was restored to 32D cells by expression of IRS-1, the principal substrate of the insulin receptor. Although 32D cells possessed receptors for both factors, they lacked the IRS-1--related protein, 4PS, which becomes phosphorylated by tyrosine in insulin- or IL-4--responsive lines after stimulation. These results indicate that factors that bind unrelated receptors can use similar mitogenic signaling pathways in hematopoietic cells and that 4PS and IRS-1 are functionally similar proteins that are essential for insulin- and IL-4--induced proliferation.

Biphasic insulin-releasing effect of BTS 67 582 in rats.

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Storey, D A; Bailey, C J

1998-12-01

BTS 67 582 (1,1-dimethyl-2(2-morpholinophenyl)guanidine fumarate) is being developed as a short-acting anti-diabetic insulin secretagogue. The effect of BTS 67 582 on the phasic pattern of insulin release was assessed in anaesthetized normal rats by measuring arterial plasma insulin concentrations while arterial glucose concentrations were fixed at 6, 8.5 and 12.5 mM. Intravenous BTS 67 582 (10 mg kg(-1)) induced an immediate but transient increase in insulin concentrations which declined by 10 min (first phase). This was followed by a smaller but sustained increase in insulin concentrations (second phase). The increment from basal to peak insulin release (0-2 min) was independent of glucose, but the first phase was maintained for longer and the second phase was greater at the highest concentration of glucose (12.5 mM). BTS 67 582 also extended the first-phase insulin response to a standard intravenous glucose challenge and enhanced the rate of glucose disappearance by approximately 12%. Thus BTS 67 582 causes biphasic stimulation of insulin release and augments the insulin-releasing effect of glucose.

Reciprocal links between metabolic and ionic events in islet cells. Their relevance to the rhythmics of insulin release.

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Malaisse, W J

1998-02-01

The notion of reciprocal links between metabolic and ionic events in islet cells and the rhythmics of insulin release is based on (i) the rhythmic pattern of hormonal release from isolated perfused rat pancreas, which supports the concept of an intrapancreatic pacemaker; (ii) the assumption that this phasic pattern is due to the integration of secretory activity in distinct functional units, e.g. distinct islets; and (iii) the fact that reciprocal coupling between metabolic and ionic events is operative in the secretory sequence.

C peptide and insulin releasing RIA test for the investigation of β cell function in diabetic patients

International Nuclear Information System (INIS)

Shi Ailan; Zhu Chengmo; Wang Qiyu; Wang Ping

1993-01-01

Results of C-peptide releasing RIA test in 15 normals, and 100 diabetes were summarized and compared with glucose tolerance test and serum insulin for investigating the characteristics in different types of diabetes and evaluating the functional state of islet β cell. In 36 cases of IDDM the fasting blood sugar was significantly increased, and further elevated after eating of bread, but its peak time delay in 2 hours (normalin 1 hour). The level of basal C-peptide is very low, but shows slightly weak on no response after bread stimulating test, all of this denotes that β cell function of islets severely injured. The increasing of fasting blood sugar in 64 cases of NIDDM was lower than those of IDDM. Fasting C-peptide and insulin was normal or increased, their peak value increased after bread stimulation with peak time delayed also at 2 hours. Above results demonstrated that the function of islets B cell decreased but not fully deprived. It is concluded that C-peptide and insulin stimulating test, together with OGTT can accurately assess the islets β cell function, and also have important significance in the pathogenesis, classification and staging, prognostic evaluation and monitoring of therapeutic effects in diabetes

Possible contribution of taurine to distorted glucagon secretion in intra-islet insulin deficiency: a metabolome analysis using a novel α-cell model of insulin-deficient diabetes.

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Megumi Bessho

Full Text Available Glycemic instability is a serious problem in patients with insulin-deficient diabetes, and it may be due in part to abnormal endogenous glucagon secretion. However, the intracellular metabolic mechanism(s involved in the aberrant glucagon response under the condition of insulin deficiency has not yet been elucidated. To investigate the metabolic traits that underlie the distortion of glucagon secretion under insulin deficient conditions, we generated an αTC1-6 cell line with stable knockdown of the insulin receptor (IRKD, i.e., an in vitro α-cell model for insulin-deficient diabetes, which exhibits an abnormal glucagon response to glucose. A comprehensive metabolomic analysis of the IRKD αTC1-6 cells (IRKD cells revealed some candidate metabolites whose levels differed markedly compared to those in control αTC1-6 cells, but also which could affect the glucagon release in IRKD cells. Of these candidates, taurine was remarkably increased in the IRKD cells and was identified as a stimulator of glucagon in αTC1-6 cells. Taurine also paradoxically exaggerated the glucagon secretion at a high glucose concentration in IRKD cells and islets with IRKD. These results indicate that the metabolic alterations induced by IRKD in α-cells, especially the increase of taurine, may lead to the distorted glucagon response in IRKD cells, suggesting the importance of taurine in the paradoxical glucagon response and the resultant glucose instability in insulin-deficient diabetes.

Evaluation of fasting state-/oral glucose tolerance test-derived measures of insulin release for the detection of genetically impaired β-cell function.

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Silke A Herzberg-Schäfer

Full Text Available BACKGROUND: To date, fasting state- and different oral glucose tolerance test (OGTT-derived measures are used to estimate insulin release with reasonable effort in large human cohorts required, e.g., for genetic studies. Here, we evaluated twelve common (or recently introduced fasting state-/OGTT-derived indices for their suitability to detect genetically determined β-cell dysfunction. METHODOLOGY/PRINCIPAL FINDINGS: A cohort of 1364 White European individuals at increased risk for type 2 diabetes was characterized by OGTT with glucose, insulin, and C-peptide measurements and genotyped for single nucleotide polymorphisms (SNPs known to affect glucose- and incretin-stimulated insulin secretion. One fasting state- and eleven OGTT-derived indices were calculated and statistically evaluated. After adjustment for confounding variables, all tested SNPs were significantly associated with at least two insulin secretion measures (p≤0.05. The indices were ranked according to their associations' statistical power, and the ranks an index obtained for its associations with all the tested SNPs (or a subset were summed up resulting in a final ranking. This approach revealed area under the curve (AUC(Insulin(0-30/AUC(Glucose(0-30 as the best-ranked index to detect SNP-dependent differences in insulin release. Moreover, AUC(Insulin(0-30/AUC(Glucose(0-30, corrected insulin response (CIR, AUC(C-Peptide(0-30/AUC(Glucose(0-30, AUC(C-Peptide(0-120/AUC(Glucose(0-120, two different formulas for the incremental insulin response from 0-30 min, i.e., the insulinogenic indices (IGI(2 and IGI(1, and insulin 30 min were significantly higher-ranked than homeostasis model assessment of β-cell function (HOMA-B; p<0.05. AUC(C-Peptide(0-120/AUC(Glucose(0-120 was best-ranked for the detection of SNPs involved in incretin-stimulated insulin secretion. In all analyses, HOMA-β displayed the highest rank sums and, thus, scored last. CONCLUSIONS/SIGNIFICANCE: With AUC(Insulin(0

Inhibition of insulin release by cyproheptadine: Effects on 3',5'-cyclic-AMP-content and /sup 45/Ca-accumulation of incubated mouse islets

Energy Technology Data Exchange (ETDEWEB)

Joost, H G; Beckmann, J; Lenzen, S; Hasselblatt, A [Goettingen Univ. (F.R. Germany)

1976-01-01

Cyproheptadine (1, 10 and 100 ..mu..m) significantly reduced insulin release from isolated mouse islets in response to glucose. In contrast, 1 mM cyproheptadine induced a large release of insulin into the incubation medium probably due to islet cell damage, since the islets had lost a considerable amount of their protein content. 3',5'-cyclic-AMP-levels of the islets were not significantly affected by 10 ..mu..M cyproheptadine in the presence as well as in the absence of theophylline (10 mM). As the inhibitory effect of cyproheptadine on insulin release was correlated with reduced accumulation of calcium-45, the agent may inhibit insulin release by interfering with the calcium handling of the ..beta..-cell.

Intracellular and extracellular adenosine triphosphate in regulation of insulin secretion from pancreatic β cells (β).

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Wang, Chunjiong; Geng, Bin; Cui, Qinghua; Guan, Youfei; Yang, Jichun

2014-03-01

Adenosine triphosphate (ATP) synthesis and release in mitochondria play critical roles in regulating insulin secretion in pancreatic β cells. Mitochondrial dysfunction is mainly characterized by a decrease in ATP production, which is a central event in the progression of pancreatic β cell dysfunction and diabetes. ATP has been demonstrated to regulate insulin secretion via several pathways: (i) Intracellular ATP directly closes ATP-sensitive potassium channel to open L-type calcium channel, leading to an increase in free cytosolic calcium levels and exocytosis of insulin granules; (ii) A decrease in ATP production is always associated with an increase in production of reactive oxygen species, which exerts deleterious effects on pancreatic β cell survival and insulin secretion; and (iii) ATP can be co-secreted with insulin from pancreatic β cells, and the released ATP functions as an autocrine signal to modulate insulin secretory process via P2 receptors on the cell membrane. In this review, the recent findings regarding the role and mechanism of ATP synthesis and release in regulation of insulin secretion from pancreatic β cells will be summarized and discussed. © 2013 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

Aβ-Induced Insulin Resistance and the Effects of Insulin on the Cholesterol Synthesis Pathway and Aβ Secretion in Neural Cells.

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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�

From Human Mesenchymal Stem Cells to Insulin-Producing Cells: Comparison between Bone Marrow- and Adipose Tissue-Derived Cells.

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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.

Role of aryl hydrocarbon receptor nuclear translocator in KATP channel-mediated insulin secretion in INS-1 insulinoma cells

International Nuclear Information System (INIS)

Kim, Ji-Seon; Zheng Haifeng; Kim, Sung Joon; Park, Jong-Wan; Park, Kyong Soo; Ho, Won-Kyung; Chun, Yang-Sook

2009-01-01

Aryl hydrocarbon receptor nuclear translocator (ARNT) has been known to participate in cellular responses to xenobiotic and hypoxic stresses, as a common partner of aryl hydrocarbon receptor and hypoxia inducible factor-1/2α. Recently, it was reported that ARNT is essential for adequate insulin secretion in response to glucose input and that its expression is downregulated in the pancreatic islets of diabetic patients. In the present study, the authors addressed the mechanism by which ARNT regulates insulin secretion in the INS-1 insulinoma cell line. In ARNT knock-down cells, basal insulin release was elevated, but insulin secretion was not further stimulated by a high-glucose challenge. Electrophysiological analyses revealed that glucose-dependent membrane depolarization was impaired in these cells. Furthermore, K ATP channel activity and expression were reduced. Of two K ATP channel subunits, Kir6.2 was found to be positively regulated by ARNT at the mRNA and protein levels. Based on these results, the authors suggest that ARNT expresses K ATP channel and by so doing regulates glucose-dependent insulin secretion.

From Human Mesenchymal Stem Cells to Insulin-Producing Cells: Comparison between Bone Marrow- and Adipose Tissue-Derived Cells

Directory of Open Access Journals (Sweden)

Mahmoud M. Gabr

2017-01-01

Full Text Available 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.

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.

Efficient Differentiation of Mouse Embryonic Stem Cells into Insulin-Producing Cells

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Szu-Hsiu Liu

2012-01-01

Full Text Available Embryonic stem (ES cells are a potential source of a variety of differentiated cells for cell therapy, drug discovery, and toxicology screening. Here, we present an efficacy strategy for the differentiation of mouse ES cells into insulin-producing cells (IPCs by a two-step differentiation protocol comprising of (i the formation of definitive endoderm in monolayer culture by activin A, and (ii this monolayer endoderm being induced to differentiate into IPCs by nicotinamide, insulin, and laminin. Differentiated cells can be obtained within approximately 7 days. The differentiation IPCs combined application of RT-PCR, ELISA, and immunofluorescence to characterize phenotypic and functional properties. In our study, we demonstrated that IPCs produced pancreatic transcription factors, endocrine progenitor marker, definitive endoderm, pancreatic β-cell markers, and Langerhans α and δ cells. The IPCs released insulin in a manner that was dose dependent upon the amount of glucose added. These techniques may be able to be applied to human ES cells, which would have very important ramifications for treating human disease.

A human beta cell line with drug inducible excision of immortalizing transgenes

Science.gov (United States)

Benazra, Marion; Lecomte, Marie-José; Colace, Claire; Müller, Andreas; Machado, Cécile; Pechberty, Severine; Bricout-Neveu, Emilie; Grenier-Godard, Maud; Solimena, Michele; Scharfmann, Raphaël; Czernichow, Paul; Ravassard, Philippe

2015-01-01

Objectives Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-βH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen. Methods We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. Results We showed that EndoC-βH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 μg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture. Conclusions EndoC βH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture. PMID:26909308

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

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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.

Multiple growth hormone-binding proteins are expressed on insulin-producing cells

DEFF Research Database (Denmark)

Møldrup, A; Billestrup, N; Thorn, N A

1989-01-01

The insulin-producing rat islet tumor cell line, RIN-5AH, expresses somatogen binding sites and responds to GH by increased proliferation and insulin production. Affinity cross-linking shows that RIN-5AH cells contain two major GH-binding subunits of Mr 100-130K (110K), which appear to exist as d....... It is concluded that the RIN-5AH cells have multiple GH-binding proteins which may mediate signals for either proliferation and/or insulin production....

Obesity-induced DNA released from adipocytes stimulates chronic adipose tissue inflammation and insulin resistance.

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Nishimoto, Sachiko; Fukuda, Daiju; Higashikuni, Yasutomi; Tanaka, Kimie; Hirata, Yoichiro; Murata, Chie; Kim-Kaneyama, Joo-Ri; Sato, Fukiko; Bando, Masahiro; Yagi, Shusuke; Soeki, Takeshi; Hayashi, Tetsuya; Imoto, Issei; Sakaue, Hiroshi; Shimabukuro, Michio; Sata, Masataka

2016-03-01

Obesity stimulates chronic inflammation in adipose tissue, which is associated with insulin resistance, although the underlying mechanism remains largely unknown. Here we showed that obesity-related adipocyte degeneration causes release of cell-free DNA (cfDNA), which promotes macrophage accumulation in adipose tissue via Toll-like receptor 9 (TLR9), originally known as a sensor of exogenous DNA fragments. Fat-fed obese wild-type mice showed increased release of cfDNA, as determined by the concentrations of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in plasma. cfDNA released from degenerated adipocytes promoted monocyte chemoattractant protein-1 (MCP-1) expression in wild-type macrophages, but not in TLR9-deficient (Tlr9 (-/-) ) macrophages. Fat-fed Tlr9 (-/-) mice demonstrated reduced macrophage accumulation and inflammation in adipose tissue and better insulin sensitivity compared with wild-type mice, whereas bone marrow reconstitution with wild-type bone marrow restored the attenuation of insulin resistance observed in fat-fed Tlr9 (-/-) mice. Administration of a TLR9 inhibitory oligonucleotide to fat-fed wild-type mice reduced the accumulation of macrophages in adipose tissue and improved insulin resistance. Furthermore, in humans, plasma ssDNA level was significantly higher in patients with computed tomography-determined visceral obesity and was associated with homeostasis model assessment of insulin resistance (HOMA-IR), which is the index of insulin resistance. Our study may provide a novel mechanism for the development of sterile inflammation in adipose tissue and a potential therapeutic target for insulin resistance.

Controlling insulin release from reverse hexagonal (HII) liquid crystalline mesophase by enzymatic lipolysis.

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Mishraki-Berkowitz, Tehila; Cohen, Guy; Aserin, Abraham; Garti, Nissim

2018-01-01

In the present study we aimed to control insulin release from the reverse hexagonal (H II ) mesophase using Thermomyces lanuginosa lipase (TLL) in the environment (outer TLL) or within the H II cylinders (inner TLL). Two insulin-loaded systems differing by the presence (or absence) of phosphatidylcholine (PC) were examined. In general, incorporation of PC into the H II interface (without TLL) increased insulin release, as a more cooperative system was formed. Addition of TLL to the systems' environments resulted in lipolysis of the H II structure. In the absence of PC, the lipolysis was more dominant and led to a significant increase in insulin release (50% after 8h). However, the presence of PC stabilized the interface, hindering the lipolysis, and therefore no impact on the release profile was detected during the first 8h. Entrapment of TLL within the H II cylinders (with and without PC) drastically increased insulin release in both systems up to 100%. In the presence of PC insulin released faster and the structure was more stable. Consequently, the presence of lipases (inner or outer) both enhanced the destruction of the carrier, and provided sustained release of the entrapped insulin. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of the growth hormone receptor gene in insulin producing cells

DEFF Research Database (Denmark)

Møldrup, Annette; Billestrup, N; Nielsen, Jens Høiriis

1990-01-01

Growth hormone (GH) plays a dual role in glucose homeostasis. On the one hand, it exerts an insulin antagonistic effect on the peripheral tissue, on the other hand, it stimulates insulin biosynthesis and beta-cell proliferation. The expression of GH-receptors on the rat insulinoma cell line RIN-5...

Structural properties and release of insulin-loaded reverse hexagonal (HII) liquid crystalline mesophase.

Science.gov (United States)

Mishraki-Berkowitz, Tehila; Aserin, Abraham; Garti, Nissim

2017-01-15

Insulin loading into the H II mesophases was examined as a function of its concentration, with addition of glycerol as a cosolvent and with addition of phosphatidylcholine (PC) as a structural stabilizer. The structural properties, the molecular interactions, the viscoelastic properties, and the dynamic behavior were investigated by SAXS, ATR-FTIR, and rheological measurements. Insulin release was then monitored and analyzed. Insulin incorporation into the H II systems shrank the cylinders as it competed with the lipids in water-bonding. Insulin interrupted the interface while increasing τ max and creating a more solid-like response. Upon addition of PC, cooperative flow behavior was detected, which is probably the reason for increase in insulin cumulative release from 28% to 52% after 300 min. In the presence of glycerol, the system was less cooperative but insulin was more compactly folded, resulting in a slight improvement in insulin release (up to 6%). Addition of both PC and glycerol caused the maximum release (55%). The addition of additives into the H II system demonstrates how structural modifications can improve insulin release, and influence future design of encapsulated drug delivery systems. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Effects of tetracaine on insulin release and calcium handling by rat pancreatic islets

International Nuclear Information System (INIS)

Abdel El Motal, S.M.A.; Pian-Smith, M.C.M.; Sharp, G.W.G.

1987-01-01

The effects of tetracaine on insulin release and 45 Ca 2+ handling by rat pancreatic islets have been studied under basal, glucose-stimulated, and 3-isobutyl-1-methylxanthine (IBMX)-stimulated conditions. Islets were isolated by the use of collagenase and used either directly (freshly isolated islets) or after a period under tissue culture conditions. Tetracaine was found to stimulate insulin release under basal conditions, to inhibit glucose-stimulated insulin release, and to potentiate insulin release stimulated by IBMX. In studies on the mechanisms underlying these effects, tetracaine was found to decrease glucose-stimulated net retention of 45 Ca 2+ (by an action to block the voltage-dependent Ca channels) and to mobilize Ca 2+ from intracellular stores. These two actions form the basis for the inhibition of glucose-stimulated insulin release, which depends heavily on Ca 2+ entry via the voltage-dependent channels and the synergism with IBMX to potentiate release. No inhibition of IBMX-stimulated release occurs because IBMX does not use the voltage-dependent channels to raise intracellular Ca 2+

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.

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

Release of immunoreactive and radioactively prelabelled endogenous (pro-)insulin from isolated islets of rat pancreas in the presence of exogenous insulin

Energy Technology Data Exchange (ETDEWEB)

Schatz, H [Giessen Univ. (Germany, F.R.). Zentrum fuer Innere Medizin; Pfeiffer, E F

1977-01-01

To study the influence of insulin on its secretion, collagenase-isolated islets of rat pancreas were prelabelled with (/sup 3/H)leucine for 2 h. After washing the islets, (pro-)insulin release was stimulated by glucose in the presence or absence of exogenous insulin (up to 2.5 mu./ml. Hormone release was unchanged by the presence of exogenous insulin as judged by determination of both immunoreactive insulin and radioactivity incorporated into the proinsulin and insulin fractions of the medium. No direct feedback mechanism for insulin secretion was apparent from this study.

Dual pathways for the intracellular processing of insulin. Relationship between retroendocytosis of intact hormone and the recycling of insulin receptors

International Nuclear Information System (INIS)

Marshall, S.

1985-01-01

Adipocytes process insulin through either of two pathways: a retroendocytotic pathway that culminates in the release of intact insulin, and a degradative pathway that terminates in the intracellular catabolism and release of degraded ligand. Mechanistically, these pathways were found to differ in several ways. First, temporal differences were found in the rate at which intact and degraded products were extruded. After 125 I-insulin was preloaded into the cell interior, intact ligand was completely released during the first 10 min (t 1/2 = 2 min), whereas degraded insulin was released at a much slower rate over 1 h (t 1/2 greater than 8 min). Secondly, it was found that chloroquine profoundly inhibited the insulin degradative pathway, resulting in the intracellular accumulation of intact ligand and a reduction in the release of degraded products. In contrast, however, chloroquine was without effect on the retroendocytotic processing of insulin. Based on the known actions of chloroquine, it appears that retroendocytosis of insulin does not involve vesicular acidification or dissociation of the insulin-receptor complex and that insulin is most likely carried to the cell exterior in the same vesicles (either receptor-bound or free) as those mediating recycling receptors. Interestingly, accumulation of undergraded insulin within chloroquine-treated cells did not result in the release of additional intact ligand, suggesting that once insulin enters the degradative compartment it is committed to catabolism and cannot exit the cell through the retroendocytotic pathway. A third difference was revealed by the finding that extracellular unlabeled insulin (100 ng/ml) markedly accelerated the rate at which preloaded 125 I-insulin was released from adipocytes (t 1/2 of 3 min versus 7 min in controls cells)

Light Control of Insulin Release and Blood Glucose Using an Injectable Photoactivated Depot.

Science.gov (United States)

Sarode, Bhagyesh R; Kover, Karen; Tong, Pei Y; Zhang, Chaoying; Friedman, Simon H

2016-11-07

In this work we demonstrate that blood glucose can be controlled remotely through light stimulated release of insulin from an injected cutaneous depot. Human insulin was tethered to an insoluble but injectable polymer via a linker, which was based on the light cleavable di-methoxy nitrophenyl ethyl (DMNPE) group. This material was injected into the skin of streptozotocin-treated diabetic rats. We observed insulin being released into the bloodstream after a 2 min trans-cutaneous irradiation of this site by a compact LED light source. Control animals treated with the same material, but in which light was blocked from the site, showed no release of insulin into the bloodstream. We also demonstrate that additional pulses of light from the light source result in additional pulses of insulin being absorbed into circulation. A significant reduction in blood glucose was then observed. Together, these results demonstrate the feasibility of using light to allow for the continuously variable control of insulin release. This in turn has the potential to allow for the tight control of blood glucose without the invasiveness of insulin pumps and cannulas.

Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic {beta} cells

Energy Technology Data Exchange (ETDEWEB)

Kumar, Divya P.; Rajagopal, Senthilkumar; Mahavadi, Sunila [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Mirshahi, Faridoddin [Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Grider, John R. [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Murthy, Karnam S., E-mail: skarnam@vcu.edu [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Sanyal, Arun J., E-mail: asanyal@mcvh-vcu.edu [Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA (United States)

2012-10-26

Highlights: Black-Right-Pointing-Pointer G protein coupled receptor TGR5 is expressed in mouse and human islets. Black-Right-Pointing-Pointer TGR5 is coupled to activation of Gs and Ca{sup 2+} release via cAMP/Epac/PLC-{epsilon} pathway. Black-Right-Pointing-Pointer Activation of TGR5 by bile salts and selective ligands causes insulin secretion. Black-Right-Pointing-Pointer TGR5 could be a potential therapeutic target to treat diabetes. -- Abstract: Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic {beta} cells. In the present study, we have identified the expression of TGR5 in pancreatic {beta} cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated G{alpha}{sub s} and caused an increase in intracellular cAMP and Ca{sup 2+}. OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective G{alpha}{sub s} inhibitor) or (U73122) (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, (U73122) or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on G{sub s}/cAMP/Ca{sup 2+} pathway. 8-pCPT-2 Prime -O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic {beta} cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic β cells

International Nuclear Information System (INIS)

Kumar, Divya P.; Rajagopal, Senthilkumar; Mahavadi, Sunila; Mirshahi, Faridoddin; Grider, John R.; Murthy, Karnam S.; Sanyal, Arun J.

2012-01-01

Highlights: ► G protein coupled receptor TGR5 is expressed in mouse and human islets. ► TGR5 is coupled to activation of Gs and Ca 2+ release via cAMP/Epac/PLC-ε pathway. ► Activation of TGR5 by bile salts and selective ligands causes insulin secretion. ► TGR5 could be a potential therapeutic target to treat diabetes. -- Abstract: Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic β cells. In the present study, we have identified the expression of TGR5 in pancreatic β cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated Gα s and caused an increase in intracellular cAMP and Ca 2+ . OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective Gα s inhibitor) or (U73122) (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, (U73122) or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on G s /cAMP/Ca 2+ pathway. 8-pCPT-2′-O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic β cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

Common elements in interleukin 4 and insulin signaling pathways in factor-dependent hematopoietic cells.

Science.gov (United States)

Wang, L M; Keegan, A D; Li, W; Lienhard, G E; Pacini, S; Gutkind, J S; Myers, M G; Sun, X J; White, M F; Aaronson, S A

1993-05-01

Interleukin 4 (IL-4), insulin, and insulin-like growth factor I (IGF-I) efficiently induced DNA synthesis in the IL-3-dependent murine myeloid cell lines FDC-P1 and FDC-P2. Although these factors could not individually sustain long-term growth of these lines, a combination of IL-4 with either insulin or IGF-I did support continuous growth. The principal tyrosine-phosphorylated substrate observed in FDC cells stimulated with IL-4, previously designated 4PS, was of the same size (170 kDa) as the major substrate phosphorylated in response to insulin or IGF-I. These substrates had phosphopeptides of the same size when analyzed by digestion with Staphylococcus aureus V8 protease, and each tightly associated with the 85-kDa component of phosphatidylinositol 3-kinase after factor stimulation. IRS-1, the principal substrate phosphorylated in response to insulin or IGF-I stimulation in nonhematopoietic cells, is similar in size to 4PS. However, anti-IRS-1 antibodies failed to efficiently precipitate 4PS, and some phosphopeptides generated by V8 protease digestion of IRS-1 were distinct in size from the phosphopeptides of 4PS. Nevertheless, IL-4, insulin, and IGF-I were capable of stimulating tyrosine phosphorylation of IRS-1 in FDC cells that expressed this substrate as a result of transfection. These findings indicate that (i) IL-4, insulin, and IGF-I use signal transduction pathways in FDC lines that have at least one major feature in common, the rapid tyrosine phosphorylation of 4PS, and (ii) insulin and IGF-I stimulation of hematopoietic cell lines leads to the phosphorylation of a substrate that may be related to but is not identical to IRS-1.

Genetic and phenotypic correlations between surrogate measures of insulin release obtained from OGTT data

DEFF Research Database (Denmark)

Gjesing, Anette Marianne Prior; Ribel-Madsen, Rasmus; Harder, Marie Neergaard

2015-01-01

closely related to fasting insulin with a genetic correlation of 0.85. The effects of 82 selected susceptibility single nucleotide polymorphisms on these insulin secretion indices supported our interpretation of the data and added insight into the biological differences between the examined traits......AIMS/HYPOTHESIS: We examined the extent to which surrogate measures of insulin release have shared genetic causes. METHODS: Genetic and phenotypic correlations were calculated in a family cohort (n = 315) in which beta cell indices were estimated based on fasting and oral glucose-stimulated plasma...... glucose, serum C-peptide and serum insulin levels. Furthermore, we genotyped a large population-based cohort (n = 6,269) for common genetic variants known to associate with type 2 diabetes, fasting plasma glucose levels or fasting serum insulin levels to examine their association with various indices...

Cell death and impairment of glucose-stimulated insulin secretion induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the β-cell line INS-1E

International Nuclear Information System (INIS)

Piaggi, Simona; Novelli, Michela; Martino, Luisa; Masini, Matilde; Raggi, Chiara; Orciuolo, Enrico; Masiello, Pellegrino; Casini, Alessandro; De Tata, Vincenzo

2007-01-01

The aim of this research was to characterize 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity on the insulin-secreting β-cell line INS-1E. A sharp decline of cell survival (below 20%) was observed after 1 h exposure to TCDD concentrations between 12.5 and 25 nM. Ultrastructurally, β-cell death was characterized by extensive degranulation, appearance of autophagic vacuoles, and peripheral nuclear condensation. Cytotoxic concentrations of TCDD rapidly induced a dose-dependent increase in intracellular calcium concentration. Blocking calcium entry by EGTA significantly decreased TCDD cytotoxicity. TCDD was also able to rapidly induce mitochondrial depolarization. Interestingly, 1 h exposition of INS-1E cells to very low TCDD concentrations (0.05-1 nM) dramatically impaired glucose-stimulated but not KCl-stimulated insulin secretion. In conclusion, our results clearly show that TCDD exerts a direct β-cell cytotoxic effect at concentrations of 15-25 nM, but also markedly impairs glucose-stimulated insulin secretion at concentrations 20 times lower than these. On the basis of this latter observation we suggest that pancreatic β-cells could be considered a specific and sensitive target for dioxin toxicity

Lyophilized insulin nanoparticles prepared from quaternized N-aryl derivatives of chitosan as a new strategy for oral delivery of insulin: in vitro, ex vivo and in vivo characterizations.

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Mahjub, Reza; Radmehr, Moojan; Dorkoosh, Farid Abedin; Ostad, Seyed Naser; Rafiee-Tehrani, Morteza

2014-12-01

The purpose of this research was the development, in vitro, ex vivo and in vivo characterization of lyophilized insulin nanoparticles prepared from quaternized N-aryl derivatives of chitosan. Insulin nanoparticles were prepared from methylated N-(4-N,N-dimethylaminobenzyl), methylated N-(4 pyridinyl) and methylated N-(benzyl). Insulin nanoparticles containing non-modified chitosan and also trimethyl chiotsan (TMC) were also prepared as control. The effects of the freeze-drying process on physico-chemical properties of nanoparticles were investigated. The release of insulin from the nanoparticles was studied in vitro. The mechanism of the release of insulin from different types of nanoparticles was determined using curve fitting. The secondary structure of the insulin released from the nanoparticles was analyzed using circular dichroism and the cell cytotoxicity of nanoparticles on a Caco-2 cell line was determined. Ex vivo studies were performed on excised rat jejunum using Frantz diffusion cells. In vivo studies were performed on diabetic male Wistar rats and blood glucose level and insulin serum concentration were determined. Optimized nanoparticles with proper physico-chemical properties were obtained. The lyophilization process was found to cause a decrease in zeta potential and an increase in PdI as well as and a decrease in entrapment efficiency (EE%) and loading efficiency (LE%) but conservation in size of nanoparticles. Atomic force microscopy (AFM) images showed non-aggregated, stable and spherical to sub-spherical nanoparticles. The in vitro release study revealed higher release rates for lyophilized compared to non-lyophilized nanoparticles. Cytotoxicity studies on Caco-2 cells revealed no significant cytotoxicity for prepared nanoparticles after 3-h post-incubation but did show the concentration-dependent cytotoxicity after 24 h. The percentage of cumulative insulin determined from ex vivo studies was significantly higher in nanoparticles prepared

Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence.

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Zachary J Farino

Full Text Available Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment.

DEFECTS IN INSULIN-SECRETION IN NIDDM - B-CELL GLUCOSE INSENSITIVITY OR GLUCOSE TOXICITY

NARCIS (Netherlands)

VANHAEFTEN, TW

In NIDDM, first-phase insulin release to glucose is (almost) absent. However, in contrast to older studies which suggested that in NIDDM the B-cell is ''blind'' for glucose, recent evidence indicates that the B-cell is not insensitive for glucose as far as second phase release is concerned. This

Insulin and leptin induce Glut4 plasma membrane translocation and glucose uptake in a human neuronal cell line by a phosphatidylinositol 3-kinase- dependent mechanism.

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Benomar, Yacir; Naour, Nadia; Aubourg, Alain; Bailleux, Virginie; Gertler, Arieh; Djiane, Jean; Guerre-Millo, Michèle; Taouis, Mohammed

2006-05-01

The insulin-sensitive glucose transporter Glut4 is expressed in brain areas that regulate energy homeostasis and body adiposity. In contrast with peripheral tissues, however, the impact of insulin on Glut4 plasma membrane (PM) translocation in neurons is not known. In this study, we examined the role of two anorexic hormones (leptin and insulin) on Glut4 translocation in a human neuronal cell line that express endogenous insulin and leptin receptors. We show that insulin and leptin both induce Glut4 translocation to the PM of neuronal cells and activate glucose uptake. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, totally abolished insulin- and leptin-dependent Glut4 translocation and stimulation of glucose uptake. Thus, Glut4 translocation is a phosphatidylinositol 3-kinase-dependent mechanism in neuronal cells. Next, we investigated the impact of chronic insulin and leptin treatments on Glut4 expression and translocation. Chronic exposure of neuronal cells to insulin or leptin down-regulates Glut4 proteins and mRNA levels and abolishes the acute stimulation of glucose uptake in response to acute insulin or leptin. In addition, chronic treatment with either insulin or leptin impaired Glut4 translocation. A cross-desensitization between insulin and leptin was apparent, where exposure to insulin affects leptin-dependent Glut4 translocation and vice versa. This cross-desensitization could be attributed to the increase in suppressor of cytokine signaling-3 expression, which was demonstrated in response to each hormone. These results provide evidence to suggest that Glut4 translocation to neuronal PM is regulated by both insulin and leptin signaling pathways. These pathways might contribute to an in vivo glucoregulatory reflex involving a neuronal network and to the anorectic effect of insulin and leptin.

Phosphatidylcholine biosynthesis and insulin release in rat islets of Langerhans

International Nuclear Information System (INIS)

Hoffman, J.M.

1988-01-01

Turnover of phosphatidylcholine (PC) has been demonstrated to play a role in glucose stimulation of insulin release by pancreatic islets of Langerhans. The activity of the islet CDP-choline pathway of PC synthesis was determined by measuring the incorporation of radiolabeled choline or 32 PO 4 into PC, phosphorylcholine and CDP-choline. Concurrently, insulin release was measured by radioimmunoassay to correlate insulin release and PC synthesis. Glucose concentrations greater than 8.5 mM stimulated CDP-choline pathway activity. However, measurement of PC lipid phosphorus tended to decrease, suggesting that stimulation of the CDP-choline pathway was a means of replenishing PC pools diminished by hydrolysis of PC. Inhibition of glucose oxidation by mannoheptulose or incubations under hypoxic conditions prevented stimulation of the CDP-choline pathway, while inhibition of phospholipase A 2 (PLA 2 ) and secretion by the removal of extracellular Ca 2+ potentiated the stimulation seen with glucose

Relationship between insulin release and 65zinc efflux from rat pancreatic islets maintained in tissue culture

International Nuclear Information System (INIS)

Formby, B.; Schmid-Formby, F.; Grodsky, G.M.

1984-01-01

In short-term batch-incubation or perfusion experiments, we studied insulin release and associated 65 Zn efflux from rat pancreatic islets loaded with 65 Zn by 24-h tissue culture in low-glucose medium. The fractional basal insulin release and 65 Zn efflux were 0.4% and 3% of total content/h/islet, respectively. Thus, basal 65 Zn efflux was much greater than that to be accounted for if zinc was released proportionally with insulin release only; extragranular zinc flux was suggested. Two millimolar glucose, with or without 1 mM 3-isobutyl-1-methylxanthine (IBMX), affected neither insulin release nor associated 65 Zn efflux. Twenty-five millimolar glucose produced a significant threefold increase in insulin release above baseline, but somewhat decreased 65 Zn efflux at marginal significance. Glucose (25 mM) plus 1 mM IBMX provoked a high increase in insulin release and an associated 30% increase in fractional 65 Zn efflux over basal. Calculations based on previous estimations of 65 Zn distribution and equilibrium with islet zinc indicated that molar zinc efflux was more than sufficient to account for a 2-zinc-insulin hexamer. L-Leucine (2 or 20 mM) plus 1 mM IBMX caused far greater 65 Zn efflux for the amount of insulin released, indicating additional 65 Zn mobilization not directly related to insulin secretion. To evaluate 65 Zn efflux during inhibited insulin secretion, batch incubations were performed in 100% D 2 O or at 27 degrees C, conditions that inhibited insulin release stimulated by high glucose plus IBMX. These agents decreased the 65 Zn efflux far below the basal value (35% and 50%, respectively) and greater than could be accounted for by the attendent inhibition of insulin secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Role of sialic acid in insulin action and the insulin resistance of diabetes mellitus

International Nuclear Information System (INIS)

Salhanick, A.I.; Amatruda, J.M.

1988-01-01

Adipocytes treated with neuraminidase show markedly reduced responsiveness to insulin without any alteration in insulin binding. In addition, several studies have separately demonstrated both insulin resistance and decreases in membrane sialic acid content and associated biosynthetic enzymes in diabetes mellitus. In the present study, the authors investigated the role that sialic acid residues may play in insulin action and in the hepatic insulin resistance associated with nonketotic diabetes. Primary cultures of hepatocytes from normal rats treated with neuraminidase demonstrated a dose-dependent decrease in insulin-stimulated lipogenesis. At a concentration of neuraminidase that decreases insulin action by 50%, 23% of total cellular sialic acid content was released. Neuraminidase-releasable sialic acid was significantly decreased in hepatocytes from diabetic rats and this was associated with significant insulin resistance. Treatment of hepatocytes from diabetic rats with cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) enhanced insulin responsiveness 39%. The enhanced insulin responsiveness induced by CMP-NANA was blocked by cytidine 5'-monophosphate (CMP) suggesting that the CMP-NANA effect was catalyzed by a cell surface sialyl-transferase. CMP reduced neuraminidase-releasable [ 14 C]sialic acid incorporation into hepatocytes by 43%. The data demonstrate a role for cell surface sialic acid residues in hepatic insulin action and support a role for decreased cell surface sialic acid residues in the insulin resistance of diabetes mellitus

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

Metabolomic and proteomic analysis of a clonal insulin-producing beta-cell line (INS-1 832/13).

Science.gov (United States)

Fernandez, Céline; Fransson, Ulrika; Hallgard, Elna; Spégel, Peter; Holm, Cecilia; Krogh, Morten; Wårell, Kristofer; James, Peter; Mulder, Hindrik

2008-01-01

Metabolites generated from fuel metabolism in pancreatic beta-cells control exocytosis of insulin, a process which fails in type 2 diabetes. To identify and quantify these metabolites, global and unbiased analysis of cellular metabolism is required. To this end, polar metabolites, extracted from the clonal 832/13 beta-cell line cultured at 2.8 and 16.7 mM glucose for 48 h, were derivatized followed by identification and quantification, using gas chromatography (GC) and mass spectrometry (MS). After culture at 16.7 mM glucose for 48 h, 832/13 beta-cells exhibited a phenotype reminiscent of glucotoxicity with decreased content and secretion of insulin. The metabolomic analysis revealed alterations in the levels of 7 metabolites derived from glycolysis, the TCA cycle and pentose phosphate shunt, and 4 amino acids. Principal component analysis of the metabolite data showed two clusters, corresponding to the cells cultured at 2.8 and 16.7 mM glucose, respectively. Concurrent changes in protein expression were analyzed by 2-D gel electrophoresis followed by LC-MS/MS. The identities of 86 spots corresponding to 75 unique proteins that were significantly different in 832/13 beta-cells cultured at 16.7 mM glucose were established. Only 5 of these were found to be metabolic enzymes that could be involved in the metabolomic alterations observed. Anticipated changes in metabolite levels in cells exposed to increased glucose were observed, while changes in enzyme levels were much less profound. This suggests that substrate availability, allosteric regulation, and/or post-translational modifications are more important determinants of metabolite levels than enzyme expression at the protein level.

A modified method of insulin producing cells' generation from bone marrow-derived mesenchymal stem cells.

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Czubak, Paweł; Bojarska-Junak, Agnieszka; Tabarkiewicz, Jacek; Putowski, Lechosław

2014-01-01

Type 1 diabetes mellitus is a result of autoimmune destruction of pancreatic insulin producing β-cells and so far it can be cured only by insulin injection, by pancreas transplantation, or by pancreatic islet cells' transplantation. The methods are, however, imperfect and have a lot of disadvantages. Therefore new solutions are needed. The best one would be the use of differentiated mesenchymal stem cells (MSCs). In the present study, we investigated the potential of the bone marrow-derived MSCs line for in vitro differentiation into insulin producing cells (IPSs). We applied an 18-day protocol to differentiate MSCs. Differentiating cells formed cell clusters some of which resembled pancreatic islet-like cells. Using dithizone we confirmed the presence of insulin in the cells. What is more, the expression of proinsulin C-peptide in differentiated IPCs was analyzed by flow cytometry. For the first time, we investigated the influence of growth factors' concentration on IPCs differentiation efficiency. We have found that an increase in the concentration of growth factors up to 60 ng/mL of β-FGF/EGF and 30 ng/mL of activin A/β-cellulin increases the percentage of IPCs. Further increase of growth factors does not show any increase of the percentage of differentiated cells. Our findings suggest that the presented protocol can be adapted for differentiation of insulin producing cells from stem cells.

ADAMTS13 expression in human chondrosarcoma cells induced by insulin

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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

In vitro phenotypic, genomic and proteomic characterization of a cytokine-resistant murine β-TC3 cell line.

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Antonina Coppola

Full Text Available Type 1 diabetes mellitus (T1DM is caused by the selective destruction of insulin-producing β-cells. This process is mediated by cells of the immune system through release of nitric oxide, free radicals and pro-inflammatory cytokines, which induce a complex network of intracellular signalling cascades, eventually affecting the expression of genes involved in β-cell survival.The aim of our study was to investigate possible mechanisms of resistance to cytokine-induced β-cell death. To this purpose, we created a cytokine-resistant β-cell line (β-TC3R by chronically treating the β-TC3 murine insulinoma cell line with IL-1β + IFN-γ. β-TC3R cells exhibited higher proliferation rate and resistance to cytokine-mediated cell death in comparison to the parental line. Interestingly, they maintained expression of β-cell specific markers, such as PDX1, NKX6.1, GLUT2 and insulin. The analysis of the secretory function showed that β-TC3R cells have impaired glucose-induced c-peptide release, which however was only moderately reduced after incubation with KCl and tolbutamide. Gene expression analysis showed that β-TC3R cells were characterized by downregulation of IL-1β and IFN-γ receptors and upregulation of SOCS3, the classical negative regulator of cytokines signaling. Comparative proteomic analysis showed specific upregulation of 35 proteins, mainly involved in cell death, stress response and folding. Among them, SUMO4, a negative feedback regulator in NF-kB and JAK/STAT signaling pathways, resulted hyper-expressed. Silencing of SUMO4 was able to restore sensitivity to cytokine-induced cell death in β-TC3R cells, suggesting it may play a key role in acquired cytokine resistance by blocking JAK/STAT and NF-kB lethal signaling.In conclusion, our study represents the first extensive proteomic characterization of a murine cytokine-resistant β-cell line, which might represent a useful tool for studying the mechanisms involved in resistance to

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

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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.

Fabrication of Glucose-Sensitive Layer-by-Layer Films for Potential Controlled Insulin Release Applications

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Talusan Timothy Jemuel E.

2015-01-01

Full Text Available Self-regulated drug delivery systems (DDS are potential alternative to the conventional method of introducing insulin to the body due to their controlled drug release mechanism. In this study, Layer-by-Layer technique was utlized to manufacture drug loaded, pH responsive thin films. Insulin was alternated with pH-sensitive, [2-(dimethyl amino ethyl aminoacrylate] (PDMAEMA and topped of with polymer/glucose oxidase (GOD layers. Similarly, films using a different polymer, namely Poly(Acrylic Acid (PAA were also fabricated. Exposure of the films to glucose solutions resulted to the production of gluconic acid causing a polymer conformation change due to protonation, thus releasing the embedded insulin. The insulin release was monitored by subjecting the dipping glucose solutions to Bradford Assay. Films exhibited a reversal in drug release profile in the presence of glucose as compared to without glucose. PAA films were also found out to release more insulin compared to that of the PDMAEMA films.The difference in the profile of the two films were due to different polymer-GOD interactions, since both films exhibited almost identical profiles when embedded with Poly(sodium 4-styrenesulfonate (PSS instead of GOD.

Direct Sensing of Nutrients via a LAT1-like Transporter in Drosophila Insulin-Producing Cells

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Gérard Manière

2016-09-01

Full Text Available Dietary leucine has been suspected to play an important role in insulin release, a hormone that controls satiety and metabolism. The mechanism by which insulin-producing cells (IPCs sense leucine and regulate insulin secretion is still poorly understood. In Drosophila, insulin-like peptides (DILP2 and DILP5 are produced by brain IPCs and are released in the hemolymph after leucine ingestion. Using Ca2+-imaging and ex vivo cultured larval brains, we demonstrate that IPCs can directly sense extracellular leucine levels via minidiscs (MND, a leucine transporter. MND knockdown in IPCs abolished leucine-dependent changes, including loss of DILP2 and DILP5 in IPC bodies, consistent with the idea that MND is necessary for leucine-dependent DILP release. This, in turn, leads to a strong increase in hemolymph sugar levels and reduced growth. GDH knockdown in IPCs also reduced leucine-dependent DILP release, suggesting that nutrient sensing is coupled to the glutamate dehydrogenase pathway.

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.

Matrix stiffness and oxigen tension modulate epigenetic conversion of mouse dermal fibroblasts into insulin producing cells.

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Alessandro Zenobi

2017-05-01

Full Text Available In vivo, cells are surrounded by a three-dimensional (3-D organization of supporting matrix, neighboring cells and a gradient of chemical and mechanical signals (Antoni, et al., 2015. However, the present understanding of many biological processes is mainly based on two-dimensional (2-D systems that typically provides a static environment. In the present study, we tested two different 3-D culture systems and apply them to the epigenetic conversion of mouse dermal fibroblasts into insulin producing-cells (Pennarossa, et al., 2013; Brevini, et al., 2015, combining also the use of two oxygen tensions. In particular, cells were differentiated using the Polytetrafluoroethylene micro-bioreactor (PTFE and the Polyacrylamide (PAA gels with different stiffness (1 kPa; 4 kPa, maintained either in the standard 20% or in the more physiological 5% oxygen tensions. Standard differentiation performed on plastic substrates was assessed as a control. Cell morphology (Fig.1A, insulin expression and release were analyzed to evaluate the role of both stiffness and oxygen tension in the process. The results obtained showed that 1 kPa PAA gel and PTFE system induced a significantly higher insulin expression and release than plastic and 4 kPa PAA gel, especially in low oxygen condition (Fig.1B. Furthermore, comparing the efficiency of the two systems tested, 1 kPa PAA gel ensured a higher insulin transcription than PTFE (Fig.1C. Recent studies show the direct influence of substrates on lineage commitment and cell differentiation (Engler, et al., 2006; Evans, et al., 2009. The evidence here presented confirm that the use of an appropriate stiffness (similar to the pancreatic tissue, combined with a physiological oxygen tension, promote β-cell differentiation, with beneficial effects on cell functional activity and insulin release. The present results highlight the importance of 3-D cell rearrangement and oxigen tension to promote in vitro epigenetic conversion of

Generation and functional analysis of T cell lines and clones specific for schistosomula released products (SRP-A).

Science.gov (United States)

Damonneville, M; Velge, F; Verwaerde, C; Pestel, J; Auriault, C; Capron, A

1987-08-01

Antigens present in the products released by the larval stage of schistosome (SRP-A) were shown to induce a strong cytotoxic and protective IgE response both in the rat and the monkey. T cell lines and clones specific for SRP-A or 26 kD antigens which are the main target of the cytotoxic IgE have been derived. The passive transfer of SRP-A specific T lymphocytes into infected rats led to an increase of the IgE response, conferring a significant level of protection to the rats. In coculture assays in vitro, these cell lines significantly enhanced the production of IgE by SRP-A sensitized rat spleen cells. This helper effect on the IgE response was confirmed with 26 kD T cell clone supernatants. Moreover, supernatants obtained after stimulation with phorbol myristate acetate were able to enhance the IgE production of a hybridoma B cell line (B48-14) producing a monoclonal IgE antibody, cytotoxic for the schistosomula.

Effect of glibenclamide on insulin release at moderate and high blood glucose levels in normal man

NARCIS (Netherlands)

Ligtenberg, JJM; Venker, CE; Sluiter, WJ; VanHaeften, TW

Insulin release occurs in two phases; sulphonylurea derivatives may have different potencies in stimulating first-and second-phase insulin release. We studied the effect of glibenclamide on insulin secretion at submaximally and maximally stimulating blood glucose levels with a primed hyperglycaemic

The potential role of SOCS-3 in the interleukin-1beta-induced desensitization of insulin signaling in pancreatic beta-cells

DEFF Research Database (Denmark)

Emanuelli, Brice; Glondu, Murielle; Filloux, Chantal

2004-01-01

insulin signaling is required for the optimal beta-cell function, we assessed the effect of IL-1beta on the insulin pathway in a rat pancreatic beta-cell line. We show that IL-1beta decreases insulin-induced tyrosine phosphorylation of the insulin receptor (IR) and insulin receptor substrate (IRS...

Potentiation of insulin release in response to amino acid methyl esters correlates to activation of islet glutamate dehydrogenase activity

DEFF Research Database (Denmark)

Kofod, Hans; Lernmark, A; Hedeskov, C J

1986-01-01

Column perifusion of mouse pancreatic islets was used to study the ability of amino acids and their methyl esters to influence insulin release and activate islet glutamate dehydrogenase activity. In the absence of L-glutamine, L-serine and the methyl ester of L-phenylalanine, but neither L...... glutamate dehydrogenase activity showed that only the two methyl esters of L-phenylalanine and L-serine activated the enzyme. It is concluded that the mechanism by which methyl esters of amino acids potentiate insulin release is most likely to be mediated by the activation of pancreatic beta-cell glutamate...

Retroendocytosis of insulin in rat adipocytes

International Nuclear Information System (INIS)

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

1986-01-01

A variety of ligands internalized by receptor-mediated endocytosis follow a short circuit pathway that does not lead to degradation but results in rapid exocytosis of intact ligand, a process termed retroendocytosis. We studied the time course of [ 125 I]iodoinsulin processing and retroendocytosis after internalization in isolated rat adipocytes. After steady state binding and internalization, surface receptor-bound insulin was removed by exposing cells to a low pH at low temperatures. The cells containing internalized [ 125 I]iodoinsulin were reincubated in fresh medium; subsequently, the radioactivity remaining within the cells and released into the medium were analyzed at various times by trichloroacetic acid (TCA) precipitation, Sephadex G-50 gel filtration, and reverse phase HPLC. Cell-associated radioactivity progressively decreased after reincubation in 37 C buffer, with 50% released in 9 min and 85% by 45 min. In the media, TCA-precipitable material appeared quickly, with a t1/2 of 2 min, and plateaued by 10 min. TCA-soluble material was released continually throughout the 45-min period. The release of both TCA-precipitable and TCA-soluble material was temperature and energy dependent. Sephadex G-50 chromatography demonstrated the loss of insulin from the intracellular pool and its appearance in the medium with a time course similar to that of TCA-precipitable material. Reverse phase HPLC demonstrated that the intracellular and medium radioactivity eluting in peak II (insulin peak) on Sephadex G-50 was composed of both intact insulin and intermediates. After the internalization of insulin, rat adipocytes release not only small mol wt degradation products of insulin, but also insulin intermediates and intact insulin. The rate of retroendocytosis reported here is almost identical to the rate of insulin receptor recycling in rat adipocytes

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

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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.

The type 2 diabetes associated minor allele of rs2237895 KCNQ1 associates with reduced insulin release following an oral glucose load.

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Johan Holmkvist

Full Text Available BACKGROUND: Polymorphisms in the potassium channel, voltage-gated, KQT-like subfamily, member 1 (KCNQ1 have recently been reported to associate with type 2 diabetes. The primary aim of the present study was to investigate the putative impact of these KCNQ1 polymorphisms (rs2283228, rs2237892, rs2237895, and rs2237897 on estimates of glucose stimulated insulin release. METHODOLOGY/PRINCIPAL FINDINGS: Genotypes were examined for associations with serum insulin levels following an oral glucose tolerance test (OGTT in a population-based sample of 6,039 middle-aged and treatment-naïve individuals. Insulin release indices estimated from the OGTT and the interplay between insulin sensitivity and insulin release were investigated using linear regression and Hotelling T2 analyses. Applying an additive genetic model the minor C-allele of rs2237895 was associated with reduced serum insulin levels 30 min (mean+/-SD: (CC 277+/-160 vs. (AC 280+/-164 vs. (AA 299+/-200 pmol/l, p = 0.008 after an oral glucose load, insulinogenic index (29.6+/-17.4 vs. 30.2+/-18.7vs. 32.2+/-22.1, p = 0.007, incremental area under the insulin curve (20,477+/-12,491 vs. 20,503+/-12,386 vs. 21,810+/-14,685, p = 0.02 among the 4,568 individuals who were glucose tolerant. Adjustment for the degree of insulin sensitivity had no effect on the measures of reduced insulin release. The rs2237895 genotype had a similar impact in the total sample of treatment-naïve individuals. No association with measures of insulin release were identified for the less common diabetes risk alleles of rs2237892, rs2237897, or rs2283228. CONCLUSION: The minor C-allele of rs2237895 of KCNQ1, which has a prevalence of about 42% among Caucasians was associated with reduced measures of insulin release following an oral glucose load suggesting that the increased risk of type 2 diabetes, previously reported for this variant, likely is mediated through an impaired beta cell function.

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

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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

pH-Dependent Release of Insulin from Layer-by-Layer-Deposited Polyelectrolyte Microcapsules

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Kentaro Yoshida

2015-07-01

Full Text Available Insulin-containing microcapsules were prepared by a layer-by-layer (LbL deposition of poly(allylamine hydrochloride (PAH and polyanions, such as poly(styrenesulfonate (PSS, poly(vinyl sulfate (PVS, and dextran sulfate (DS on insulin-containing calcium carbonate (CaCO3 microparticles. The CaCO3 core was dissolved in diluted HCl solution to obtain insulin-containing hollow microcapsules. The microcapsules were characterized by scanning electron microscope (SEM and atomic force microscope (AFM images and ζ-potential. The release of insulin from the microcapsules was faster at pH 9.0 and 7.4 than in acidic solutions due to the different charge density of PAH. In addition, insulin release was suppressed when the microcapsules were constructed using PAH with a lower molecular weight, probably owing to a thicker shell of the microcapsules. The results suggested a potential use of the insulin-containing microcapsules for developing insulin delivery systems.

Targeting non-small cell lung cancer cells by dual inhibition of the insulin receptor and the insulin-like growth factor-1 receptor.

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Emma E Vincent

Full Text Available Phase III trials of the anti-insulin-like growth factor-1 receptor (IGF1R antibody figitumumab in non-small cell lung cancer (NSCLC patients have been discontinued owing to lack of survival benefit. We investigated whether inhibition of the highly homologous insulin receptor (IR in addition to the IGF1R would be more effective than inhibition of the IGF1R alone at preventing the proliferation of NSCLC cells. Signalling through IGF1R and IR in the NSCLC cell lines A549 and Hcc193 was stimulated by a combination of IGF1, IGF2 and insulin. It was inhibited by antibodies that block ligand binding, αIR3 (IGF1R and IR47-9 (IR, and by the ATP-competitive small molecule tyrosine kinase inhibitors AZ12253801 and NVPAWD742 which inhibit both IGF1R and IR tyrosine kinases. The effect of inhibitors was determined by an anchorage-independent proliferation assay and by analysis of Akt phosphorylation. In Hcc193 cells the reduction in cell proliferation and Akt phosphorylation due to anti-IGF1R antibody was enhanced by antibody-mediated inhibition of the IR whereas in A549 cells, with a relatively low IR:IGF1R expression ratio, it was not. In each cell line proliferation and Akt phosphorylation were more effectively inhibited by AZ12253801 and NVPAWD742 than by combined αIR3 and IR47-9. When the IGF1R alone is inhibited, unencumbered signalling through the IR can contribute to continued NSCLC cell proliferation. We conclude that small molecule inhibitors targeting both the IR and IGF1R more effectively reduce NSCLC cell proliferation in a manner independent of the IR:IGF1R expression ratio, providing a therapeutic rationale for the treatment of this disease.

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

The human hepatocyte cell lines IHH and HepaRG: models to study glucose, lipid and lipoprotein metabolism.

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Samanez, Carolina Huaman; Caron, Sandrine; Briand, Olivier; Dehondt, Hélène; Duplan, Isabelle; Kuipers, Folkert; Hennuyer, Nathalie; Clavey, Véronique; Staels, Bart

2012-07-01

Metabolic diseases reach epidemic proportions. A better knowledge of the associated alterations in the metabolic pathways in the liver is necessary. These studies need in vitro human cell models. Several human hepatoma models are used, but the response of many metabolic pathways to physiological stimuli is often lost. Here, we characterize two human hepatocyte cell lines, IHH and HepaRG, by analysing the expression and regulation of genes involved in glucose and lipid metabolism. Our results show that the glycolysis pathway is activated by glucose and insulin in both lines. Gluconeogenesis gene expression is induced by forskolin in IHH cells and inhibited by insulin in both cell lines. The lipogenic pathway is regulated by insulin in IHH cells. Finally, both cell lines secrete apolipoprotein B-containing lipoproteins, an effect promoted by increasing glucose concentrations. These two human cell lines are thus interesting models to study the regulation of glucose and lipid metabolism.

Numerical Modelling of Insulin and Amyloglucosidase Release from Swelling Ca-Alginate Beads

DEFF Research Database (Denmark)

Rasmussen, Michael R.; Snabe, T.; Pedersen, Lars Haastrup

2003-01-01

The release of insulin hexamer (39 kD) and amyloglucosidase (AMG, 97 kD), entrapped in spherical Ca–alginate beads, was investigated. While the release of insulin could be described solely by diffusion this was not the case for the 1.6 (r m /r m) larger AMG protein, where rm is the Stokes....... Although it should be expected that the effective diffusion coefficient of AMG is only slightly lower than that of insulin, the results show that the effective diffusions coefficient of AMG was found to be much smaller. In the case of AMG, it was shown that including bead size changes and the resulting...

Application of insulin-like growth factor-I and insulin release test in diabetes mellitus

International Nuclear Information System (INIS)

Chen Dong; Ma Yongxiu; Duan Wenruo

2003-01-01

The purpose of this study was to determine the role of insulin-like growth factor-I (IGF-I) and insulin release test (IRT) in understanding the extent of damage to ability of reducing blood sugar in different types of diabetes mellitus (DM) and in selection of treatment plan and adjustment of using drugs. OGTT, IRT and determination of IGF-I level of 67 normal subjects and 217 DM patients were performed. The result was analyzed comparatively. The level of IGF-I was negatively correlated with the level of fasting blood sugar, and positively correlated with the level of fasting insulin. Our conclusions are: There are two ways of reducing blood sugar: one is by insulin, and the other is by IGF-I. IRT can reflect the former better, and IGF-I the latter. The combination of these two is of significant value in diagnosis and treatment of DM

Insulin-loaded poly(epsilon-caprolactone) nanoparticles: efficient, sustained and safe insulin delivery system.

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de Araújo, Thiago M; Teixeira, Zaine; Barbosa-Sampaio, Helena C; Rezende, Luiz F; Boschero, Antonio C; Durán, Nelson; Höehr, Nelci F

2013-06-01

The aim of this work was to develop an efficient, biodegradable, biocompatible and safe controlled release system using insulin-loaded poly(epsilon-caprolactone) (PCL) nanoparticles. The insulin-loaded PCL nanoparticles were prepared by double emulsion method (water-in-oil-in-water) using Pluronic F68 as emulsifier. Using the double emulsion method a high insulin encapsulation efficiency (90.6 +/-1.6%) with a zeta potential of -29 +/-2.7 mV and average particle size of 796 +/-10.5 nm was obtained. Insulin-loaded PCL nanoparticles showed no toxicity to MIN6 cells. Insulin nanoparticles administered subcutaneously and intraperitoneally in rats reduced glycaemia of basal levels after 15 minutes, and presented a sustainable hypoglycemic effect on insulin-dependent type 1 diabetic rats, showing to be more efficient than unencapsulated insulin. Furthermore, these nanoparticles were not hepatotoxic, as evaluated by the effect over liver cell-death and oxidative stress scavenger system in rats. These results suggest that insulin-loaded PCL nanoparticles prepared by water-in-oil-in-water emulsion method are biocompatible, efficient and safe insulin-delivering system with controlled insulin release, which indicates that it may be a powerful tool for insulin-dependent patients care.

Glucose-mediated control of ghrelin release from primary cultures of gastric mucosal cells

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Sakata, Ichiro; Park, Won-Mee; Walker, Angela K.; Piper, Paul K.; Chuang, Jen-Chieh; Osborne-Lawrence, Sherri

2012-01-01

The peptide hormone ghrelin is released from a distinct group of gastrointestinal cells in response to caloric restriction, whereas its levels fall after eating. The mechanisms by which ghrelin secretion is regulated remain largely unknown. Here, we have used primary cultures of mouse gastric mucosal cells to investigate ghrelin secretion, with an emphasis on the role of glucose. Ghrelin secretion from these cells upon exposure to different d-glucose concentrations, the glucose antimetabolite 2-deoxy-d-glucose, and other potential secretagogues was assessed. The expression profile of proteins involved in glucose transport, metabolism, and utilization within highly enriched pools of mouse ghrelin cells and within cultured ghrelinoma cells was also determined. Ghrelin release negatively correlated with d-glucose concentration. Insulin blocked ghrelin release, but only in a low d-glucose environment. 2-Deoxy-d-glucose prevented the inhibitory effect of high d-glucose exposure on ghrelin release. mRNAs encoding several facilitative glucose transporters, hexokinases, the ATP-sensitive potassium channel subunit Kir6.2, and sulfonylurea type 1 receptor were expressed highly within ghrelin cells, although neither tolbutamide nor diazoxide exerted direct effects on ghrelin secretion. These findings suggest that direct exposure of ghrelin cells to low ambient d-glucose stimulates ghrelin release, whereas high d-glucose and glucose metabolism within ghrelin cells block ghrelin release. Also, low d-glucose sensitizes ghrelin cells to insulin. Various glucose transporters, channels, and enzymes that mediate glucose responsiveness in other cell types may contribute to the ghrelin cell machinery involved in regulating ghrelin secretion under these different glucose environments, although their exact roles in ghrelin release remain uncertain. PMID:22414807

Stress Hyperglycemia, Insulin Treatment, and Innate Immune Cells

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Fangming Xiu

2014-01-01

Full Text Available Hyperglycemia (HG and insulin resistance are the hallmarks of a profoundly altered metabolism in critical illness resulting from the release of cortisol, catecholamines, and cytokines, as well as glucagon and growth hormone. Recent studies have proposed a fundamental role of the immune system towards the development of insulin resistance in traumatic patients. A comprehensive review of published literatures on the effects of hyperglycemia and insulin on innate immunity in critical illness was conducted. This review explored the interaction between the innate immune system and trauma-induced hypermetabolism, while providing greater insight into unraveling the relationship between innate immune cells and hyperglycemia. Critical illness substantially disturbs glucose metabolism resulting in a state of hyperglycemia. Alterations in glucose and insulin regulation affect the immune function of cellular components comprising the innate immunity system. Innate immune system dysfunction via hyperglycemia is associated with a higher morbidity and mortality in critical illness. Along with others, we hypothesize that reduction in morbidity and mortality observed in patients receiving insulin treatment is partially due to its effect on the attenuation of the immune response. However, there still remains substantial controversy regarding moderate versus intensive insulin treatment. Future studies need to determine the integrated effects of HG and insulin on the regulation of innate immunity in order to provide more effective insulin treatment regimen for these patients.

Glucose tolerance, insulin sensitivity and insulin release in European non-diabetic carriers of a polymorphism upstream of CDKN2A and CDKN2B

DEFF Research Database (Denmark)

Hribal, M L; Presta, I; Procopio, T

2011-01-01

The aim of this study was to investigate the association of the rs10811661 polymorphism near the CDKN2B/CDKN2A genes with glucose tolerance, insulin sensitivity and insulin release in three samples of white people with European ancestry.......The aim of this study was to investigate the association of the rs10811661 polymorphism near the CDKN2B/CDKN2A genes with glucose tolerance, insulin sensitivity and insulin release in three samples of white people with European ancestry....

Generation and functional analysis of T cell lines and clones specific for schistosomula released products (SRP-A).

Science.gov (United States)

Damonneville, M; Velge, F; Verwaerde, C; Pestel, J; Auriault, C; Capron, A

1987-01-01

Antigens present in the products released by the larval stage of schistosome (SRP-A) were shown to induce a strong cytotoxic and protective IgE response both in the rat and the monkey. T cell lines and clones specific for SRP-A or 26 kD antigens which are the main target of the cytotoxic IgE have been derived. The passive transfer of SRP-A specific T lymphocytes into infected rats led to an increase of the IgE response, conferring a significant level of protection to the rats. In coculture assays in vitro, these cell lines significantly enhanced the production of IgE by SRP-A sensitized rat spleen cells. This helper effect on the IgE response was confirmed with 26 kD T cell clone supernatants. Moreover, supernatants obtained after stimulation with phorbol myristate acetate were able to enhance the IgE production of a hybridoma B cell line (B48-14) producing a monoclonal IgE antibody, cytotoxic for the schistosomula. PMID:3498590

The F-actin modifier villin regulates insulin granule dynamics and exocytosis downstream of islet cell autoantigen 512

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Hassan Mziaut

2016-08-01

Full Text Available Objective: Insulin release from pancreatic islet β cells should be tightly controlled to avoid hypoglycemia and insulin resistance. The cortical actin cytoskeleton is a gate for regulated exocytosis of insulin secretory granules (SGs by restricting their mobility and access to the plasma membrane. Prior studies suggest that SGs interact with F-actin through their transmembrane cargo islet cell autoantigen 512 (Ica512 (also known as islet antigen 2/Ptprn. Here we investigated how Ica512 modulates SG trafficking and exocytosis. Methods: Transcriptomic changes in Ica512−/− mouse islets were analyzed. Imaging as well as biophysical and biochemical methods were used to validate if and how the Ica512-regulated gene villin modulates insulin secretion in mouse islets and insulinoma cells. Results: The F-actin modifier villin was consistently downregulated in Ica512−/− mouse islets and in Ica512-depleted insulinoma cells. Villin was enriched at the cell cortex of β cells and dispersed villin−/− islet cells were less round and less deformable. Basal mobility of SGs in villin-depleted cells was enhanced. Moreover, in cells depleted either of villin or Ica512 F-actin cages restraining cortical SGs were enlarged, basal secretion was increased while glucose-stimulated insulin release was blunted. The latter changes were reverted by overexpressing villin in Ica512-depleted cells, but not vice versa. Conclusion: Our findings show that villin controls the size of the F-actin cages restricting SGs and, thus, regulates their dynamics and availability for exocytosis. Evidence that villin acts downstream of Ica512 also indicates that SGs directly influence the remodeling properties of the cortical actin cytoskeleton for tight control of insulin secretion. Keywords: F-actin, Granules, Ica512, Insulin, Secretion, Villin

Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells.

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Laura Bordone

2006-02-01

Full Text Available Sir2 and insulin/IGF-1 are the major pathways that impinge upon aging in lower organisms. In Caenorhabditis elegans a possible genetic link between Sir2 and the insulin/IGF-1 pathway has been reported. Here we investigate such a link in mammals. We show that Sirt1 positively regulates insulin secretion in pancreatic beta cells. Sirt1 represses the uncoupling protein (UCP gene UCP2 by binding directly to the UCP2 promoter. In beta cell lines in which Sirt1 is reduced by SiRNA, UCP2 levels are elevated and insulin secretion is blunted. The up-regulation of UCP2 is associated with a failure of cells to increase ATP levels after glucose stimulation. Knockdown of UCP2 restores the ability to secrete insulin in cells with reduced Sirt1, showing that UCP2 causes the defect in glucose-stimulated insulin secretion. Food deprivation induces UCP2 in mouse pancreas, which may occur via a reduction in NAD (a derivative of niacin levels in the pancreas and down-regulation of Sirt1. Sirt1 knockout mice display constitutively high UCP2 expression. Our findings show that Sirt1 regulates UCP2 in beta cells to affect insulin secretion.

Tripeptide amide L-pyroglutamyl-histidyl-L-prolineamide (L-PHP-thyrotropin-releasing hormone, TRH) promotes insulin-producing cell proliferation.

Science.gov (United States)

Luo, LuGuang; Luo, John Z Q; Jackson, Ivor

2013-02-01

A very small tripeptide amide L-pyroglutamyl-L-histidyl-L-prolineamide (L-PHP, Thyrotropin-Releasing Hormone, TRH), was first identified in the brain hypothalamus area. Further studies found that L-PHP was expressed in pancreas. The biological role of pancreatic L-PHP is still not clear. Growing evidence indicates that L-PHP expression in the pancreas may play a pivotal role for pancreatic development in the early prenatal period. However, the role of L-PHP in adult pancreas still needs to be explored. L-PHP activation of pancreatic β cell Ca2+ flow and stimulation of β-cell insulin synthesis and release suggest that L-PHP involved in glucose metabolism may directly act on the β cell separate from any effects via the central nervous system (CNS). Knockout L-PHP animal models have shown that loss of L-PHP expression causes hyperglycemia, which cannot be reversed by administration of thyroid hormone, suggesting that the absence of L-PHP itself is the cause. L-PHP receptor type-1 has been identified in pancreas which provides a possibility for L-PHP autocrine and paracrine regulation in pancreatic function. During pancreatic damage in adult pancreas, L-PHP may protect beta cell from apoptosis and initiate its regeneration through signal pathways of growth hormone in β cells. L-PHP has recently been discovered to affect a broad array of gene expression in the pancreas including growth factor genes. Signal pathways linked between L-PHP and EGF receptor phosphorylation suggest that L-PHP may be an important factor for adult β-cell regeneration, which could involve adult stem cell differentiation. These effects suggest that L-PHP may benefit pancreatic β cells and diabetic therapy in clinic.

A novel cell growth-promoting factor identified in a B cell leukemia cell line, BALL-1

International Nuclear Information System (INIS)

Dao, T.; Holan, V.; Minowada, J.

1993-01-01

A novel leukemia cell growth-promoting activity has been identified in the culture supernatant from a human B cell leukemia cell line, BALL-1. The supernatant from unstimulated cultures of the BALL-1 cells significantly promoted the growth of 16 out of 24 leukemia/lymphoma cell lines of different lineages (T, B and non-lymphoid) in a minimal concentration of fetal bovine serum (FBS), and 5 out of 12 cases of fresh leukemia cells in FBS-free medium. The growth-promoting sieve filtration and dialysis. The MW of the factor was less than 10 kDa. The growth-promoting activity was heat and acid stable and resistant to trypsin treatment. The factor isolated from the BALL-1 supernatant was distinct from known polypeptide growth factors with MW below 10 kDa, such as epidermal growth factor, transforming growth factor α, insulin-like growth factor I (IGF-I), IGF-II and insulin, as determine by specific antibodies and by cell-growth-promoting tests. The factor is the BALL-1 supernatant did not promote the proliferation of normal human fresh peripheral blood lymphocytes or mouse fibroblast cell line, BALB/C 3T3. In addition to the BALL-1 supernatant, a similar growth-promoting activity was found in the culture supernatant from 13 of 17 leukemia/lymphoma cell lines tested. The activity in these culture supernatant promoted the growth of leukemia/lymphoma cell lines in autocrine and/or paracrine fashions. These observations suggest that the low MW cell growth-promoting activity found in the BALL-1 culture supernatant is mediated by a novel factor which may be responsible for the clonal expansion of particular leukemic clones. (author)

Mathematical modeling and statistical analysis of calcium-regulated insulin granule exocytosis in ß-cells from mice and humans

DEFF Research Database (Denmark)

Pedersen, Morten Gram; Cortese, Giuliana; Eliasson, Lena

2011-01-01

Insulin is released from pancreatic ß-cells as a result of Ca2+-evoked exocytosis of dense-core granules. Secretion is biphasic, which has been suggested to correspond to the release of different granule pools. Here we review and carefully reanalyze previously published patch-clamp data on depola......Insulin is released from pancreatic ß-cells as a result of Ca2+-evoked exocytosis of dense-core granules. Secretion is biphasic, which has been suggested to correspond to the release of different granule pools. Here we review and carefully reanalyze previously published patch-clamp data...... on depolarization-evoked Ca2+-currents and corresponding capacitance measurements. Using a statistical mixed-effects model, we show that the data indicate that pool depletion is negligible in response to short depolarizations in mouse ß-cells. We then review mathematical models of granule dynamics and exocytosis...

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

Cross-Linked Dependency of Boronic Acid-Conjugated Chitosan Nanoparticles by Diols for Sustained Insulin Release

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Nabil A. Siddiqui

2016-10-01

Full Text Available Boronic acids have been widely investigated for their potential use as glucose sensors in glucose responsive polymeric insulin delivery systems. Interactions between cyclic diols and boronic acids, anchored to polymeric delivery systems, may result in swelling of the delivery system, releasing the drug. In this study, 4-formylphenylboronic acid conjugated chitosan was formulated into insulin containing nanoparticles via polyelectrolyte complexation. The nanoparticles had an average diameter of 140 ± 12.8 nm, polydispersity index of 0.17 ± 0.1, zeta potential of +19.1 ± 0.69 mV, encapsulation efficiency of 81% ± 1.2%, and an insulin loading capacity of 46% ± 1.8% w/w. Changes in size of the nanoparticles and release of insulin were type of sugar- and concentration-dependent. High concentration of diols resulted in a sustained release of insulin due to crosslink formation with boronic acid moieties within the nanoparticles. The formulation has potential to be developed into a self-regulated insulin delivery system for the treatment of diabetes.

Arginase-II Promotes Tumor Necrosis Factor-α Release From Pancreatic Acinar Cells Causing β-Cell Apoptosis in Aging.

Science.gov (United States)

Xiong, Yuyan; Yepuri, Gautham; Necetin, Sevil; Montani, Jean-Pierre; Ming, Xiu-Fen; Yang, Zhihong

2017-06-01

Aging is associated with glucose intolerance. Arginase-II (Arg-II), the type-II L -arginine-ureahydrolase, is highly expressed in pancreas. However, its role in regulation of pancreatic β-cell function is not known. Here we show that female (not male) mice deficient in Arg-II (Arg-II -/- ) are protected from age-associated glucose intolerance and reveal greater glucose induced-insulin release, larger islet size and β-cell mass, and more proliferative and less apoptotic β-cells compared with the age-matched wild-type (WT) controls. Moreover, Arg-II is mainly expressed in acinar cells and is upregulated with aging, which enhances p38 mitogen-activated protein kinase (p38 MAPK) activation and release of tumor necrosis factor-α (TNF-α). Accordingly, conditioned medium of isolated acinar cells from old WT (not Arg-II -/- ) mice contains higher TNF-α levels than the young mice and stimulates β-cell apoptosis and dysfunction, which are prevented by a neutralizing anti-TNF-α antibody. In acinar cells, our study demonstrates an age-associated Arg-II upregulation, which promotes TNF-α release through p38 MAPK leading to β-cell apoptosis, insufficient insulin secretion, and glucose intolerance in female rather than male mice. © 2017 by the American Diabetes Association.

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.

Detection of transketolase in bone marrow-derived insulin-producing cells: benfotiamine enhances insulin synthesis and glucose metabolism.

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Oh, Seh-Hoon; Witek, Rafal P; Bae, Si-Hyun; Darwiche, Houda; Jung, Youngmi; Pi, Liya; Brown, Alicia; Petersen, Bryon E

2009-01-01

Adult bone marrow (BM)-derived insulin-producing cells (IPCs) are capable of regulating blood glucose levels in chemically induced hyperglycemic mice. Using cell transplantation therapy, fully functional BM-derived IPCs help to mediate treatment of diabetes mellitus. Here, we demonstrate the detection of the pentose phosphate pathway enzyme, transketolase (TK), in BM-derived IPCs cultured under high-glucose conditions. Benfotiamine, a known activator of TK, was not shown to affect the proliferation of insulinoma cell line, INS-1; however, when INS-1 cells were cultured with oxythiamine, an inhibitor of TK, cell proliferation was suppressed. Treatment with benfotiamine activated glucose metabolism in INS-1 cells in high-glucose culture conditions, and appeared to maximize the BM-derived IPCs ability to synthesize insulin. Benfotiamine was not shown to induce the glucose receptor Glut-2, however it was shown to activate glucokinase, the enzyme responsible for conversion of glucose to glucose-6-phosphate. Furthermore, benfotiamine-treated groups showed upregulation of the downstream glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH). However, in cells where the pentose phosphate pathway was blocked by oxythiamine treatment, there was a clear downregulation of Glut-2, glucokinase, insulin, and GAPDH. When benfotiamine was used to treat mice transplanted with BM-derived IPCs transplanted, their glucose level was brought to a normal range. The glucose challenge of normal mice treated with benfotiamine lead to rapidly normalized blood glucose levels. These results indicate that benfotiamine activates glucose metabolism and insulin synthesis to prevent glucose toxicity caused by high concentrations of blood glucose in diabetes mellitus.

Detection of Transketolase in Bone Marrow—Derived Insulin-Producing Cells: Benfotiamine Enhances Insulin Synthesis and Glucose Metabolism

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Witek, Rafal P.; Bae, Si-Hyun; Darwiche, Houda; Jung, Youngmi; Pi, Liya; Brown, Alicia; Petersen, Bryon E.

2009-01-01

Adult bone marrow (BM)-derived insulin-producing cells (IPCs) are capable of regulating blood glucose levels in chemically induced hyperglycemic mice. Using cell transplantation therapy, fully functional BM-derived IPCs help to mediate treatment of diabetes mellitus. Here, we demonstrate the detection of the pentose phosphate pathway enzyme, transketolase (TK), in BM-derived IPCs cultured under high-glucose conditions. Benfotiamine, a known activator of TK, was not shown to affect the proliferation of insulinoma cell line, INS-1; however, when INS-1 cells were cultured with oxythiamine, an inhibitor of TK, cell proliferation was suppressed. Treatment with benfotiamine activated glucose metabolism in INS-1 cells in high-glucose culture conditions, and appeared to maximize the BM-derived IPCs ability to synthesize insulin. Benfotiamine was not shown to induce the glucose receptor Glut-2, however it was shown to activate glucokinase, the enzyme responsible for conversion of glucose to glucose-6-phosphate. Furthermore, benfotiamine-treated groups showed upregulation of the downstream glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH). However, in cells where the pentose phosphate pathway was blocked by oxythiamine treatment, there was a clear downregulation of Glut-2, glucokinase, insulin, and GAPDH. When benfotiamine was used to treat mice transplanted with BM-derived IPCs transplanted, their glucose level was brought to a normal range. The glucose challenge of normal mice treated with benfotiamine lead to rapidly normalized blood glucose levels. These results indicate that benfotiamine activates glucose metabolism and insulin synthesis to prevent glucose toxicity caused by high concentrations of blood glucose in diabetes mellitus. PMID:18393672

Repression of COUP-TFI Improves Bone Marrow-Derived Mesenchymal Stem Cell Differentiation into Insulin-Producing Cells

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Tao Zhang

2017-09-01

Full Text Available Identifying molecular mechanisms that regulate insulin expression in bone marrow-derived mesenchymal stem cells (bmMSCs can provide clues on how to stimulate the differentiation of bmMSCs into insulin-producing cells (IPCs, which can be used as a therapeutic approach against type 1 diabetes (T1D. As repression factors may inhibit differentiation, the efficiency of this process is insufficient for cell transplantation. In this study, we used the mouse insulin 2 (Ins2 promoter sequence and performed a DNA affinity precipitation assay combined with liquid chromatography-mass spectrometry to identify the transcription factor, chicken ovalbumin upstream promoter transcriptional factor I (COUP-TFI. Functionally, bmMSCs were reprogrammed into IPCs via COUP-TFI suppression and MafA overexpression. The differentiated cells expressed higher levels of genes specific for islet endocrine cells, and they released C-peptide and insulin in response to glucose stimulation. Transplantation of IPCs into streptozotocin-induced diabetic mice caused a reduction in hyperglycemia. Mechanistically, COUP-TFI bound to the DR1 (direct repeats with 1 spacer element in the Ins2 promoter, thereby negatively regulating promoter activity. Taken together, the data provide a novel mechanism by which COUP-TFI acts as a negative regulator in the Ins2 promoter. The differentiation of bmMSCs into IPCs could be improved by knockdown of COUP-TFI, which may provide a novel stem cell-based therapy for T1D. Keywords: siRNAs, differentiation, stem cell transplantation, diabetes, mesenchymal stem cells

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...

Polymer encapsulated dopaminergic cell lines as "alternative neural grafts".

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Jaeger, C B; Greene, L A; Tresco, P A; Winn, S R; Aebischer, P

1990-01-01

Our preliminary findings (Jaeger et al., 1988; Aebischer et al., 1989; Tresco et al., 1989) and the studies in progress show that encapsulated dopaminergic cell lines survive enclosure within a semi-permeable membrane. The encapsulated cells remained viable for extended time periods when maintained in vitro. Moreover, encapsulated PC12 and T28 cells have the potential to survive following their implantation into the forebrain of rats. Cell lines are essentially "immortal" because they continue to divide indefinitely. This property allows perpetual "self-renewal" of a given cell population. However, the capacity of continuous uncontrolled cell division may also lead to tumor formation. This in fact is the case for unencapsulated PC12 cell implants placed into the brain of young Sprague Dawley rats (Jaeger, 1985). Cell line encapsulation has the potential to prevent tumor growth (Jaeger et al., 1988). Survival for 6 months in vitro suggests that encapsulation does not preclude long-term maintenance of an homogeneous cell line like PC12 cells. The presence of mitotic figures in the capsules further supports the likelihood of propagation and self renewal of the encapsulated population. Another significant property of cell lines is that they consist of a single, genetically homogeneous cell type. They do not require specific synaptic interactions for their survival. In the case of PC12 and T28 lines, the cells synthesize and release neurotransmitters. Our data show that PC12 and T28 cells continue to release dopamine spontaneously and to express specific transmitters and enzymes following encapsulation. Thus, cell lines such as these may constitute relatively simple "neural implants" exerting their function via humoral release.(ABSTRACT TRUNCATED AT 250 WORDS)

Measuring phospholipase D activity in insulin-secreting pancreatic beta-cells and insulin-responsive muscle cells and adipocytes.

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Cazzolli, Rosanna; Huang, Ping; Teng, Shuzhi; Hughes, William E

2009-01-01

Phospholipase D (PLD) is an enzyme producing phosphatidic acid and choline through hydrolysis of phosphatidylcholine. The enzyme has been identified as a member of a variety of signal transduction cascades and as a key regulator of numerous intracellular vesicle trafficking processes. A role for PLD in regulating glucose homeostasis is emerging as the enzyme has recently been identified in events regulating exocytosis of insulin from pancreatic beta-cells and also in insulin-stimulated glucose uptake through controlling GLUT4 vesicle exocytosis in muscle and adipose tissue. We present methodologies for assessing cellular PLD activity in secretagogue-stimulated insulin-secreting pancreatic beta-cells and also insulin-stimulated adipocyte and muscle cells, two of the principal insulin-responsive cell types controlling blood glucose levels.

The level of menadione redox-cycling in pancreatic β-cells is proportional to the glucose concentration: role of NADH and consequences for insulin secretion.

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Heart, Emma; Palo, Meridith; Womack, Trayce; Smith, Peter J S; Gray, Joshua P

2012-01-15

Pancreatic β-cells release insulin in response to elevation of glucose from basal (4-7mM) to stimulatory (8-16mM) levels. Metabolism of glucose by the β-cell results in the production of low levels of reactive oxygen intermediates (ROI), such as hydrogen peroxide (H(2)O(2)), a newly recognized coupling factor linking glucose metabolism to insulin secretion. However, high and toxic levels of H(2)O(2) inhibit insulin secretion. Menadione, which produces H(2)O(2) via redox cycling mechanism in a dose-dependent manner, was investigated for its effect on β-cell metabolism and insulin secretion in INS-1 832/13, a rat β-cell insulinoma cell line, and primary rodent islets. Menadione-dependent redox cycling and resulting H(2)O(2) production under stimulatory glucose exceeded several-fold those reached at basal glucose. This was paralleled by a differential effect of menadione (0.1-10μM) on insulin secretion, which was enhanced at basal, but inhibited at stimulatory glucose. Redox cycling of menadione and H(2)O(2) formation was dependent on glycolytically-derived NADH, as inhibition of glycolysis and application of non-glycogenic insulin secretagogues did not support redox cycling. In addition, activity of plasma membrane electron transport, a system dependent in part on glycolytically-derived NADH, was also inhibited by menadione. Menadione-dependent redox cycling was sensitive to the NQO1 inhibitor dicoumarol and the flavoprotein inhibitor diphenylene iodonium, suggesting a role for NQO1 and other oxidoreductases in this process. These data may explain the apparent dichotomy between the stimulatory and inhibitory effects of H(2)O(2) and menadione on insulin secretion. Published by Elsevier Inc.

Histone deacetylase regulates insulin signaling via two pathways in pancreatic β cells.

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Yukina Kawada

Full Text Available Recent studies demonstrated that insulin signaling plays important roles in the regulation of pancreatic β cell mass, the reduction of which is known to be involved in the development of diabetes. However, the mechanism underlying the alteration of insulin signaling in pancreatic β cells remains unclear. The involvement of epigenetic control in the onset of diabetes has also been reported. Thus, we analyzed the epigenetic control of insulin receptor substrate 2 (IRS2 expression in the MIN6 mouse insulinoma cell line. We found concomitant IRS2 up-regulation and enhanced insulin signaling in MIN6 cells, which resulted in an increase in cell proliferation. The H3K9 acetylation status of the Irs2 promoter was positively associated with IRS2 expression. Treatment of MIN6 cells with histone deacetylase inhibitors led to increased IRS2 expression, but this occurred in concert with low insulin signaling. We observed increased IRS2 lysine acetylation as a consequence of histone deacetylase inhibition, a modification that was coupled with a decrease in IRS2 tyrosine phosphorylation. These results suggest that insulin signaling in pancreatic β cells is regulated by histone deacetylases through two novel pathways affecting IRS2: the epigenetic control of IRS2 expression by H3K9 promoter acetylation, and the regulation of IRS2 activity through protein modification. The identification of the histone deacetylase isoform(s involved in these mechanisms would be a valuable approach for the treatment of type 2 diabetes.

Analysis of results of oral glucose tolerance test (OGTT) and insulin releasing test in hepatogenic diabetics

International Nuclear Information System (INIS)

He Haoming; Fu Qiang; Tian Xiaoping; Su Cainu

2001-01-01

Objective: To explore the clinical values of OGTT and insulin releasing test in hepatogenic diabetics. Method: OGTT was performed by enzymes method and insulin releasing test by RIA in 30 patients with hepatogenic diabetes, 31 cases with II diabetes and 35 controls. Results: During OGTT, blood glucose levels at various time were about the same in hepatogenic diabetics and II diabetics (P < 0.05), except at 180 min (P < 0.01). Basal hyperinsulinemia was present is hepatogenic diabetics. Conclusion: OGTT and insulin releasing test had a definite clinical value in the differential diagnosis of hepatogenic diabetics

Older Subjects with β-cell Dysfunction have an Accentuated Incretin Release.

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Garduno-Garcia, José de Jesús; Gastaldelli, Amalia; DeFronzo, Ralph A; Lertwattanarak, Raweewan; Holst, Jens J; Musi, Nicolas

2018-04-16

Insulin secretion declines with age and this contributes to the increased risk of developing impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2DM) in older subjects. Insulin secretion is regulated by the incretin hormones glucagon-like peptide (GLP) 1 and glucose-dependent insulinotropic peptide (GIP). Here we tested the hypotheses that incretin release is reduced in older subjects, and that this decline is associated with β-cell dysfunction. 40 young (25±3 y) and 53 older (74±7 y) lean non-diabetic subjects underwent a 2 h oral glucose tolerance test (OGTT). Based on the OGTT, subjects were divided in 3 groups: young normal glucose tolerant (Y-NGT, n=40), older with NGT (O-NGT, n=32), and older with IGT (O-IGT, n=21). Plasma insulin, C-peptide, GLP-1, and GIP concentrations were measured every 15-30 min. We quantitated insulin sensitivity (Matsuda index) and insulin secretory rate (ISR) by deconvolution of C-peptide with the calculation of β-cell glucose sensitivity. Matsuda index, early phase ISR (0-30min) and parameters of β-cell function were reduced in O-IGT vs. Y-NGT, but not in O-NGT. GLP-1 concentrations were elevated in both older groups [GLP-1_AUC0-120 was 2.8±0.1 in Y-NGT, 3.8±0.5 in O-NGT, and 3.7±0.4 nmol/l∙120 min in O-IGT (P<0.05)] while GIP secretion was elevated in O-NGT vs. Y-NGT [GIP_AUC0-120 was 4.7±0.3 in Y-NGT, 6.0±0.4 in O-NGT, and 4.8±0.3 nmol/l∙120 min in O-IGT (P<0.05)]. Aging is associated with an exaggerated GLP-1 secretory response. However, this was not sufficient to increase insulin first phase release in O-IGT and overcome insulin resistance.

The Type 2 Diabetes Associated Minor Allele of rs2237895 KCNQ1 Associates with Reduced Insulin Release Following an Oral Glucose Load

DEFF Research Database (Denmark)

Brunak, Søren; Holmkvist, J; Banasik, K

2009-01-01

, and rs2237897) on estimates of glucose stimulated insulin release. METHODOLOGY/PRINCIPAL FINDINGS: Genotypes were examined for associations with serum insulin levels following an oral glucose tolerance test (OGTT) in a population-based sample of 6,039 middle-aged and treatment-naïve individuals. Insulin...... release indices estimated from the OGTT and the interplay between insulin sensitivity and insulin release were investigated using linear regression and Hotelling T2 analyses. Applying an additive genetic model the minor C-allele of rs2237895 was associated with reduced serum insulin levels 30 min (mean......,568 individuals who were glucose tolerant. Adjustment for the degree of insulin sensitivity had no effect on the measures of reduced insulin release. The rs2237895 genotype had a similar impact in the total sample of treatment-naïve individuals. No association with measures of insulin release were identified...

Multicompartmental, multilayered probucol microcapsules for diabetes mellitus: Formulation characterization and effects on production of insulin and inflammation in a pancreatic β-cell line.

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Mooranian, Armin; Negrulj, Rebecca; Arfuso, Frank; Al-Salami, Hani

2016-11-01

We have shown that the primary bile acid, cholic acid (CA), has anti-diabetic effects in vivo. Probucol (PB) is a lipophilic drug with potential applications in type 2 diabetes (T2D). This study aimed to encapsulate CA with PB and examine the formulation and surface characteristics of the microcapsules. We also tested the microcapsules' biological effects on pancreatic β-cells. Using the polymer, sodium alginate (SA), two formulations were prepared: PB-SA (control), and PB-CA-SA (test). Complete characterizations of the morphology, shape, size, chemical, thermal, and rheological properties, swelling and mechanical strength, cross-sectional imaging (Micro CT), stability, Zeta-potential, drug contents, and PB release profile were carried out, at different temperature and pH values. The microcapsules were applied to a NIT-1 cell culture and the supernatant was analyzed for insulin and TNF-α concentrations. CA incorporation optimized the PB microcapsules, which exhibited pseudoplastic-thixotropic rheological characteristics. The size of the microcapsules remained similar after CA addition, and the microcapsules showed even drug distribution and no chemical alterations of the excipients. Micro-CT imaging, differential scanning calorimetry, Fourier transform infrared spectroscopy, scanning electron microscopy, and energy-dispersive X-ray spectroscopy showed consistent microcapsules with uniform shape and morphology. PB-CA-SA microcapsules enhanced NIT-1 cell viability under hyperglycemic states and resulted in improved insulin release as well as reduced cytokine production at the physiological glucose levels. The addition of the primary bile acid, CA, improved the physical properties of the microcapsules and enhanced their pharmacological activity in vitro, suggesting potential applications in diabetes treatment.

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

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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.

**-Postprandial pancreatic ["1"1C]methionine uptake after pancreaticoduodenectomy mirrors basal beta cell function and insulin release

International Nuclear Information System (INIS)

Steiner, Emanuel; Kazianka, Lukas; Breuer, Robert; Miholic, Johannes; Hacker, Marcus; Wadsak, Wolfgang; Mitterhauser, Markus; Stimpfl, Thomas; Reiter, Birgit; Karanikas, Georgios

2017-01-01

[S-methyl-"1"1C]-L-methionine (["1"1C]MET) uptake in the pancreas might be a central indicator of beta cell function. Since gastric emptying was recently shown to influence glycemic control in subjects after pancreaticoduodenectomy (PD, the surgical treatment of neoplasms of the pancreas head), we looked for imaginable relationships between gastric emptying, pre- and postprandial insulin concentrations, and ["1"1C]MET uptake. Nineteen tumor-free survivors after PD (age mean ± SD: 61 ± 8.7 yrs.; 10 male, 9 female) and 10 healthy controls (age: 27 ± 8.7 yrs.; 7 male, 3 female) were given a mixed test meal. One gram of paracetamol was ingested with the meal to evaluate the speed of gastric emptying. Insulin, glucose, and paracetamol plasma concentrations were measured before and over 180 minutes after ingestion. Beta cell function was calculated from fasting glucose and insulin plasma concentrations. Simultaneously, 800 MBq of ["1"1C]MET were administered and the activity (maximum tissue standardized uptake values [SUVmax]) over the pancreas was measured at 15, 30, and 60 minutes after injection. Total integrated SUVmax (area under the curve [AUC]) and incremental SUVmax were calculated. The uptake of ["1"1C]MET in the pancreas was significantly higher (p < 0.0001) in controls compared to the PD group. Gastric emptying was significantly slower in controls compared to pancreatectomy subjects (p < 0.0001). Paracetamol AUC_3_0 correlated with the SUVmax increment between 15 and 30 minutes (R"2 = 0.27, p = 0.0263), suggesting a relationship between gastric emptying and the uptake of ["1"1C]MET. Total integrated SUVmax correlated with insulin AUC_6_0 (R"2 = 0.66,p < 0.0001) in patients after PD. Multivariate regression analysis revealed insulin AUC_6_0 and beta cell function, calculated from the fasting insulin to glucose ratio, as independent predictors of "1"1C-methionine uptake, i.e. total integrated SUVmax, in patients after PD (R"2 = 0.78, p < 0.0001). Postprandial

**-Postprandial pancreatic [{sup 11}C]methionine uptake after pancreaticoduodenectomy mirrors basal beta cell function and insulin release

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Steiner, Emanuel; Kazianka, Lukas; Breuer, Robert; Miholic, Johannes [Medical University of Vienna, Department of Surgery, Vienna (Austria); Hacker, Marcus; Wadsak, Wolfgang; Mitterhauser, Markus [Medical University of Vienna, Department of Biomedical Imaging and Image-Guided Therapy, Division of Nuclear Medicine, Vienna (Austria); Stimpfl, Thomas; Reiter, Birgit [Medical University of Vienna, Clinical Institute of Laboratory Medicine, Forensic Toxicology, Vienna (Austria); Karanikas, Georgios [Medical University of Vienna, Department of Biomedical Imaging and Image-Guided Therapy, Division of Nuclear Medicine, Divisional Head PET-PET/CT (Nuclear Medicine), Vienna (Austria)

2017-03-15

[S-methyl-{sup 11}C]-L-methionine ([{sup 11}C]MET) uptake in the pancreas might be a central indicator of beta cell function. Since gastric emptying was recently shown to influence glycemic control in subjects after pancreaticoduodenectomy (PD, the surgical treatment of neoplasms of the pancreas head), we looked for imaginable relationships between gastric emptying, pre- and postprandial insulin concentrations, and [{sup 11}C]MET uptake. Nineteen tumor-free survivors after PD (age mean ± SD: 61 ± 8.7 yrs.; 10 male, 9 female) and 10 healthy controls (age: 27 ± 8.7 yrs.; 7 male, 3 female) were given a mixed test meal. One gram of paracetamol was ingested with the meal to evaluate the speed of gastric emptying. Insulin, glucose, and paracetamol plasma concentrations were measured before and over 180 minutes after ingestion. Beta cell function was calculated from fasting glucose and insulin plasma concentrations. Simultaneously, 800 MBq of [{sup 11}C]MET were administered and the activity (maximum tissue standardized uptake values [SUVmax]) over the pancreas was measured at 15, 30, and 60 minutes after injection. Total integrated SUVmax (area under the curve [AUC]) and incremental SUVmax were calculated. The uptake of [{sup 11}C]MET in the pancreas was significantly higher (p < 0.0001) in controls compared to the PD group. Gastric emptying was significantly slower in controls compared to pancreatectomy subjects (p < 0.0001). Paracetamol AUC{sub 30} correlated with the SUVmax increment between 15 and 30 minutes (R{sup 2} = 0.27, p = 0.0263), suggesting a relationship between gastric emptying and the uptake of [{sup 11}C]MET. Total integrated SUVmax correlated with insulin AUC{sub 60} (R{sup 2} = 0.66,p < 0.0001) in patients after PD. Multivariate regression analysis revealed insulin AUC{sub 60} and beta cell function, calculated from the fasting insulin to glucose ratio, as independent predictors of {sup 11}C-methionine uptake, i.e. total integrated SUVmax, in

Human umbilical cord-derived mesenchymal stem cells ameliorate insulin resistance by suppressing NLRP3 inflammasome-mediated inflammation in type 2 diabetes rats

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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

Molecular basis for vulnerability to mitochondrial and oxidative stress in a neuroendocrine CRI-G1 cell line.

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Natasha Chandiramani

2011-01-01

thus represent potential therapeutic targets for diabetes and neurodegenerative diseases. In addition, the newly identified CRI-G1-RS cell line represents a new experimental model for investigating how endogenous antioxidants affect glucose sensing and insulin release by pancreatic β cells.

Insulin-loaded polymeric mucoadhesive nanoparticles: development, characterization and cytotoxicity evaluation

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Tiago Henrique Honorato Gatti

2018-06-01

Full Text Available Abstract Mucoadhesive nanoparticles are particularly interesting for delivery through nasal or pulmonary routes, as an approach to overcome the mucociliary clearance. Moreover, these nanoparticles are attractive for peptide and protein delivery, particularly for insulin to treat diabetes, as an alternative to conventional parenteral administration. Thus, chitosan, a cationic mucoadhesive polysaccharide found in shells of crustaceans, and the negatively-charged dextran sulfate are able to form nanoparticles through ionic condensation, representing a potential insulin carrier. Herein, chitosan/dextran sulfate nanoparticles at various ratios were prepared for insulin loading. Formulations were characterized for particle size, zeta potential, encapsulation efficiency, scanning electron microscopy, differential scanning calorimetry, and in vitro drug release. Moreover, the interaction with mucin and the cytotoxicity against a lung cell line were studied, which altogether have not been addressed before. Results evidenced that a proper selection of polyelectrolytes is necessary for smaller particle size formation and also the composition and zeta potential impact encapsulation efficiency, which is benefited by the positive charge of chitosan. Insulin remained stable after encapsulation as evidenced by calorimetric assays, and was released in a sustained manner in the first 10 h. Positively-charged nanoparticles based on chitosan/dextran-sulfate at the ratio of 6:4 successfully interacted with mucin, which is a prerequisite for delivery to mucus-containing tissues. Finally, insulin-loaded nanoparticles displayed no cytotoxicity effect against lung cells at tested concentrations, suggesting the potential for further in vivo studies.

Insulin is essential for in vitro chondrogenesis of mesenchymal progenitor cells and influences chondrogenesis in a dose-dependent manner.

Science.gov (United States)

Mueller, Michael B; Blunk, Torsten; Appel, Bernhard; Maschke, Angelika; Goepferich, Achim; Zellner, Johannes; Englert, Carsten; Prantl, Lukas; Kujat, Richard; Nerlich, Michael; Angele, Peter

2013-01-01

Insulin is a commonly used additive in chondrogenic media for differentiating mesenchymal stem cells (MSCs). The indispensability of other bioactive factors like TGF-β or dexamethasone in these medium formulations has been shown, but the role of insulin is unclear. The purpose of this study was to investigate whether insulin is essential for MSC chondrogenesis and if there is a dose-dependent effect of insulin on MSC chondrogenesis. We cultivated human MSCs in pellet culture in serum-free chondrogenic medium with insulin concentrations between 0 and 50 μg/ml and assessed the grade of chondrogenic differentiation by histological evaluation and determination of glycosaminoglycan (GAG), total collagen and DNA content. We further tested whether insulin can be delivered in an amount sufficient for MSC chondrogenesis via a drug delivery system in insulin-free medium. Chondrogenesis was not induced by standard chondrogenic medium without insulin and the expression of cartilage differentiation markers was dose-dependent at insulin concentrations between 0 and 10 μg/ml. An insulin concentration of 50 μg/ml had no additional effect compared with 10 μg/ml. Insulin was delivered by a release system into the cell culture under insulin-free conditions in an amount sufficient to induce chondrogenesis. Insulin is essential for MSC chondrogenesis in this system and chondrogenic differentiation is influenced by insulin in a dose-dependent manner. Insulin can be provided in a sufficient amount by a drug delivery system. Therefore, insulin is a suitable and inexpensive indicator substance for testing drug release systems in vitro.

DPP-4 inhibitor des-F-sitagliptin treatment increased insulin exocytosis from db/db mice {beta} cells

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Nagamatsu, Shinya, E-mail: shinya@ks.kyorin-u.ac.jp [Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611 (Japan); Ohara-Imaizumi, Mica; Nakamichi, Yoko; Aoyagi, Kyota; Nishiwaki, Chiyono [Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611 (Japan)

2011-09-09

Highlights: {yields} Anti-diabetic new drug, DPP-4 inhibitor, can affect the insulin exocytosis. {yields} DPP-4 inhibitor treatment altered syntaxin 1 expression. {yields} Treatment of db/db mice with DPP-4 inhibitor increased insulin release. -- Abstract: Incretin promotes insulin secretion acutely. Recently, orally-administered DPP-4 inhibitors represent a new class of anti-hyperglycemic agents. Indeed, inhibitors of dipeptidyl peptidase-IV (DPP-4), sitagliptin, has just begun to be widely used as therapeutics for type 2 diabetes. However, the effects of sitagliptin-treatment on insulin exocytosis from single {beta}-cells are yet unknown. We therefore investigated how sitagliptin-treatment in db/db mice affects insulin exocytosis by treating db/db mice with des-F-sitagliptin for 2 weeks. Perfusion studies showed that 2 weeks-sitagliptin treatment potentiated insulin secretion. We then analyzed insulin granule motion and SNARE protein, syntaxin 1, by TIRF imaging system. TIRF imaging of insulin exocytosis showed the increased number of docked insulin granules and increased fusion events from them during first-phase release. In accord with insulin exocytosis data, des-F-sitagliptin-treatment increased the number of syntaxin 1 clusters on the plasma membrane. Thus, our data demonstrated that 2-weeks des-F-sitagliptin-treatment increased the fusion events of insulin granules, probably via increased number of docked insulin granules and that of syntaxin 1 clusters.

In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum.

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Bruserud, Oystein; Tronstad, Karl Johan; Berge, Rolf

2005-06-01

Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines. The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS). Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the

Intracellular serotonin modulates insulin secretion from pancreatic beta-cells by protein serotonylation.

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Nils Paulmann

2009-10-01

Full Text Available While serotonin (5-HT co-localization with insulin in granules of pancreatic beta-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is still unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1-/- and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1-/- beta-cells, which clearly showed that the secretory defect is downstream of Ca(2+-signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in beta-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic beta-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.

Insulin resistance and chronic inflammation

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Natalia Matulewicz

2016-12-01

Full Text Available Insulin resistance is a condition of reduced biological response to insulin. Growing evidence indicates the role of the chronic low-grade inflammatory response in the pathogenesis of insulin resistance. Adipose tissue in obesity is characterized by increased lipolysis with the excessive release of free fatty acids, and is also a source of proinflammatory cytokines. Both these factors may inhibit insulin action. Proinflammatory cytokines exert their effect by stimulating major inflammatory NFκB and JNK pathways within the cells. Inflammatory processes in other insulin responsive tissues may also play a role in inducing insulin resistance. This paper is an overview of the chronic low-grade inflammation in adipose tissue, skeletal muscle, liver and endothelial cells during the development of insulin resistance.

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

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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.

Insulin-like growth factor stimulation increases radiosensitivity of a pancreatic cancer cell line through endoplasmic reticulum stress under hypoxic conditions

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Isohashi, Fumiaki; Endo, Hiroko; Mukai, Mutsuko; Inoue, Masahiro; Inoue, Takehiro

2008-01-01

Tumor hypoxia is an obstacle to radiotherapy. Radiosensitivity under hypoxic conditions is determined by molecular oxygen levels, as well as by various biological cellular responses. The insulin-like growth factor (IGF) signaling pathway is a widely recognized survival signal that confers radioresistance. However, under hypoxic conditions the role of IGF signaling in radiosensitivity is still poorly understood. Here, we demonstrate that IGF-II stimulation decreases clonogenic survival under hypoxic conditions in the pancreatic cancer cell lines AsPC-1 and Panc-1, and in the human breast cancer cell line MCF-7. IGF treatment under hypoxic conditions suppressed increased radiation sensitivity in these cell lines by pharmacologically inhibiting the phosphoinositide 3-kinase-mammalian target of rapamycin pathway, a major IGF signal-transduction pathway. Meanwhile, IGF-II induced the endoplasmic reticulum stress response under hypoxia, including increased protein levels of CHOP and ATF4, mRNA levels of CHOP, GADD34, and BiP as well as splicing levels of XBP-1. The response was suppressed by inhibiting phosphoinositide 3-kinase and mammalian target of rapamycin activity. Overexpression of CHOP in AsPC-1 cells increased radiation sensitivity by IGF-II simulation under hypoxic conditions, whereas suppression of CHOP expression levels with small hairpin RNA or a dominant negative form of a proline-rich extensin-like receptor protein kinase in hypoxia decreased IGF-induced radiosensitivity. IGF-induced endoplasmic reticulum stress contributed to radiosensitization independent of cell cycle status. Taken together, IGF stimulation increased radiosensitivity through the endoplasmic reticulum stress response under hypoxic conditions. (author)

In vitro evaluation of (99m)Tc-EDDA/tricine-HYNIC-Q-Litorin in gastrin-releasing peptide receptor positive tumor cell lines.

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Yurt Lambrecht, Fatma; Durkan, Kübra; Ozgür, Aykut; Gündüz, Cumhur; Avcı, Cığır Biray; Susluer, Sunde Yılmaz

2013-05-01

Bombesin and its derivatives exhibit a high affinity for gastrin-releasing peptide receptor (GRPr), which is over-expressed in a variety of human cancers (prostate, pancreatic, lung, etc.). The aim of this study was to investigate the in vitro potential of the hydrazinonicotinamide (HYNIC)-Q-Litorin. (99m)Tc labeling was performed by using different co-ligands: tricine and ethylenediamine diacetic acid (EDDA). The radiochemical stability of radiolabeled peptide conjugates was checked at room temperature and in cysteine solution up to 24 h. The in vitro cell uptake of (99m)Tc-EDDA-HYNIC-Q-Litorin and (99m)Tc-tricine-HYNIC-Q-Litorin were evaluated on pancreatic tumor and control cell lines. Optimum specific activity and incubation time were determined for all the cell lines. The results showed that the cell uptake of the radiolabeled peptide conjugates in tumor cell lines were higher than in the control cell line. The findings of this study indicated the need for further development of in vivo study as a radiopharmaceutical for pancreatic tumor imaging.

Doxycycline-regulated 3T3-L1 preadipocyte cell line with inducible, stable expression of adenoviral E4orf1 gene: a cell model to study insulin-independent glucose disposal.

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Krishnapuram, Rashmi; Dhurandhar, Emily J; Dubuisson, Olga; Hegde, Vijay; Dhurandhar, Nikhil V

2013-01-01

Impaired glycemic control and excessive adiposity are major risk factors for Type 2 Diabetes mellitus. In rodent models, Ad36, a human adenovirus, improves glycemic control, independent of dietary fat intake or adiposity. It is impractical to use Ad36 for therapeutic action. Instead, we identified that E4orf1 protein of Ad36, mediates its anti-hyperglycemic action independent of insulin signaling. To further evaluate the therapeutic potential of E4orf1 to improve glycemic control, we established a stable 3T3-L1 cell system in which E4orf1 expression can be regulated. The development and characterization of this cell line is described here. Full-length adenoviral-36 E4orf1 cDNA obtained by PCR was cloned into a tetracycline responsive element containing vector (pTRE-Tight-E4orf1). Upon screening dozens of pTRE-Tight-E4orf1 clones, we identified the one with the highest expression of E4orf1 in response to doxycycline treatment. Furthermore, using this inducible system we characterized the ability of E4orf1 to improve glucose disposal in a time dependent manner. This stable cell line offers a valuable resource to carefully study the novel signaling pathways E4orf1 uses to enhance cellular glucose disposal independent of insulin.

Doxycycline-regulated 3T3-L1 preadipocyte cell line with inducible, stable expression of adenoviral E4orf1 gene: a cell model to study insulin-independent glucose disposal.

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Rashmi Krishnapuram

Full Text Available Impaired glycemic control and excessive adiposity are major risk factors for Type 2 Diabetes mellitus. In rodent models, Ad36, a human adenovirus, improves glycemic control, independent of dietary fat intake or adiposity. It is impractical to use Ad36 for therapeutic action. Instead, we identified that E4orf1 protein of Ad36, mediates its anti-hyperglycemic action independent of insulin signaling. To further evaluate the therapeutic potential of E4orf1 to improve glycemic control, we established a stable 3T3-L1 cell system in which E4orf1 expression can be regulated. The development and characterization of this cell line is described here. Full-length adenoviral-36 E4orf1 cDNA obtained by PCR was cloned into a tetracycline responsive element containing vector (pTRE-Tight-E4orf1. Upon screening dozens of pTRE-Tight-E4orf1 clones, we identified the one with the highest expression of E4orf1 in response to doxycycline treatment. Furthermore, using this inducible system we characterized the ability of E4orf1 to improve glucose disposal in a time dependent manner. This stable cell line offers a valuable resource to carefully study the novel signaling pathways E4orf1 uses to enhance cellular glucose disposal independent of insulin.

Acetyl-CoA carboxylase in Reuber hepatoma cells: variation in enzyme activity, insulin regulation, and cellular lipid content.

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Bianchi, A; Evans, J L; Nordlund, A C; Watts, T D; Witters, L A

1992-01-01

Reuber hepatoma cells are useful cultured lines for the study of insulin action, lipid and lipoprotein metabolism, and the regulation of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid biosynthesis. During investigations in different clonal lines of these cells, we have uncovered marked intercellular variability in the activity, enzyme content, and insulin regulation of ACC paralleled by differences in cellular neutral lipid (triglyceride) content. Two contrasting clonal lines, Fao and H356A-1, have been studied in detail. Several features distinguish these two lines, including differences in ACC activity and enzyme kinetics, the content of the two major hepatic ACC isozymes (Mr 280,000 and 265,000 Da) and their heteroisozymic complex, the extent of ACC phosphorylation, and the ability of ACC to be activated on stimulation by insulin and insulinomimetic agonists. As studied by Nile Red staining and fluorescence-activated cell sorting, these two lines also display marked differences in neutral lipid content, which correlates with both basal levels of ACC activity and inhibition of ACC by the fatty acid analog, 5-(tetradecyloxy)-2-furoic acid (TOFA). These results emphasize the importance of characterization of any particular clonal line of Reuber cells for studies of enzyme regulation, substrate metabolism, and hormone action. With respect to ACC, studies in contrasting clonal lines of Reuber cells could provide valuable clues to understanding both the complex mechanisms of intracellular ACC regulation in the absence and presence of hormones and its regulatory role(s) in overall hepatic lipid metabolism.

Aging and insulin signaling differentially control normal and tumorous germline stem cells.

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Kao, Shih-Han; Tseng, Chen-Yuan; Wan, Chih-Ling; Su, Yu-Han; Hsieh, Chang-Che; Pi, Haiwei; Hsu, Hwei-Jan

2015-02-01

Aging influences stem cells, but the processes involved remain unclear. Insulin signaling, which controls cellular nutrient sensing and organismal aging, regulates the G2 phase of Drosophila female germ line stem cell (GSC) division cycle in response to diet; furthermore, this signaling pathway is attenuated with age. The role of insulin signaling in GSCs as organisms age, however, is also unclear. Here, we report that aging results in the accumulation of tumorous GSCs, accompanied by a decline in GSC number and proliferation rate. Intriguingly, GSC loss with age is hastened by either accelerating (through eliminating expression of Myt1, a cell cycle inhibitory regulator) or delaying (through mutation of insulin receptor (dinR) GSC division, implying that disrupted cell cycle progression and insulin signaling contribute to age-dependent GSC loss. As flies age, DNA damage accumulates in GSCs, and the S phase of the GSC cell cycle is prolonged. In addition, GSC tumors (which escape the normal stem cell regulatory microenvironment, known as the niche) still respond to aging in a similar manner to normal GSCs, suggesting that niche signals are not required for GSCs to sense or respond to aging. Finally, we show that GSCs from mated and unmated females behave similarly, indicating that female GSC-male communication does not affect GSCs with age. Our results indicate the differential effects of aging and diet mediated by insulin signaling on the stem cell division cycle, highlight the complexity of the regulation of stem cell aging, and describe a link between ovarian cancer and aging. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Release of volatile organic compounds (VOCs from the lung cancer cell line CALU-1 in vitro

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Schubert Jochen

2008-11-01

Full Text Available Abstract Background The aim of this work was to confirm the existence of volatile organic compounds (VOCs specifically released or consumed by lung cancer cells. Methods 50 million cells of the human non-small cell lung cancer (NSCLC cell line CALU-1 were incubated in a sealed fermenter for 4 h or over night (18 hours. Then air samples from the headspace of the culture vessel were collected and preconcentrated by adsorption on solid sorbents with subsequent thermodesorption and analysis by means of gas chromatography mass spectrometry (GC-MS. Identification of altogether 60 compounds in GCMS measurement was done not only by spectral library match, but also by determination of retention times established with calibration mixtures of the respective pure compounds. Results The results showed a significant increase in the concentrations of 2,3,3-trimethylpentane, 2,3,5-trimethylhexane, 2,4-dimethylheptane and 4-methyloctane in the headspace of CALU-1 cell culture as compared to medium controls after 18 h. Decreased concentrations after 18 h of incubation were found for acetaldehyde, 3-methylbutanal, butyl acetate, acetonitrile, acrolein, methacrolein, 2-methylpropanal, 2-butanone, 2-methoxy-2-methylpropane, 2-ethoxy-2-methylpropane, and hexanal. Conclusion Our findings demonstrate that certain volatile compounds can be cancer-cell derived and thus indicative of the presence of a tumor, whereas other compounds are not released but seem to be consumed by CALU-1 cells.

Restitution of defective glucose-stimulated insulin secretion in diabetic GK rat by acetylcholine uncovers paradoxical stimulatory effect of beta-cell muscarinic receptor activation on cAMP production.

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Dolz, Manuel; Bailbé, Danielle; Giroix, Marie-Hélène; Calderari, Sophie; Gangnerau, Marie-Noelle; Serradas, Patricia; Rickenbach, Katharina; Irminger, Jean-Claude; Portha, Bernard

2005-11-01

Because acetylcholine (ACh) is a recognized potentiator of glucose-stimulated insulin release in the normal beta-cell, we have studied ACh's effect on islets of the Goto-Kakizaki (GK) rat, a spontaneous model of type 2 diabetes. We first verified that ACh was able to restore the insulin secretory glucose competence of the GK beta-cell. Then, we demonstrated that in GK islets 1) ACh elicited a first-phase insulin release at low glucose, whereas it had no effect in Wistar; 2) total phospholipase C activity, ACh-induced inositol phosphate production, and intracellular free calcium concentration ([Ca2+]i) elevation were normal; 3) ACh triggered insulin release, even in the presence of thapsigargin, which induced a reduction of the ACh-induced [Ca2+]i response (suggesting that ACh produces amplification signals that augment the efficacy of elevated [Ca2+]i on GK exocytosis); 4) inhibition of protein kinase C did not affect [Ca2+]i nor the insulin release responses to ACh; and 5) inhibition of cAMP-dependent protein kinases (PKAs), adenylyl cyclases, or cAMP generation, while not affecting the [Ca2+]i response, significantly lowered the insulinotropic response to ACh (at low and high glucose). In conclusion, ACh acts mainly through activation of the cAMP/PKA pathway to potently enhance Ca2+-stimulated insulin release in the GK beta-cell and, in doing so, normalizes its defective glucose responsiveness.

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

The GTPase Rab37 Participates in the Control of Insulin Exocytosis.

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Sanda Ljubicic

Full Text Available Rab37 belongs to a subclass of Rab GTPases regulating exocytosis, including also Rab3a and Rab27a. Proteomic studies indicate that Rab37 is associated with insulin-containing large dense core granules of pancreatic β-cells. In agreement with these observations, we detected Rab37 in extracts of β-cell lines and human pancreatic islets and confirmed by confocal microscopy the localization of the GTPase on insulin-containing secretory granules. We found that, as is the case for Rab3a and Rab27a, reduction of Rab37 levels by RNA interference leads to impairment in glucose-induced insulin secretion and to a decrease in the number of granules in close apposition to the plasma membrane. Pull-down experiments revealed that, despite similar functional effects, Rab37 does not interact with known Rab3a or Rab27a effectors and is likely to operate through a different mechanism. Exposure of insulin-secreting cells to proinflammatory cytokines, fatty acids or oxidized low-density lipoproteins, mimicking physiopathological conditions that favor the development of diabetes, resulted in a decrease in Rab37 expression. Our data identify Rab37 as an additional component of the machinery governing exocytosis of β-cells and suggest that impaired expression of this GTPase may contribute to defective insulin release in pre-diabetic and diabetic conditions.

The comprehensive electrophysiological study of curcuminoids on delayed-rectifier K+ currents in insulin-secreting cells.

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Kuo, Ping-Chung; Yang, Chia-Jung; Lee, Yu-Chi; Chen, Pei-Chun; Liu, Yen-Chin; Wu, Sheng-Nan

2018-01-15

Curcumin (CUR) has been demonstrated to induce insulin release from pancreatic β-cells; however, how curcuminoids (including demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC)) exert any possible effects on membrane ion currents inherently in insulin-secreting cells remains largely unclear. The effects of CUR and other structurally similar curcuminoids on ion currents in rat insulin-secreting (INS-1) insulinoma cells were therefore investigated in this study. The effects of these compounds on ionic currents and membrane potential were studied by patch-clamp technique. CUR suppressed the amplitude of delayed-rectifier K + current (I K(DR) ) in a time-, state- and concentration-dependent manner in these cells and the inhibition was not reversed by diazoxide, nicorandil or chlorotoxin. The value of dissociation constant for CUR-induced suppression of I K(DR) in INS-1 cells was 1.26μM. Despite the inability of CUR to alter the activation rate of I K(DR) , it accelerated current inactivation elicited by membrane depolarization. Increasing CUR concentrations shifted the inactivation curve of I K(DR) to hyperpolarized potential and slowed the recovery of I K(DR) inactivation. CUR, DMC, and BDMC all exerted depressant actions on I K(DR) amplitude to a similar magnitude, although DMC and BDMC did not increase current inactivation clearly. CUR slightly suppressed the peak amplitude of voltage-gated Na + current. CUR, DMC and BDMC depolarized the resting potential and increased firing frequency of action potentials. The CUR-mediated decrease of I K(DR) and the increase of current inactivation also occurred in βTC-6 INS-1 cells. Taken these results together, these effects may be one of the possible mechanisms contributing their insulin-releasing effect. Copyright © 2017 Elsevier B.V. All rights reserved.

The level of menadione redox-cycling in pancreatic β-cells is proportional to the glucose concentration: Role of NADH and consequences for insulin secretion

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Heart, Emma [Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, MA, 02543 (United States); Palo, Meridith; Womack, Trayce [Department of Science, United States Coast Guard Academy, New London, CT, 06320 (United States); Smith, Peter J.S. [Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, MA, 02543 (United States); Institute for Life Sciences, University of Southampton (United Kingdom); Gray, Joshua P., E-mail: Joshua.p.gray@uscga.edu [Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, MA, 02543 (United States); Department of Science, United States Coast Guard Academy, New London, CT, 06320 (United States)

2012-01-15

Pancreatic β-cells release insulin in response to elevation of glucose from basal (4–7 mM) to stimulatory (8–16 mM) levels. Metabolism of glucose by the β-cell results in the production of low levels of reactive oxygen intermediates (ROI), such as hydrogen peroxide (H{sub 2}O{sub 2}), a newly recognized coupling factor linking glucose metabolism to insulin secretion. However, high and toxic levels of H{sub 2}O{sub 2} inhibit insulin secretion. Menadione, which produces H{sub 2}O{sub 2} via redox cycling mechanism in a dose-dependent manner, was investigated for its effect on β-cell metabolism and insulin secretion in INS-1 832/13, a rat β-cell insulinoma cell line, and primary rodent islets. Menadione-dependent redox cycling and resulting H{sub 2}O{sub 2} production under stimulatory glucose exceeded several-fold those reached at basal glucose. This was paralleled by a differential effect of menadione (0.1–10 μM) on insulin secretion, which was enhanced at basal, but inhibited at stimulatory glucose. Redox cycling of menadione and H{sub 2}O{sub 2} formation was dependent on glycolytically-derived NADH, as inhibition of glycolysis and application of non-glycogenic insulin secretagogues did not support redox cycling. In addition, activity of plasma membrane electron transport, a system dependent in part on glycolytically-derived NADH, was also inhibited by menadione. Menadione-dependent redox cycling was sensitive to the NQO1 inhibitor dicoumarol and the flavoprotein inhibitor diphenylene iodonium, suggesting a role for NQO1 and other oxidoreductases in this process. These data may explain the apparent dichotomy between the stimulatory and inhibitory effects of H{sub 2}O{sub 2} and menadione on insulin secretion. -- Highlights: ► Menadione stimulation or inhibition of insulin secretion is dependent upon applied glucose levels. ► Menadione-dependent H{sub 2}O{sub 2} production is proportional to applied glucose levels. ► Quinone-mediated redox cycling

The level of menadione redox-cycling in pancreatic β-cells is proportional to the glucose concentration: Role of NADH and consequences for insulin secretion

International Nuclear Information System (INIS)

Heart, Emma; Palo, Meridith; Womack, Trayce; Smith, Peter J.S.; Gray, Joshua P.

2012-01-01

Pancreatic β-cells release insulin in response to elevation of glucose from basal (4–7 mM) to stimulatory (8–16 mM) levels. Metabolism of glucose by the β-cell results in the production of low levels of reactive oxygen intermediates (ROI), such as hydrogen peroxide (H 2 O 2 ), a newly recognized coupling factor linking glucose metabolism to insulin secretion. However, high and toxic levels of H 2 O 2 inhibit insulin secretion. Menadione, which produces H 2 O 2 via redox cycling mechanism in a dose-dependent manner, was investigated for its effect on β-cell metabolism and insulin secretion in INS-1 832/13, a rat β-cell insulinoma cell line, and primary rodent islets. Menadione-dependent redox cycling and resulting H 2 O 2 production under stimulatory glucose exceeded several-fold those reached at basal glucose. This was paralleled by a differential effect of menadione (0.1–10 μM) on insulin secretion, which was enhanced at basal, but inhibited at stimulatory glucose. Redox cycling of menadione and H 2 O 2 formation was dependent on glycolytically-derived NADH, as inhibition of glycolysis and application of non-glycogenic insulin secretagogues did not support redox cycling. In addition, activity of plasma membrane electron transport, a system dependent in part on glycolytically-derived NADH, was also inhibited by menadione. Menadione-dependent redox cycling was sensitive to the NQO1 inhibitor dicoumarol and the flavoprotein inhibitor diphenylene iodonium, suggesting a role for NQO1 and other oxidoreductases in this process. These data may explain the apparent dichotomy between the stimulatory and inhibitory effects of H 2 O 2 and menadione on insulin secretion. -- Highlights: ► Menadione stimulation or inhibition of insulin secretion is dependent upon applied glucose levels. ► Menadione-dependent H 2 O 2 production is proportional to applied glucose levels. ► Quinone-mediated redox cycling is dependent on glycolysis

Comparison of two insulin assays for first-phase insulin release in type 1 diabetes prediction and prevention studies.

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Mahon, Jeffrey L; Beam, Craig A; Marcovina, Santica M; Boulware, David C; Palmer, Jerry P; Winter, William E; Skyler, Jay S; Krischer, Jeffrey P

2011-11-20

Detection of below-threshold first-phase insulin release or FPIR (1+3 minute insulin concentrations during an intravenous glucose tolerance test [IVGTT]) is important in type 1 diabetes prediction and prevention studies including the TrialNet Oral Insulin Prevention Trial. We assessed whether an insulin immunoenzymometric assay (IEMA) could replace the less practical but current standard of a radioimmunoassay (RIA) for FPIR. One hundred thirty-three islet autoantibody positive relatives of persons with type 1 diabetes underwent 161 IVGTTs. Insulin concentrations were measured by both assays in 1056 paired samples. A rule classifying FPIR (below-threshold, above-threshold, uncertain) by the IEMA was derived and validated against FPIR by the RIA. The insulin IEMA-based rule accurately classified below- and above-threshold FPIRs by the RIA in 110/161 (68%) IVGTTs, but was uncertain in 51/161 (32%) tests for which FPIR by RIA is needed. An uncertain FPIR by the IEMA was more likely among below-threshold vs above-threshold FPIRs by the RIA (64% [30/47] vs. 18% [21/114], respectively; pTrialNet is limiting the insulin RIA for FPIR to the latter given the practical advantages of the more specific IEMA. Copyright © 2011 Elsevier B.V. All rights reserved.

Characterisation of insulin-producing cells differentiated from tonsil derived mesenchymal stem cells.

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Kim, So-Yeon; Kim, Ye-Ryung; Park, Woo-Jae; Kim, Han Su; Jung, Sung-Chul; Woo, So-Youn; Jo, Inho; Ryu, Kyung-Ha; Park, Joo-Won

2015-01-01

Tonsil-derived (T-) mesenchymal stem cells (MSCs) display mutilineage differentiation potential and self-renewal capacity and have potential as a banking source. Diabetes mellitus is a prevalent disease in modern society, and the transplantation of pancreatic progenitor cells or various stem cell-derived insulin-secreting cells has been suggested as a novel therapy for diabetes. The potential of T-MSCs to trans-differentiate into pancreatic progenitor cells or insulin-secreting cells has not yet been investigated. We examined the potential of human T-MSCs to trans-differentiate into pancreatic islet cells using two different methods based on β-mercaptoethanol and insulin-transferin-selenium, respectively. First, we compared the efficacy of the two methods for inducing differentiation into insulin-producing cells. We demonstrated that the insulin-transferin-selenium method is more efficient for inducing differentiation into insulin-secreting cells regardless of the source of the MSCs. Second, we compared the differentiation potential of two different MSC types: T-MSCs and adipose-derived MSCs (A-MSCs). T-MSCs had a differentiation capacity similar to that of A-MSCs and were capable of secreting insulin in response to glucose concentration. Islet-like clusters differentiated from T-MSCs had lower synaptotagmin-3, -5, -7, and -8 levels, and consequently lower secreted insulin levels than cells differentiated from A-MSCs. These results imply that T-MSCs can differentiate into functional pancreatic islet-like cells and could provide a novel, alternative cell therapy for diabetes mellitus. Copyright © 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Insulin binding and stimulation of hexose and amino acid transport by normal and receptor-defective human fibroblasts

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Longo, N.; Nagata, N.; Danner, D.; Priest, J.; Elsas, L.

1986-01-01

The authors analyzed insulin receptors in cells cultured from a sibship of related parents who had two offspring with severe insulin resistance (Leprechaunism). 124 I-Insulin (1 ng/ml) binding to skin fibroblasts from the proband, mother, and father was 9, 60 and 62% of control cells, respectively, at equilibrium, Non-linear regression analysis, utilizing a two receptors model, of curvilinear Scatchard plots indicated a reduced number of high-affinity binding sites in both parents. Influx of L-Proline (System A), L-Serine (ASC) and L-Leucine (L) was similar in control and mutant cells. Similarly, during the depletion of intracellular amino acid pools, there was a release from transinhibition for System A and a decrease of transstimulation of Systems ASC and L in both cell lines. Surprisingly, insulin augmented, normally, A system influx with an ED 50 = 70 ng/ml at 24 0 C and 7 ng/ml at 37 0 C. By contrast insulin failed to simulated 3-0-methyl-D-glucose influx into the proband's cells, while normal cells were stimulated 30% with an ED 50 of 6 ng/ml. These results indicate that defective high-affinity insulin binding is inherited as an autosomal recessive trait; that general membrane functions are intact; that insulin regulates A system amino acid and hexose transport by two different mechanisms; and, that the latter mechanism is impaired by this family's receptor mutation

Branched chain amino acid suppressed insulin-initiated proliferation of human cancer cells through induction of autophagy.

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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.

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.

Insulin promotes cell migration by regulating PSA-NCAM

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Monzo, Hector J.; Coppieters, Natacha [Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Department of Anatomy and Medical Imaging, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Park, Thomas I.H. [Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Department of Pharmacology, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Dieriks, Birger V.; Faull, Richard L.M. [Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Department of Anatomy and Medical Imaging, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Dragunow, Mike [Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Department of Pharmacology, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Curtis, Maurice A., E-mail: m.curtis@auckland.ac.nz [Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Department of Anatomy and Medical Imaging, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand)

2017-06-01

Cellular interactions with the extracellular environment are modulated by cell surface polysialic acid (PSA) carried by the neural cell adhesion molecule (NCAM). PSA-NCAM is involved in cellular processes such as differentiation, plasticity, and migration, and is elevated in Alzheimer's disease as well as in metastatic tumour cells. Our previous work demonstrated that insulin enhances the abundance of cell surface PSA by inhibiting PSA-NCAM endocytosis. In the present study we have identified a mechanism for insulin-dependent inhibition of PSA-NCAM turnover affecting cell migration. Insulin enhanced the phosphorylation of the focal adhesion kinase leading to dissociation of αv-integrin/PSA-NCAM clusters, and promoted cell migration. Our results show that αv-integrin plays a key role in the PSA-NCAM turnover process. αv-integrin knockdown stopped PSA-NCAM from being endocytosed, and αv-integrin/PSA-NCAM clusters co-labelled intracellularly with Rab5, altogether indicating a role for αv-integrin as a carrier for PSA-NCAM during internalisation. Furthermore, inhibition of p-FAK caused dissociation of αv-integrin/PSA-NCAM clusters and counteracted the insulin-induced accumulation of PSA at the cell surface and cell migration was impaired. Our data reveal a functional association between the insulin/p-FAK-dependent regulation of PSA-NCAM turnover and cell migration through the extracellular matrix. Most importantly, they identify a novel mechanism for insulin-stimulated cell migration. - Highlights: • Insulin modulates PSA-NCAM turnover through upregulation of p-FAK. • P-FAK modulates αv-integrin/PSA-NCAM clustering. • αv-integrin acts as a carrier for PSA-NCAM endocytosis. • Cell migration is promoted by cell surface PSA. • Insulin promotes PSA-dependent migration in vitro.

Insulin promotes cell migration by regulating PSA-NCAM

International Nuclear Information System (INIS)

Monzo, Hector J.; Coppieters, Natacha; Park, Thomas I.H.; Dieriks, Birger V.; Faull, Richard L.M.; Dragunow, Mike; Curtis, Maurice A.

2017-01-01

Cellular interactions with the extracellular environment are modulated by cell surface polysialic acid (PSA) carried by the neural cell adhesion molecule (NCAM). PSA-NCAM is involved in cellular processes such as differentiation, plasticity, and migration, and is elevated in Alzheimer's disease as well as in metastatic tumour cells. Our previous work demonstrated that insulin enhances the abundance of cell surface PSA by inhibiting PSA-NCAM endocytosis. In the present study we have identified a mechanism for insulin-dependent inhibition of PSA-NCAM turnover affecting cell migration. Insulin enhanced the phosphorylation of the focal adhesion kinase leading to dissociation of αv-integrin/PSA-NCAM clusters, and promoted cell migration. Our results show that αv-integrin plays a key role in the PSA-NCAM turnover process. αv-integrin knockdown stopped PSA-NCAM from being endocytosed, and αv-integrin/PSA-NCAM clusters co-labelled intracellularly with Rab5, altogether indicating a role for αv-integrin as a carrier for PSA-NCAM during internalisation. Furthermore, inhibition of p-FAK caused dissociation of αv-integrin/PSA-NCAM clusters and counteracted the insulin-induced accumulation of PSA at the cell surface and cell migration was impaired. Our data reveal a functional association between the insulin/p-FAK-dependent regulation of PSA-NCAM turnover and cell migration through the extracellular matrix. Most importantly, they identify a novel mechanism for insulin-stimulated cell migration. - Highlights: • Insulin modulates PSA-NCAM turnover through upregulation of p-FAK. • P-FAK modulates αv-integrin/PSA-NCAM clustering. • αv-integrin acts as a carrier for PSA-NCAM endocytosis. • Cell migration is promoted by cell surface PSA. • Insulin promotes PSA-dependent migration in vitro.

Reductive methylation of insulin. Production of a biologically active tritiated insulin

Energy Technology Data Exchange (ETDEWEB)

Marsh, J W; Nahum, A; Steiner, D F [Department of Biochemistry, University of Chicago, Illinois, USA

1983-01-01

Reductive methylation of the three amino groups of porcine insulin was accomplished by incubation with formaldehyde and sodium cyanoborohydride. The two amino termini and the epsilon amino group of B29 lysine were each dimethylated within 1 h of incubation. The fully methylated insulin bound more tightly to a reverse phase column than did native insulin, had a slightly more acid isoelectric point, and maintained approximately 50% biological activity when examined with an insulin sensitive cultured cell line. Reductive methylation with sodium cyanoboro (/sup 3/H) hydride resulted in a (/sup 3/H) methylated insulin with a specific activity of 6 Ci/mmol.

Regulation of dendritic cell function by insulin/IGF-1/PI3K/Akt signaling through klotho expression.

Science.gov (United States)

Xuan, Nguyen Thi; Hoang, Nguyen Huy; Nhung, Vu Phuong; Duong, Nguyen Thuy; Ha, Nguyen Hai; Hai, Nong Van

2017-06-01

Insulin or insulin-like growth factor 1 (IGF-1) promotes the activation of phosphoinositide 3 kinase (PI3K)/Akt signaling in immune cells including dendritic cells (DCs), the most potent professional antigen-presenting cells for naive T cells. Klotho, an anti-aging protein, participates in the regulation of the PI3K/Akt signaling, thus the Ca 2+ -dependent migration is reduced in klotho-deficient DCs. The present study explored the effects of insulin/IGF-1 on DC function through klotho expression. To this end, the mouse bone marrow cells were isolated and cultured with GM-CSF to attain bone marrow-derived DCs (BMDCs). Cells were treated with insulin or IGF-1 and followed by stimulating with lipopolysaccharides (LPS). Tumor necrosis factor (TNF)-α formation was examined by enzyme-linked immunosorbent assay (ELISA). Phagocytosis was analyzed by FITC-dextran uptake assay. The expression of klotho was determined by quantitative PCR, immunoprecipitation and western blotting. As a result, treatment of the cells with insulin/IGF-1 resulted in reducing the klotho expression as well as LPS-stimulated TNF-α release and increasing the FITC-dextran uptake but unaltering reactive oxygen species (ROS) production in BMDCs. The effects were abolished by using pharmacological inhibition of PI3K/Akt with LY294002 and paralleled by transfecting DCs with klotho siRNA. In conclusion, the regulation of klotho sensitive DC function by IGF-1 or insulin is mediated through PI3K/Akt signaling pathway in BMDCs.

The voltage-gated proton channel Hv1 is expressed in pancreatic islet β-cells and regulates insulin secretion

Energy Technology Data Exchange (ETDEWEB)

Zhao, Qing [Department of Biophysics, School of Physics Science, The Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, Tianjin 300071 (China); Che, Yongzhe [School of Medicine, Nankai University, Tianjin 300071 (China); Li, Qiang; Zhang, Shangrong [Department of Biophysics, School of Physics Science, The Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, Tianjin 300071 (China); Gao, Ying-Tang [Key Laboratory of Artificial Cell, Third Central Clinical College of Tianjin Medical University, Tianjin 300170 (China); Wang, Yifan; Wang, Xudong; Xi, Wang; Zuo, Weiyan [Department of Biophysics, School of Physics Science, The Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, Tianjin 300071 (China); Li, Shu Jie, E-mail: shujieli@nankai.edu.cn [Department of Biophysics, School of Physics Science, The Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, Tianjin 300071 (China)

2015-12-25

The voltage-gated proton channel Hv1 is a potent acid extruder that participates in the extrusion of the intracellular acid. Here, we showed for the first time, Hv1 is highly expressed in mouse and human pancreatic islet β-cells, as well as β-cell lines. Imaging studies demonstrated that Hv1 resides in insulin-containing granules in β-cells. Knockdown of Hv1 with RNA interference significantly reduces glucose- and K{sup +}-induced insulin secretion in isolated islets and INS-1 (832/13) β-cells and has an impairment on glucose- and K{sup +}-induced intracellular Ca{sup 2+} homeostasis. Our data demonstrated that the expression of Hv1 in pancreatic islet β-cells regulates insulin secretion through regulating Ca{sup 2+} homeostasis.

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

Effects of turtle oil on insulin sensitivity and glucose metabolism in insulin resistant cell model

International Nuclear Information System (INIS)

Bai Jing; Tian Yaping; Guo Duo

2007-01-01

To evaluate the effects of turtle oil on insulin sensitivity and glucose metabolism in an insulin-resistant (IR) cell model which was established by the way of high concentration of insulin induction with HepG 2 cell in vitro culture. The IR cells were treated by turtle oil, the glucose consumption and 3 H-D-glucose incorporation rate in IR cells were detected by the way of glucose oxidase and 3 H-D-glucose incorporation assay respectively. The state of cell proliferation was tested by MTT method. The results showed that the incorporation rate of 3 H-D-glucose in IR cells was significantly lower than that in the control cells(P 3 H-D-glucose incorporation rate in either IR cells or control cells was increased with the increase of insulin concentration. Moreover, the 3 H-D-glucose incorporation rate of IR cells increased slower than that of control cells. The MTT assay showed that turtle oil can promote the proliferation of IR cell and control cell. The glucose uptake and glucose consumption in IR cell which treated with turtle oil was significantly increase than that in the control cells (P<0.05). Turtle oil can improve the insulin sensitivity and glucose metabolism in the IR cell model. (authors)

Brain pericyte-derived soluble factors enhance insulin sensitivity in GT1-7 hypothalamic neurons.

Science.gov (United States)

Takahashi, Hiroyuki; Takata, Fuyuko; Matsumoto, Junichi; Machida, Takashi; Yamauchi, Atsushi; Dohgu, Shinya; Kataoka, Yasufumi

2015-02-20

Insulin signaling in the hypothalamus plays an important role in food intake and glucose homeostasis. Hypothalamic neuronal functions are modulated by glial cells; these form an extensive network connecting the neurons and cerebral vasculature, known as the neurovascular unit (NVU). Brain pericytes are periendothelial accessory structures of the blood-brain barrier and integral members of the NVU. However, the interaction between pericytes and neurons is largely unexplored. Here, we investigate whether brain pericytes could affect hypothalamic neuronal insulin signaling. Our immunohistochemical observations demonstrated the existence of pericytes in the mouse hypothalamus, exhibiting immunoreactivity of platelet-derived growth factor receptor β (a pericyte marker), and laminin, a basal lamina marker. We then exposed a murine hypothalamic neuronal cell line, GT1-7, to conditioned medium obtained from primary cultures of rat brain pericytes. Pericyte-conditioned medium (PCM), but not astrocyte- or aortic smooth muscle cell-conditioned medium, increased the insulin-stimulated phosphorylation of Akt in GT1-7 cells in a concentration-dependent manner. PCM also enhanced insulin-stimulated tyrosine phosphorylation of insulin receptor β without changing its expression or localization in cytosolic or plasma membrane fractions. These results suggest that pericytes, rather than astrocytes, increase insulin sensitivity in hypothalamic neurons by releasing soluble factors under physiological conditions in the NVU. Copyright © 2015 Elsevier Inc. All rights reserved.

Insulin-secretagogue activity and cytoprotective role of the traditional antidiabetic plant Scoparia dulcis (Sweet Broomweed).

Science.gov (United States)

Latha, Muniappan; Pari, Leelavinothan; Sitasawad, Sandhya; Bhonde, Ramesh

2004-09-03

Scoparia dulcis (Sweet Broomweed) has been documented as a traditional treatment of diabetes. The administration of an aqueous extract of Scoparia dulcis at a dose of 200 mg/kg body weight significantly decreased the blood glucose with significant increase in plasma insulin level in streptozotocin diabetic rats at the end of 15 days treatment. The insulin secretagogue action of Scoparia dulcis plant extract (SPEt) was further investigated using isolated pancreatic islets from mice. SPEt at a dose of 10 microg/ml evoked 6.0 fold stimulation of insulin secretion from isolated islets indicating its insulin secretagogue activity. In addition the effect of SPEt on streptozotocin induced cell death and nitric oxide (NO) in terms of nitrite production were also examined. SPEt protected against streptozotocin- mediated cytotoxicity (88%) and NO production in rat insulinoma cell line (RINm5F). Above results suggest the glucose lowering effect of SPEt to be associated with potentiation of insulin release from pancreatic islets. Our results revealed the possible therapeutic value of Scoparia dulcis for the better control, management and prevention of diabetes mellitus progression.

19 CFR 142.50 - Line Release data base corrections or changes.

Science.gov (United States)

2010-04-01

... 19 Customs Duties 2 2010-04-01 2010-04-01 false Line Release data base corrections or changes. 142...; DEPARTMENT OF THE TREASURY (CONTINUED) ENTRY PROCESS Line Release § 142.50 Line Release data base corrections... numbers or bond information on a Line Release Data Loading Sheet as soon as possible. Notification shall...

The EndoC-βH1 cell line is a valid model of human beta cells and applicable for screenings to identify novel drug target candidates

Directory of Open Access Journals (Sweden)

Violeta Georgieva Tsonkova

2018-02-01

Full Text Available Objective: To characterize the EndoC-βH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates. Methods: EndoC-βH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation. Results: Transplantation of EndoC-βH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-βH1 pseudoislets compared to monolayer cultures for both glucose and incretins.Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate.By screening of various proteins and peptides, we found Bombesin (BB receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation.ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion. Conclusions: Overall, the EndoC-βH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated

Fibronectin and laminin promote differentiation of human mesenchymal stem cells into insulin producing cells through activating Akt and ERK

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Chiou Shih-Hwa

2010-07-01

Full Text Available Abstract Background Islet transplantation provides a promising cure for Type 1 diabetes; however it is limited by a shortage of pancreas donors. Bone marrow-derived multipotent mesenchymal stem cells (MSCs offer renewable cells for generating insulin-producing cells (IPCs. Methods We used a four-stage differentiation protocol, containing neuronal differentiation and IPC-conversion stages, and combined with pellet suspension culture to induce IPC differentiation. Results Here, we report adding extracellular matrix proteins (ECM such as fibronectin (FN or laminin (LAM enhances pancreatic differentiation with increases in insulin and Glut2 gene expressions, proinsulin and insulin protein levels, and insulin release in response to elevated glucose concentration. Adding FN or LAM induced activation of Akt and ERK. Blocking Akt or ERK by adding LY294002 (PI3K specific inhibitor, PD98059 (MEK specific inhibitor or knocking down Akt or ERK failed to abrogate FN or LAM-induced enhancement of IPC differentiation. Only blocking both of Akt and ERK or knocking down Akt and ERK inhibited the enhancement of IPC differentiation by adding ECM. Conclusions These data prove IPC differentiation by MSCs can be modulated by adding ECM, and these stimulatory effects were mediated through activation of Akt and ERK pathways.

Exaggerated release and preserved insulinotropic action of glucagon-like peptide-1 underlie insulin hypersecretion in glucose-tolerant individuals after Roux-en-Y gastric bypass

DEFF Research Database (Denmark)

Dirksen, Carsten; Bojsen-Møller, Kirstine N; Jørgensen, Nils Bruun

2013-01-01

Roux-en-Y gastric bypass (RYGB) improves glycaemic control in part by increasing postprandial insulin secretion through exaggerated glucagon-like peptide (GLP)-1 release. However, it is unknown whether islet cell responsiveness to i.v. glucose, non-glucose (arginine) and incretin hormones...

Endogenous pyrogen production by Hodgkin's disease and human histiocytic lymphoma cell lines in vitro.

Science.gov (United States)

Bodel, P; Ralph, P; Wenc, K; Long, J C

1980-02-01

Fever not explained by infection may occur in patients with malignant lymphoma presumably caused by a release of endogenous pyrogen. Although pyrogen has been found in some tumors with a mixed cell population, production of endogenous pyrogen by the neoplastic cells has not been demonstrated. This report documents the apparently spontaneous synthesis and release of such pyrogen by two human tumor cell lines derived from patients with Hodgkin's disease and histiocytic lymphoma. The endogenous pyrogen from the two cell lines was similar and closely resembled that produced by normal human monocytes in antigenic properties as well as heat and pronase sensitivity. The Hodgkin's disease and histiocytic lymphoma cell lines do not require specific stimulation for the production of endogenous pyrogen suggesting that the mechanism of pyrogen release by neoplastic macrophage-related cells differs from that of normal phagocytic cells. The tumor-associated fever in some patients with malignant lymphoma may be caused by a release of endogenous pyrogen by proliferating neoplastic cells.

Studies on binding and mitogenic effect of insulin and insulin-like growth factor I in glomerular mesangial cells

International Nuclear Information System (INIS)

Conti, F.G.; Striker, L.J.; Lesniak, M.A.; MacKay, K.; Roth, J.; Striker, G.E.

1988-01-01

The mesangial cells are actively involved in regulating glomerular hemodynamics. Their overlying endothelium is fenestrated; therefore, these cells are directly exposed to plasma substances, including hormones such as insulin and insulin-like growth factor I (IGF-I). These peptides may contribute to the mesangial sclerosis and cellular hyperplasia that characterize diabetic glomerulopathy. We report herein the characterization of the receptors and the mitogenic effects of IGF-I and insulin on mouse glomerular mesangial cells in culture. The IGF-I receptor was characterized on intact cells. The Kd of the IGF-I receptor was 1.47 X 10(-9) M, and the estimated number of sites was 64,000 receptors/cell. The binding was time, temperature, and pH dependent, and the receptor showed down-regulation after exposure to serum. The expression of the receptor did not change on cells at different densities. The specific binding for insulin was too low to allow characterization of the insulin receptor on intact cells. However, it was possible to identify the insulin receptor in a wheat germ agglutinin-purified preparation of solubilized mesangial cells. This receptor showed the characteristic features of the insulin receptor, including pH dependence of binding and a curvilinear Scatchard plot. The mitogenic effects of insulin and IGF-I on mesangial cells were measured by the incorporation of [3H]thymidine into DNA. IGF-I was more potent than insulin. The half-maximal response to IGF-I stimulation occurred at 1.3 X 10(-10) M, and a similar increase with insulin was observed at concentrations in the range of 10(-7) M, suggesting that this insulin action was mediated through the IGF-I receptor. These data show that the mouse microvascular smooth muscle cells of the glomerulus express a cell surface receptor for IGF-I in vitro and that this peptide is a potent mitogen for these mesangial cells

Mechanism of palytoxin-induced [3H]norepinephrine release from a rat pheochromocytoma cell line

International Nuclear Information System (INIS)

Tatsumi, M.; Takahashi, M.; Ohizumi, Y.

1984-01-01

Palytoxin, isolated from the zoanthid Palytoha species, is one of the most potent marine toxins. Palytoxin caused a release of [ 3 H]norepinephrine from clonal rat pheochromocytoma cells in a concentration-dependent manner. This releasing action of palytoxin was markedly inhibited or abolished by Co 2+ or Ca 2+ -free medium, but was not modified by tetrodotoxin. The release of [ 3 H]norepinephrine induced by a low concentration of palytoxin was abolished in sodium-free medium and increased as the external Na+ concentrations were increased, but the release induced by a high concentration was unaffected by varying the concentration of external Na + . The release of [ 3 H]norepinephrine induced by both concentrations of palytoxin increased with increasing Ca 2+ concentrations. Palytoxin caused a concentration-dependent increase in 22 Na and 45 Ca influxes into pheochromocytoma cells. The palytoxin-induced 45 Ca influx was markedly inhibited by Co 2+ , whereas the palytoxin-induced 22 Na influx was not affected by tetrodotoxin. These results suggest that in pheochromocytoma cells the [ 3 H]norepinephrine release induced by lower concentrations of palytoxin is primarily brought about by increasing tetrodotoxin-insensitive Na + permeability across the cell membrane, whereas that induced by higher concentrations is mainly caused by a direct increase in Ca 2+ influx into them

Insulin-induced activation of glycerol-3-phosphate acyltransferase by a chiro-inositol-containing insulin mediator is defective in adipocytes of insulin-resistant, type II diabetic, Goto-Kakizaki rats.

Science.gov (United States)

Farese, R V; Standaert, M L; Yamada, K; Huang, L C; Zhang, C; Cooper, D R; Wang, Z; Yang, Y; Suzuki, S; Toyota, T

1994-11-08

Type II diabetic Goto-Kakizaki (GK) rats were insulin-resistant in euglycemic-hyperinsulinemic clamp studies. We therefore examined insulin signaling systems in control Wistar and diabetic GK rats. Glycerol-3-phosphate acyltransferase (G3PAT), which is activated by headgroup mediators released from glycosyl-phosphatidylinositol (GPI), was activated by insulin in intact and cell-free adipocyte preparations of control, but not diabetic, rats. A specific chiro-inositol-containing inositol phosphoglycan (IPG) mediator, prepared from beef liver, bypassed this defect and comparably activated G3PAT in cell-free adipocyte preparations of both diabetic GK and control rats. A myo-inositol-containing IPG mediator did not activate G3PAT. Relative to control adipocytes, labeling of GPI by [3H]glucosamine was diminished by 50% and insulin failed to stimulate GPI hydrolysis in GK adipocytes. In contrast to GPI-dependent G3PAT activation, insulin-stimulated hexose transport was intact in adipocytes and soleus and gastrocnemius muscles of the GK rat, as was insulin-induced activation of mitogen-activated protein kinase and protein kinase C. We conclude that (i) chiro-inositol-containing IPG mediator activates G3PAT during insulin action, (ii) diabetic GK rats have a defect in synthesizing or releasing functional chiro-inositol-containing IPG, and (iii) defective IPG-regulated intracellular glucose metabolism contributes importantly to insulin resistance in diabetic GK rats.

L-leucine methyl ester stimulates insulin secretion and islet glutamate dehydrogenase

DEFF Research Database (Denmark)

Knudsen, P; Kofod, Hans; Lernmark, A

1983-01-01

Column perifusion of collagenase-isolated mouse pancreatic islets was used to study the dynamics of insulin release in experiments lasting for several hours. The methyl esters of L-leucine and L-arginine were synthesized. Whereas L-arginine methyl ester (L-arginine OMe) had no effect, L-leucine OMe...... stimulated the release of insulin. The effect of L-leucine OMe was maximal at 5 mmol/liter. Whereas the Km for glucose-stimulated insulin release was unaffected by 1 mmol/liter L-leucine OMe, the maximal release of D-glucose was increased by the amino acid derivative that appeared more effective than L......-leucine. L-Leucine OMe was also a potent stimulus of insulin release from the perfused mouse pancreas. In the presence of 10 mmol/liter L-glutamine, 1 mmol/liter L-leucine OMe induced a 50- to 75-fold increase in insulin release. A similar stimulatory effect was also observed in column-perifused RIN 5F cells...

[Anti-tumor effects of DDP-PLLA-CNTs on human cholangiocarcinoma cell line in vitro].

Science.gov (United States)

Li, Maolan; Lu, Wei; Zhang, Fei; Ding, Qichen; Wu, Xiangsong; Tan, Zhujun; Wu, Wenguang; Weng, Hao; Wang, Xuefeng; Shi, Weibin; Dong, Ping; Gu, Jun; Liu, Yingbin

2014-11-04

To explore the antitumor effects of DDP-PLLA-CNTs on human cholangiocarcinoma cell line. DDP-PLLA-CNTs were prepared with the method of ultrasound emulsification. The morphology of DDP-PLLA-CNTs was determined by scanning electron microscope (SEM). And its drug loading and drug release curve in vitro was detected by UV-Vis-NIR spectrophotometer. CCK8 was used to test the cytotoxic effects of DDP-PLLA-CNTs at different concentrations on QBC939 cell proliferation.Flow cytometry was employed to measure the changes of apoptotic rate. With excellent controlled-release characteristic of in vitro drug release, DDP-PLLA-CNTs inhibited the proliferation and significantly increased the apoptotic rate of QBC939 cell line. DDP-PLLA-CNTs have drug sustained-release characteristics and can significantly inhibit the proliferation of QBC939 cell line.

Variations in insulin responsiveness in rat fat cells are due to metabolic differences rather than insulin binding

DEFF Research Database (Denmark)

Hansen, Finn Mølgård; Nilsson, Poul; Sonne, Ole

1983-01-01

-insulin to fat cells. Insulin binding was not correlated to the plasma insulin level which however was reflected in the lipoprotein lipase activity in the adipose tissue. In conclusion, these results indicate that variations in insulin responsiveness in fat cells are due to alterations in cellular metabolism...

Differential Cell Sensitivity between OTA and LPS upon Releasing TNF-α

Directory of Open Access Journals (Sweden)

Lauy Al-Anati

2010-06-01

Full Text Available The release of tumor necrosis factor α (TNF-α by ochratoxin A (OTA was studied in various macrophage and non-macrophage cell lines and compared with E. coli lipopolysaccharide (LPS as a standard TNF-α release agent. Cells were exposed either to 0, 2.5 or 12.5 µmol/L OTA, or to 0.1 µg/mL LPS, for up to 24 h. OTA at 2.5 µmol/L and LPS at 0.1 µg/mL were not toxic to the tested cells as indicated by viability markers. TNF-a was detected in the incubated cell medium of rat Kupffer cells, peritoneal rat macrophages, and the mouse monocyte macrophage cell line J774A.1: TNF-a concentrations were 1,000 pg/mL, 1,560 pg/mL, and 650 pg/mL, respectively, for 2.5 µmol/L OTA exposure and 3,000 pg/mL, 2,600 pg/mL, and 2,115 pg/mL, respectively, for LPS exposure. Rat liver sinusoidal endothelial cells, rat hepatocytes, human HepG2 cells, and mouse L929 cells lacked any cytokine response to OTA, but showed a significant release of TNF-a after LPS exposure, with the exception of HepG2 cells. In non-responsive cell lines, OTA lacked both any activation of NF-κB or the translocation of activated NF-κB to the cell nucleus, i.e., in mouse L929 cells. In J774A.1 cells, OTA mediated TNF-a release via the pRaf/MEK 1/2–NF-κB and p38-NF-κB pathways, whereas LPS used pRaf/MEK 1/2-NF-κB, but not p38-NF-κB pathways. In contrast, in L929 cells, LPS used other pathways to activate NF-κB. Our data indicate that only macrophages and macrophage derived cells respond to OTA and are considered as sources for TNF-a release upon OTA exposure.

Insulin-induced activation of glycerol-3-phosphate acyltransferase by a chiro-inositol-containing insulin mediator is defective in adipocytes of insulin-resistant, type II diabetic, Goto-Kakizaki rats.

OpenAIRE

Farese, R V; Standaert, M L; Yamada, K; Huang, L C; Zhang, C; Cooper, D R; Wang, Z; Yang, Y; Suzuki, S; Toyota, T

1994-01-01

Type II diabetic Goto-Kakizaki (GK) rats were insulin-resistant in euglycemic-hyperinsulinemic clamp studies. We therefore examined insulin signaling systems in control Wistar and diabetic GK rats. Glycerol-3-phosphate acyltransferase (G3PAT), which is activated by headgroup mediators released from glycosyl-phosphatidylinositol (GPI), was activated by insulin in intact and cell-free adipocyte preparations of control, but not diabetic, rats. A specific chiro-inositol-containing inositol phosph...

The EndoC-βH1 cell line is a valid model of human beta cells and applicable for screenings to identify novel drug target candidates.

Science.gov (United States)

Tsonkova, Violeta Georgieva; Sand, Fredrik Wolfhagen; Wolf, Xenia Asbæk; Grunnet, Lars Groth; Kirstine Ringgaard, Anna; Ingvorsen, Camilla; Winkel, Louise; Kalisz, Mark; Dalgaard, Kevin; Bruun, Christine; Fels, Johannes Josef; Helgstrand, Charlotte; Hastrup, Sven; Öberg, Fredrik Kryh; Vernet, Erik; Sandrini, Michael Paolo Bastner; Shaw, Allan Christian; Jessen, Carsten; Grønborg, Mads; Hald, Jacob; Willenbrock, Hanni; Madsen, Dennis; Wernersson, Rasmus; Hansson, Lena; Jensen, Jan Nygaard; Plesner, Annette; Alanentalo, Tomas; Petersen, Maja Borup Kjær; Grapin-Botton, Anne; Honoré, Christian; Ahnfelt-Rønne, Jonas; Hecksher-Sørensen, Jacob; Ravassard, Philippe; Madsen, Ole D; Rescan, Claude; Frogne, Thomas

2018-02-01

To characterize the EndoC-βH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates. EndoC-βH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation. Transplantation of EndoC-βH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-βH1 pseudoislets compared to monolayer cultures for both glucose and incretins. Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate. By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation. ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion. Overall, the EndoC-βH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active

Type 2 diabetes risk allele near CENTD2 is associated with decreased glucose-stimulated insulin release

DEFF Research Database (Denmark)

Nielsen, Trine; Sparsø, T; Grarup, N

2011-01-01

By combining multiple genome-wide association (GWA) studies and comprehensive replication efforts, 12 novel type 2 diabetes associated loci have recently been discovered. Here we evaluate the effect of lead variants of these loci on estimates of insulin release and insulin resistance derived from...

Aerosolized liposomes with dipalmitoyl phosphatidylcholine enhance pulmonary absorption of encapsulated insulin compared with co-administered insulin.

Science.gov (United States)

Chono, Sumio; Togami, Kohei; Itagaki, Shirou

2017-11-01

We have previously shown that aerosolized liposomes with dipalmitoyl phosphatidylcholine (DPPC) enhance the pulmonary absorption of encapsulated insulin. In this study, we aimed to compare insulin encapsulated into the liposomes versus co-administration of empty liposomes and unencapsulated free insulin, where the DPCC liposomes would serve as absorption enhancer. The present study provides the useful information for development of noninvasive treatment of diabetes. Co-administration of empty DPPC liposomes and unencapsulated free insulin was investigated in vivo to assess the potential enhancement in protein pulmonary absorption. Co-administration was compared to DPPC liposomes encapsulating insulin, and free insulin. DPPC liposomes enhanced the pulmonary absorption of unencapsulated free insulin; however, the enhancing effect was lower than that of the DPPC liposomes encapsulating insulin. The mechanism of the pulmonary absorption of unencapsulated free insulin by DPPC liposomes involved the opening of epithelial cell space in alveolar mucosa, and not mucosal cell damage, similar to that of the DPPC liposomes encapsulating insulin. In an in vitro stability test, insulin in the alveolar mucus layer that covers epithelial cells was stable. These findings suggest that, although unencapsulated free insulin spreads throughout the alveolar mucus layer, the concentration of insulin released near the absorption surface is increased by the encapsulation of insulin into DPPC liposomes and the absorption efficiency is also increased. We revealed that the encapsulation of insulin into DPPC liposomes is more effective for pulmonary insulin absorption than co-administration of DPPC liposomes and unencapsulated free insulin.

Rapid internalization of the insulin receptor in rat hepatoma cells

International Nuclear Information System (INIS)

Backer, J.M.; White, M.F.; Kahn, C.R.

1987-01-01

The authors have studied the internalization of the insulin receptor (IR) in rat hepatoma cells (Fao). The cells were surface-iodinated at 4 0 C, stimulated with insulin at 37 0 C, and then cooled rapidly, trypsinized at 4 0 C and solubilized. The IR was immunoprecipitated with a specific antibody, and internalization of the IR was assessed by the appearance of trypsin-resistant bands on SDS-PAGE. Insulin induced the internalization of surface receptors with a t 1/2 of 9-10 mins; cells not exposed to insulin internalized less than 20% of the IR during 1 h at 37 0 C. Further experiments demonstrated that the accumulation of trypsin-resistant IR paralleled a loss of receptor from the cell surface. Insulin-stimulated cells were chilled and iodinated at 4 0 C, followed by solubilization, immunoprecipitation and SDS-PAGE; alternatively, insulin-stimulated cells were chilled, surface-bound ligand removed by washing the cells at pH 4.2, and specific [ 125 I]insulin binding measured at 4 0 C. Both techniques confirmed the disappearance of IR from the cell surface at rates comparable to the insulin-stimulated internalization described above. The total amount of phosphotyrosine-containing IR, as assessed by immunoprecipitation with an anti-phosphotyrosine antibody, remained constant during this time interval, suggesting that active kinase is translocated into the cell. In summary, the authors data indicate that insulin binding increases the rate of IR internalization of Fao cells. This relocation may facilitate the interaction of the activated tyrosine kinase in the IR with intracellular substrates, thus transmitting the insulin signal to metabolic pathways

Synthesis and characterization of insulin/zirconium phosphate@TiO2 hybrid composites for enhanced oral insulin delivery applications.

Science.gov (United States)

Safari, Mostafa; Kamari, Younes; Ghiaci, Mehran; Sadeghi-Aliabadi, Hojjat; Mirian, Mina

2017-05-01

In this work, a series of composites of insulin (Ins)/zirconium phosphate (ZrP) were synthesized by intercalation method, then, these composites were coated with TiO 2 by sol-gel method to prepare Ins/ZrP@TiO 2 hybrid composites and the drug release of the composites was investigated by using UV-Vis spectroscopy. Ins/ZrP (10, 30, 60 wt%) composites were prepared by intercalation of insulin into the ZrP layers in water. Then Ins/ZrP composites were coated with different amounts of TiO 2 (30, 50, 100 wt %) by using titanium tetra n-butoxide, as precursor. Formation of intercalated Ins/ZrP and Ins/ZrP@TiO 2 hybrid composites was characterized by FT-IR, FE-SEM, BET and XRD analysis. Zeta potential of the optimized Ins/ZrP@TiO 2 hybrid composite was determined -27.2 mV. Cytotoxic effects of the optimized Ins/ZrP@TiO 2 hybrid composite against HeLa and Hek293T cell lines were evaluated using MTT assay and the results showed that designed drug delivery system was not toxic in biological environment. Compared to the Ins/ZrP composites, incorporation of TiO 2 coating enhanced the drug entrapment considerably, and reduced the drug release. The Ins/ZrP composites without TiO 2 coating released the whole drug after 30 min in pH 7.4 (phosphate buffer solution) while the TiO 2 -coated composites released the entrapped drug after 20 h. In addition to increasing the shelf life of hormone, this nanoencapsulation and nanocoating method can convert the insulin utilization from injection to oral and present a painless and more comfortable treatment for diabetics.

The insulinotropic effect of endothelin-1 is mediated by glucagon release from the islet alpha cells

DEFF Research Database (Denmark)

Brock, B; Gregersen, S; Kristensen, K

1999-01-01

AIMS/HYPOTHESIS: The circulating concentrations of endothelin-1 (ET-1), a peptide derived from endothelium, are increased in hypertension and diabetes. Endothelin-1 has recently been shown to be an insulinotropic agent. The mechanism of action of endothelin-1 on the endocrine pancreas has not yet...... been clarified. METHODS: We investigated the action of endothelin-1 on the insulin secretion, the binding of (125)I-ET-1 to beta cells as well as its effects on purified beta and non-beta cells from normal rats. The expression of endothelin receptors in alpha- and beta-cell lines and in normal rat...... from purified beta cells. Endothelin-1-(100 nmol/l) increased, however, both insulin and glucagon secretion from a mixture of purified beta and non-beta cells indicating that alpha cells seem to have a key role for the action of ET-1 on insulin secretion. CONCLUSION/INTERPRETATION: The insulinotropic...

Cell surface alteration in Epstein-Barr virus-transformed cells from patients with extreme insulin resistance

International Nuclear Information System (INIS)

Gorden, D.L.; Robert, A.; Moncada, V.Y.; Taylor, S.I.; Muehlhauser, J.C.; Carpentier, J.L.

1990-01-01

An abnormality was detected in the morphology of the cell surface of Epstein-Barr virus-transformed lymphocytes of patients with genetic forms of insulin resistance. In cells from two patients with leprechaunism and two patients with type A extreme insulin resistance, scanning electron microscopy demonstrated a decrease in the percentage of the cell surface occupied by microvilli in cells from the patients with leprechaunism and type A insulin resistance compared with control cells. When cells from a healthy control subject and one of the patients with leprechaunism (Lep/Ark-1) were incubated with 125 I-labeled insulin, there was a decrease in the percentage of 125 I-insulin associated with microvilli on the cell surface. Thus, the decreased localization of insulin receptors with the microvillous region of the cell surface was in proportion to the decrease in microvilli

Evaluation of Insulin Medium or Chondrogenic Medium on Proliferation and Chondrogenesis of ATDC5 Cells

OpenAIRE

Yao, Yongchang; Zhai, Zhichen; Wang, Yingjun

2014-01-01

Background. The ATDC5 cell line is regarded as an excellent cell model for chondrogenesis. In most studies with ATDC5 cells, insulin medium (IM) was used to induce chondrogenesis while chondrogenic medium (CM), which was usually applied in chondrogenesis of mesenchymal stem cells (MSCs), was rarely used for ATDC5 cells. This study was mainly designed to investigate the effect of IM, CM, and growth medium (GM) on chondrogenesis of ATDC5 cells. Methods. ATDC5 cells were, respectively, cultured ...

Extrinsic factors promoting insulin producing cell-differentiation and insulin expression enhancement-hope for diabetics.

Science.gov (United States)

Dave, Shruti

2013-11-01

Diabetes mellitus (DM) is considered to be an autoimmune disorder leading to destruction of beta-cells resulting in to a loss of blood sugar control. Attempts using many pharmacological compositions including exogenous insulin have failed to show tight control of glycemia and associated manifestations. Stem cells are considered a potential tool for the supply of insulin-producing cells (IPC) generation in vitro. Stem cell differentiation in to pancreatic lineages requires influence of both intrinsic and extrinsic factors. Application of islet growth factors is considered to be potential for enhancement of beta-cell replication, function and survival. Use of certain extrinsic factors is known to facilitate expression of transcription factors known to be important for beta-cell differentiation and production of insulin enabling IPC generation. Hierarchies of secreted signals and transcription factors have been identified by studies from several laboratories that guide cell differentiation in to IPC. This knowledge provides insights for in vitro IPC differentiation from stem cells. Current advancement in medical knowledge promises an insulin independency for DM patients. The review sheds light on few specific extrinsic factors which facilitate differentiation of stem cells in to IPC in vitro have been discussed; which can be proven as a potential therapeutic option for treatment of DM and associated diseases.

Hypoxic enhancement of exosome release by breast cancer cells

International Nuclear Information System (INIS)

King, Hamish W; Michael, Michael Z; Gleadle, Jonathan M

2012-01-01

Exosomes are nanovesicles secreted by tumour cells which have roles in paracrine signalling during tumour progression, including tumour-stromal interactions, activation of proliferative pathways and bestowing immunosuppression. Hypoxia is an important feature of solid tumours which promotes tumour progression, angiogenesis and metastasis, potentially through exosome-mediated signalling. Breast cancer cell lines were cultured under either moderate (1% O 2 ) or severe (0.1% O 2 ) hypoxia. Exosomes were isolated from conditioned media and quantitated by nanoparticle tracking analysis (NTA) and immunoblotting for the exosomal protein CD63 in order to assess the impact of hypoxia on exosome release. Hypoxic exosome fractions were assayed for miR-210 by real-time reverse transcription polymerase chain reaction and normalised to exogenous and endogenous control genes. Statistical significance was determined using the Student T test with a P value of < 0.05 considered significant. Exposure of three different breast cancer cell lines to moderate (1% O 2 ) and severe (0.1% O 2 ) hypoxia resulted in significant increases in the number of exosomes present in the conditioned media as determined by NTA and CD63 immunoblotting. Activation of hypoxic signalling by dimethyloxalylglycine, a hypoxia-inducible factor (HIF) hydroxylase inhibitor, resulted in significant increase in exosome release. Transfection of cells with HIF-1α siRNA prior to hypoxic exposure prevented the enhancement of exosome release by hypoxia. The hypoxically regulated miR-210 was identified to be present at elevated levels in hypoxic exosome fractions. These data provide evidence that hypoxia promotes the release of exosomes by breast cancer cells, and that this hypoxic response may be mediated by HIF-1α. Given an emerging role for tumour cell-derived exosomes in tumour progression, this has significant implications for understanding the hypoxic tumour phenotype, whereby hypoxic cancer cells may release

Insulin utilizes the PI 3-kinase pathway to inhibit SP-A gene expression in lung epithelial cells

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Snyder Jeanne M

2002-10-01

Full Text Available Abstract Background It has been proposed that high insulin levels may cause delayed lung development in the fetuses of diabetic mothers. A key event in lung development is the production of adequate amounts of pulmonary surfactant. Insulin inhibits the expression of surfactant protein A (SP-A, the major surfactant-associated protein, in lung epithelial cells. In the present study, we investigated the signal transduction pathways involved in insulin inhibition of SP-A gene expression. Methods H441 cells, a human lung adenocarcinoma cell line, or human fetal lung explants were incubated with or without insulin. Transcription run-on assays were used to determine SP-A gene transcription rates. Northern blot analysis was used to examine the effect of various signal transduction inhibitors on SP-A gene expression. Immunoblot analysis was used to evaluate the levels and phosphorylation states of signal transduction protein kinases. Results Insulin decreased SP-A gene transcription in human lung epithelial cells within 1 hour. Insulin did not affect p44/42 mitogen-activated protein kinase (MAPK phosphorylation and the insulin inhibition of SP-A mRNA levels was not affected by PD98059, an inhibitor of the p44/42 MAPK pathway. In contrast, insulin increased p70 S6 kinase Thr389 phosphorylation within 15 minutes. Wortmannin or LY294002, both inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase, or rapamycin, an inhibitor of the activation of p70 S6 kinase, a downstream effector in the PI 3-kinase pathway, abolished or attenuated the insulin-induced inhibition of SP-A mRNA levels. Conclusion Insulin inhibition of SP-A gene expression in lung epithelial cells probably occurs via the rapamycin-sensitive PI 3-kinase signaling pathway.

Deletion of glutamate dehydrogenase in beta-cells abolishes part of the insulin secretory response not required for glucose homeostasis

DEFF Research Database (Denmark)

Carobbio, Stefania; Frigerio, Francesca; Rubi, Blanca

2009-01-01

Insulin exocytosis is regulated in pancreatic ss-cells by a cascade of intracellular signals translating glucose levels into corresponding secretory responses. The mitochondrial enzyme glutamate dehydrogenase (GDH) is regarded as a major player in this process, although its abrogation has not been...... tested yet in animal models. Here, we generated transgenic mice, named betaGlud1(-/-), with ss-cell-specific GDH deletion. Our results show that GDH plays an essential role in the full development of the insulin secretory response. In situ pancreatic perfusion revealed that glucose-stimulated insulin...... secretion was reduced by 37% in betaGlud1(-/-). Furthermore, isolated islets with either constitutive or acute adenovirus-mediated knock-out of GDH showed a 49 and 38% reduction in glucose-induced insulin release, respectively. Adenovirus-mediated re-expression of GDH in betaGlud1(-/-) islets fully restored...

Heterogeneous response of isolated adult rat heart cells to insulin

International Nuclear Information System (INIS)

Haworth, R.A.; Hunter, D.R.; Berkoff, H.A.

1984-01-01

3-O-Methylglucose uptake by Ca2+-resistant adult rat heart cells in suspension was measured, free of artifactual inhibitor-insensitive uptake, and with an accuracy of +/- 1.9% pellet water. (Ca2+-resistant cells are cells which retain their original rod-shaped morphology in the presence of physiological levels of Ca2+.) High levels of insulin (10(-6) M) stimulated the rate of 3-O-methylglucose uptake approximately 10-fold. In the presence of low levels of insulin (3 X 10(-11) M, 10(-10) M) uptake was biphasic; it could not be described by a single exponential function within experimental error, but required the sum of two exponentials. Deviation from a single exponential function was not so great with high levels of insulin (10(-6) M) or no insulin. Cell sugar uptake was also investigated using autoradiography of cells which had accumulated [2-14C]deoxyglucose under similar conditions. This showed considerable heterogeneity of 2-deoxyglucose uptake by cells treated with low levels of insulin, but significantly less heterogeneity of 2-deoxyglucose uptake by cells treated with high levels of insulin. It is concluded that the deviation of 3-O-methylglucose uptake from a single exponential observed at low insulin levels can be accounted for in terms of a heterogeneous response of cells to insulin

Receptor-mediated endocytosis and intracellular trafficking of insulin and low-density lipoprotein by retinal vascular endothelial cells.

Science.gov (United States)

Stitt, A W; Anderson, H R; Gardiner, T A; Bailie, J R; Archer, D B

1994-08-01

The authors investigated the receptor-mediated endocytosis (RME) and intracellular trafficking of insulin and low-density lipoprotein (LDL) in cultured retinal vascular endothelial cells (RVECs). Low-density lipoprotein and insulin were conjugated to 10 nm colloidal gold, and these ligands were added to cultured bovine RVECs for 20 minutes at 4 degrees C. The cultures were then warmed to 37 degrees C and fixed after incubation times between 30 seconds and 1 hour. Control cells were incubated with unconjugated gold colloid at times and concentrations similar to those of the ligands. Additional control cells were exposed to several concentrations of anti-insulin receptor antibody or a saturating solution of unconjugated insulin before incubation with gold insulin. Using transmission electron microscopy, insulin gold and LDL gold were both observed at various stages of RME. Insulin-gold particles were first seen to bind to the apical plasma membrane (PM) before clustering in clathrin-coated pits and internalization in coated vesicles. Gold was later visualized in uncoated cytoplasmic vesicles, corresponding to early endosomes and multivesicular bodies (MVBs) or late endosomes. In several instances, localized regions of the limiting membrane of the MVBs appeared coated, a feature of endosomal membranes not previously described. After RME at the apical PM and passage through the endosomal system, the greater part of both insulin- and LDL-gold conjugates was seen to accumulate in large lysosome-like compartments. However, a small but significant proportion of the internalized ligands was transcytosed and released as discrete membrane-associated quanta at the basal cell surface. The uptake of LDL gold was greatly increased in highly vacuolated, late-passage RVECs. In controls, anti-insulin receptor antibody and excess unconjugated insulin caused up to 89% inhibition in gold-insulin binding and internalization. These results illustrate the internalization and intracellular

Role of insulin in regulation of Na+-/K+-dependent ATPase activity and pump function in corneal endothelial cells.

Science.gov (United States)

Hatou, Shin; Yamada, Masakazu; Akune, Yoko; Mochizuki, Hiroshi; Shiraishi, Atsushi; Joko, Takeshi; Nishida, Teruo; Tsubota, Kazuo

2010-08-01

The Na(+)-/K(+)-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. The role of insulin in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells was investigated. Confluent monolayers of mouse corneal endothelial cells were exposed to insulin. ATPase activity was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate; Na,K-ATPase activity was defined as the portion of total ATPase activity sensitive to ouabain. Pump function was measured with the use of a Ussing chamber; pump function attributable to Na,K-ATPase activity was defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis and immunocytochemistry were performed to measure the expression of the Na,K-ATPase alpha(1)-subunit. Insulin increased the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were blocked by protein kinase C (PKC) inhibitors and protein phosphatases 1 and 2A inhibitor. Western blot analysis indicated that insulin decreased the ratio of the inactive Na,K-ATPase alpha(1)-subunit. Immunocytochemistry indicated that insulin increased the cell surface expression of the Na,K-ATPase alpha(1)-subunit. These results suggest that insulin increases the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. The effect of insulin is mediated by PKC and presumably results in the activation of PP1, 2A, or both, which are essential for activating Na,K-ATPase by alpha(1)-subunit dephosphorylation.

Valsartan Improves β-Cell Function and Insulin Sensitivity in Subjects With Impaired Glucose Metabolism

Science.gov (United States)

van der Zijl, Nynke J.; Moors, Chantalle C.M.; Goossens, Gijs H.; Hermans, Marc M.H.; Blaak, Ellen E.; Diamant, Michaela

2011-01-01

OBJECTIVE Recently, the Nateglinide and Valsartan in Impaired Glucose Tolerance Outcomes Research Trial demonstrated that treatment with the angiotensin receptor blocker (ARB) valsartan for 5 years resulted in a relative reduction of 14% in the incidence of type 2 diabetes in subjects with impaired glucose metabolism (IGM). We investigated whether improvements in β-cell function and/or insulin sensitivity underlie these preventive effects of the ARB valsartan in the onset of type 2 diabetes. RESEARCH DESIGN AND METHODS In this randomized controlled, double-blind, two-center study, the effects of 26 weeks of valsartan (320 mg daily; n = 40) or placebo (n = 39) on β-cell function and insulin sensitivity were assessed in subjects with impaired fasting glucose and/or impaired glucose tolerance, using a combined hyperinsulinemic-euglycemic and hyperglycemic clamp with subsequent arginine stimulation and a 2-h 75-g oral glucose tolerance test (OGTT). Treatment effects were analyzed using ANCOVA, adjusting for center, glucometabolic status, and sex. RESULTS Valsartan increased first-phase (P = 0.028) and second-phase (P = 0.002) glucose-stimulated insulin secretion compared with placebo, whereas the enhanced arginine-stimulated insulin secretion was comparable between groups (P = 0.25). In addition, valsartan increased the OGTT-derived insulinogenic index (representing first-phase insulin secretion after an oral glucose load; P = 0.027). Clamp-derived insulin sensitivity was significantly increased with valsartan compared with placebo (P = 0.049). Valsartan treatment significantly decreased systolic and diastolic blood pressure compared with placebo (P valsartan treatment increased glucose-stimulated insulin release and insulin sensitivity in normotensive subjects with IGM. These findings may partly explain the beneficial effects of valsartan in the reduced incidence of type 2 diabetes. PMID:21330640

Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

Science.gov (United States)

Pruett, W; Yuan, Y; Rose, E; Batzer, A G; Harada, N; Skolnik, E Y

1995-03-01

Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context

Human interleukin 1β stimulates islet insulin release by a mechanism not dependent on changes in phospholipase C and protein kinase C activities or Ca2+ handling

International Nuclear Information System (INIS)

Welsh, N.; Nilsson, T.; Hallberg, A.; Arkhammar, P.; Berggren, P.-O.; Sandler, S.

1989-01-01

Isolated islets from adult rats or obese hyperglycemic (ob/ob) mice were incubated with human recombinant interleukin 1β in order to study whether the acute effects of the cytokine on islet insulin release are associated with changes in islet phospholipase C activity, Ca 2+ handling or protein phosphorylation. The cytokine stimulated insulin release both at low and high glucose concentrations during one hour incubations. In shortterm incubations ( 2+ concentration at rest nor that observed subsequent to stimulation with a high concentration of glucose. Furthermore, the endogenous protein kinase C activity, as visualized by immunoprecipitation of a 32 P-labelled substrate for this enzyme, was not altered by interleukin 1β. Separation of 32 P-labelled proteins by means of 2-dimensional gel electrophoresis failed to reveal any specific effects of the cytokine on the total protein phosphorylation activity. These results suggest that the stimulatory effects on insulin release exerted by interleukin 1β are not caused by acute activation of phospholipase C and protein kinase C or by an alternation of islet Ca 2+ handling of the B-cells. (author)

Expression of growth factor receptors and targeting of EGFR in cholangiocarcinoma cell lines

International Nuclear Information System (INIS)

Xu, Ling; Hausmann, Martin; Dietmaier, Wolfgang; Kellermeier, Silvia; Pesch, Theresa; Stieber-Gunckel, Manuela; Lippert, Elisabeth; Klebl, Frank; Rogler, Gerhard

2010-01-01

Cholangiocarcinoma (CC) is a malignant neoplasm of the bile ducts or the gallbladder. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. The aim of this study was to determine the expression of seven growth factor receptors in CC cell lines and to assess the effect of blocking the EGFR receptor in vitro. Expression of EGFR (epithelial growth factor receptor), HGFR (hepatocyte growth factor receptor) IGF1R (insulin-like growth factor 1 receptor), IGF2R (insulin-like growth factor 2 receptor) and VEGFR1-3 (vascular endothelial growth factor receptor 1-3) were examined in four human CC cell lines (EGI-1, HuH28, OZ and TFK-1). The effect of the anti-EGFR-antibody cetuximab on cell growth and apoptosis was studied and cell lines were examined for KRAS mutations. EGFR, HGFR and IGFR1 were present in all four cell lines tested. IGFR2 expression was confirmed in EGI-1 and TFK-1. No growth-inhibitory effect was found in EGI-1 cells after incubation with cetuximab. Cetuximab dose-dependently inhibited growth in TFK-1. Increased apoptosis was only seen in TFK-1 cells at the highest cetuximab dose tested (1 mg/ml), with no dose-response-relationship at lower concentrations. In EGI-1 a heterozygous KRAS mutation was found in codon 12 (c.35G>A; p.G12D). HuH28, OZ and TFK-1 lacked KRAS mutation. CC cell lines express a pattern of different growth receptors in vitro. Growth factor inhibitor treatment could be affected from the KRAS genotype in CC. The expression of EGFR itself does not allow prognoses on growth inhibition by cetuximab

Expression of growth factor receptors and targeting of EGFR in cholangiocarcinoma cell lines

Directory of Open Access Journals (Sweden)

Kellermeier Silvia

2010-06-01

Full Text Available Abstract Background Cholangiocarcinoma (CC is a malignant neoplasm of the bile ducts or the gallbladder. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. The aim of this study was to determine the expression of seven growth factor receptors in CC cell lines and to assess the effect of blocking the EGFR receptor in vitro. Methods Expression of EGFR (epithelial growth factor receptor, HGFR (hepatocyte growth factor receptor IGF1R (insulin-like growth factor 1 receptor, IGF2R (insulin-like growth factor 2 receptor and VEGFR1-3 (vascular endothelial growth factor receptor 1-3 were examined in four human CC cell lines (EGI-1, HuH28, OZ and TFK-1. The effect of the anti-EGFR-antibody cetuximab on cell growth and apoptosis was studied and cell lines were examined for KRAS mutations. Results EGFR, HGFR and IGFR1 were present in all four cell lines tested. IGFR2 expression was confirmed in EGI-1 and TFK-1. No growth-inhibitory effect was found in EGI-1 cells after incubation with cetuximab. Cetuximab dose-dependently inhibited growth in TFK-1. Increased apoptosis was only seen in TFK-1 cells at the highest cetuximab dose tested (1 mg/ml, with no dose-response-relationship at lower concentrations. In EGI-1 a heterozygous KRAS mutation was found in codon 12 (c.35G>A; p.G12D. HuH28, OZ and TFK-1 lacked KRAS mutation. Conclusion CC cell lines express a pattern of different growth receptors in vitro. Growth factor inhibitor treatment could be affected from the KRAS genotype in CC. The expression of EGFR itself does not allow prognoses on growth inhibition by cetuximab.

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

Inkjet printing of insulin microneedles for transdermal delivery.

Science.gov (United States)

Ross, Steven; Scoutaris, Nicolaos; Lamprou, Dimitrios; Mallinson, David; Douroumis, Dennis

2015-08-01

Inkjet printing technology was used to apply insulin polymeric layers on metal microneedles for transdermal delivery. A range of various polymers such as gelatin (GLN), polyvinyl caprolactame-polyvinyl acetate-polyethylene glycol (SOL), poly(2-ethyl-2-oxazoline) (POX) and trehalose (THL) were assessed for their capacity to form thin uniform and homogeneous layers that preserve insulin intact. Atomic force microscopy (AFM) showed homogeneous insulin-polymer layers without any phase separation while SOL demonstrated the best performance. Circular discroism (CD) analysis of rehydrated films showed that insulin's alpha helices and β-sheet were well preserved for THL and SOL. In contrast, GLN and POX insulin layers revealed small band shifts indicating possible conformational changes. Insulin release in Franz diffusion cells from MNs inserted into porcine skin showed rapid release rates for POX and GLN within the first 20 min. Inkjet printing was proved an effective approach for transdermal delivery of insulin in solid state.

Insulin sensitivity, insulin release and glucagon-like peptide-1 levels in persons with impaired fasting glucose and/or impaired glucose tolerance in the EUGENE2 study

DEFF Research Database (Denmark)

Laakso, M; Zilinskaite, J; Hansen, T

2008-01-01

AIMS/HYPOTHESIS: We examined the phenotype of individuals with impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT) with regard to insulin release and insulin resistance. METHODS: Non-diabetic offspring (n=874; mean age 40+/-10.4 years; BMI 26.6+/-4.9 kg/m(2)) of type 2 diabetic...

Effects of immediate-release niacin and dietary fatty acids on acute insulin and lipid status in individuals with metabolic syndrome.

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Montserrat-de la Paz, Sergio; Lopez, Sergio; Bermudez, Beatriz; Guerrero, Juan M; Abia, Rocio; Muriana, Francisco Jg

2018-04-01

The nature of dietary fats profoundly affects postprandial hypertriglyceridemia and glucose homeostasis. Niacin is a potent lipid-lowering agent. However, limited data exist on postprandial triglycerides and glycemic control following co-administration of high-fat meals with a single dose of niacin in subjects with metabolic syndrome (MetS). The aim of the study was to explore whether a fat challenge containing predominantly saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) or MUFAs plus omega-3 long-chain polyunsaturated (LCPUFAs) fatty acids together with a single dose of immediate-release niacin have a relevant role in postprandial insulin and lipid status in subjects with MetS. In a randomized crossover within-subject design, 16 men with MetS were given a single dose of immediate-release niacin (2 g) and ∼15 cal kg -1 body weight meals containing either SFAs, MUFAs, MUFAs plus omega-3 LCPUFAs or no fat. At baseline and hourly over 6 h, plasma glucose, insulin, C-peptide, triglycerides, free fatty acids (FFAs), total cholesterol, and both high- and low-density lipoprotein cholesterol were assessed. Co-administered with niacin, high-fat meals significantly increased the postprandial concentrations of glucose, insulin, C-peptide, triglycerides, FFAs and postprandial indices of β-cell function. However, postprandial indices of insulin sensitivity were significantly decreased. These effects were significantly attenuated with MUFAs or MUFAs plus omega-3 LCPUFAs when compared with SFAs. In the setting of niacin co-administration and compared to dietary SFAs, MUFAs limit the postprandial insulin, triglyceride and FFA excursions, and improve postprandial glucose homeostasis in MetS. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

[In vitro generation of insulin-producing cells from the neonatal rat bone marrow mesenchymal stem cells].

Science.gov (United States)

Li, Xiaohu; Huang, Haiyan; Liu, Xirong; Xia, Hongxia; Li, Mincai

2015-03-01

To observe the differentiation of the neonatal rat bone marrow mesenchymal stem cells (MSCs) into insulin-producing cells and detect the expressions of insulin, pancreatic duodenal homebox-1 (PDX-1) and nestin. MSCs were isolated from the neonatal rats and cultured in the modified medium composed of 10 μg/L human epidermal growth factor (EGF), 10 μg/L basic fibroblast growth factor (bFGF), 10 μg/L hepatocyte growth factor (HGF), 10 μg/L human B cell regulin, 20 mmol/L nicotinamide and 20 g/L B27. After the induction, the mRNA expressions of insulin, PDX-1 and nestin were examined by reverse transcription-PCR, and the insulin, PDX-1 and nestin protein levels were detected by immunocytochemistry. The insulin and PDX-1 mRNA expressions increased and the nestin mRNA expression decreased in the differentiation of the neonatal rat MSCs into insulin-producing cells. The nestin, PDX-1 and insulin proteins were co-expressed in insulin-producing cells. MSCs can be induced to differentiate into insulin-producing cells.

The glial cell line-derived neurotrophic factor (GDNF) does not acutely change acetylcholine release in developing and adult neuromuscular junction.

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Garcia, Neus; Santafé, Manel M; Tomàs, Marta; Lanuza, Maria A; Besalduch, Nuria; Priego, Merche; Tomàs, Josep

2010-08-16

We use immunocytochemistry to show that the trophic molecule glial cell line-derived neurotrophic factor (GDNF) and its receptor GDNF family receptor alpha-1 (GFRalpha-1) are present in both neonatal (P6) and adult (P45) rodent neuromuscular junctions (NMJ) colocalized with several synaptic markers. However, incubation with exogenous GDNF (10-200ng/ml, 1-3h), does not affect spontaneous ACh release. Moreover, GDNF does not change the size of the evoked ACh release from the weak and the strong axonal inputs on dually innervated postnatal endplates nor in the most developed singly-innervated synapses at P6 and P45. Our findings indicate that GDNF (unlike neurotrophins) does not acutely modulate transmitter release during the developmental process of synapse elimination nor as the NMJ matures. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Biosynthesis of 10 kDa and 7.5 kDa insulin-like growth factor II in a human rhabdomyosarcoma cell line

DEFF Research Database (Denmark)

Nielsen, F C; Haselbacher, G; Christiansen, Jan

1993-01-01

In the present study we have analysed the expression of insulin-like growth factor II (IGF-II) in the human rhabdomyosarcoma cell line IN157.IN157 cells express high levels of three IGF-II mRNAs of 6.0 kb, 4.8 kb and 4.2 kb. In contrast, normal skeletal muscle expresses a negligible amount of IGF......-II mRNA. Two forms of IGF-II with molecular masses of 7.5 kDa and 10 kDa, corresponding to the mature IGF-II and IGF-II with a C-terminal extension of 21 amino acids (IGF-IIE21), were secreted into the culture medium at amounts of 17 ng/ml (2.3 nM) and 15 ng/ml (1.5 nM), respectively. IN157 cells also......-II and IGF-IIE21 with Kd values of 0.5 nM and 2 nM, respectively, and IGF-I with about 500 times lower affinity. IGF-II and IGF-IIE21 stimulated DNA synthesis via the IGF-I receptor, whereas the IGF-II/Man 6-P receptor mediated their rapid internalization and inactivation. During culture of IN157 cells about...

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...

Arsenite reduces insulin secretion in rat pancreatic β-cells by decreasing the calcium-dependent calpain-10 proteolysis of SNAP-25

International Nuclear Information System (INIS)

Diaz-Villasenor, Andrea; Burns, Anna L.; Salazar, Ana Maria; Sordo, Monserrat; Hiriart, Marcia; Cebrian, Mariano E.; Ostrosky-Wegman, Patricia

2008-01-01

An increase in the prevalence of type 2 diabetes has been consistently observed among residents of high arsenic exposure areas. We have previously shown that in rat pancreatic β-cells, low arsenite doses impair the secretion of insulin without altering its synthesis. To further study the mechanism by which arsenite reduces insulin secretion, we evaluated the effects of arsenite on the calcium-calpain pathway that triggers insulin exocytosis in RINm5F cells. Cell cycle and proliferation analysis were also performed to complement the characterization. Free [Ca 2+ ]i oscillations needed for glucose-stimulated insulin secretion were abated in the presence of subchronic low arsenite doses (0.5-2 μM). The global activity of calpains increased with 2 μM arsenite. However, during the secretion of insulin stimulated with glucose (15.6 mM), 1 μM arsenite decreased the activity of calpain-10, measured as SNAP-25 proteolysis. Both proteins are needed to fuse insulin granules with the membrane to produce insulin exocytosis. Arsenite also induced a slowdown in the β cell line proliferation in a dose-dependent manner, reflected by a reduction of dividing cells and in their arrest in G2/M. Data obtained showed that one of the mechanisms by which arsenite impairs insulin secretion is by decreasing the oscillations of free [Ca 2+ ]i, thus reducing calcium-dependent calpain-10 partial proteolysis of SNAP-25. The effects in cell division and proliferation observed with arsenite exposure can be an indirect consequence of the decrease in insulin secretion

Mycoplasma hyorhinis-Contaminated Cell Lines Activate Primary Innate Immune Cells via a Protease-Sensitive Factor.

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Simon Heidegger

Full Text Available Mycoplasma are a frequent and occult contaminant of cell cultures, whereby these prokaryotic organisms can modify many aspects of cell physiology, rendering experiments that are conducted with such contaminated cells problematic. Chronic Mycoplasma contamination in human monocytic cells lines has been associated with suppressed Toll-like receptor (TLR function. In contrast, we show here that components derived from a Mycoplasma hyorhinis-infected cell line can activate innate immunity in non-infected primary immune cells. Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2. Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment. We further show that in plasmacytoid dendritic cells, soluble factors from Mycoplasma hyorhinis-infected cells induce the production of large amounts of IFN-α. We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments.

Neuronal calcium sensor synaptotagmin-9 is not involved in the regulation of glucose homeostasis or insulin secretion.

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Natalia Gustavsson

Full Text Available BACKGROUND: Insulin secretion is a complex and highly regulated process. It is well established that cytoplasmic calcium is a key regulator of insulin secretion, but how elevated intracellular calcium triggers insulin granule exocytosis remains unclear, and we have only begun to define the identities of proteins that are responsible for sensing calcium changes and for transmitting the calcium signal to release machineries. Synaptotagmins are primarily expressed in brain and endocrine cells and exhibit diverse calcium binding properties. Synaptotagmin-1, -2 and -9 are calcium sensors for fast neurotransmitter release in respective brain regions, while synaptotagmin-7 is a positive regulator of calcium-dependent insulin release. Unlike the three neuronal calcium sensors, whose deletion abolished fast neurotransmitter release, synaptotagmin-7 deletion resulted in only partial loss of calcium-dependent insulin secretion, thus suggesting that other calcium-sensors must participate in the regulation of insulin secretion. Of the other synaptotagmin isoforms that are present in pancreatic islets, the neuronal calcium sensor synaptotagmin-9 is expressed at the highest level after synaptotagmin-7. METHODOLOGY/PRINCIPAL FINDINGS: In this study we tested whether synaptotagmin-9 participates in the regulation of glucose-stimulated insulin release by using pancreas-specific synaptotagmin-9 knockout (p-S9X mice. Deletion of synaptotagmin-9 in the pancreas resulted in no changes in glucose homeostasis or body weight. Glucose tolerance, and insulin secretion in vivo and from isolated islets were not affected in the p-S9X mice. Single-cell capacitance measurements showed no difference in insulin granule exocytosis between p-S9X and control mice. CONCLUSIONS: Thus, synaptotagmin-9, although a major calcium sensor in the brain, is not involved in the regulation of glucose-stimulated insulin release from pancreatic β-cells.

Neuronal calcium sensor synaptotagmin-9 is not involved in the regulation of glucose homeostasis or insulin secretion.

Science.gov (United States)

Gustavsson, Natalia; Wang, Xiaorui; Wang, Yue; Seah, Tingting; Xu, Jun; Radda, George K; Südhof, Thomas C; Han, Weiping

2010-11-09

Insulin secretion is a complex and highly regulated process. It is well established that cytoplasmic calcium is a key regulator of insulin secretion, but how elevated intracellular calcium triggers insulin granule exocytosis remains unclear, and we have only begun to define the identities of proteins that are responsible for sensing calcium changes and for transmitting the calcium signal to release machineries. Synaptotagmins are primarily expressed in brain and endocrine cells and exhibit diverse calcium binding properties. Synaptotagmin-1, -2 and -9 are calcium sensors for fast neurotransmitter release in respective brain regions, while synaptotagmin-7 is a positive regulator of calcium-dependent insulin release. Unlike the three neuronal calcium sensors, whose deletion abolished fast neurotransmitter release, synaptotagmin-7 deletion resulted in only partial loss of calcium-dependent insulin secretion, thus suggesting that other calcium-sensors must participate in the regulation of insulin secretion. Of the other synaptotagmin isoforms that are present in pancreatic islets, the neuronal calcium sensor synaptotagmin-9 is expressed at the highest level after synaptotagmin-7. In this study we tested whether synaptotagmin-9 participates in the regulation of glucose-stimulated insulin release by using pancreas-specific synaptotagmin-9 knockout (p-S9X) mice. Deletion of synaptotagmin-9 in the pancreas resulted in no changes in glucose homeostasis or body weight. Glucose tolerance, and insulin secretion in vivo and from isolated islets were not affected in the p-S9X mice. Single-cell capacitance measurements showed no difference in insulin granule exocytosis between p-S9X and control mice. Thus, synaptotagmin-9, although a major calcium sensor in the brain, is not involved in the regulation of glucose-stimulated insulin release from pancreatic β-cells.

Insulin signaling in Caenorhabditis elegans regulates both endocrine-like and cell-autonomous outputs.

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Iser, Wendy B; Gami, Minaxi S; Wolkow, Catherine A

2007-03-15

In C. elegans, insulin signaling affects development, lifespan and stress resistance. Several studies have shown that insulin signaling affects lifespan in an endocrine-like manner from different cells, while the major downstream target of insulin, the FOXO transcription factor encoded by daf-16, may act preferentially in intestinal cells to prolong lifespan. This discrepancy raised the possibility that insulin may have both endocrine and cell-intrinsic outputs. Here, we further investigated the types of cells capable of producing endocrine outputs of insulin and also identified a new cell-intrinsic insulin output. We found that insulin signaling within groups of neurons promoted wildtype lifespan, showing that the endocrine outputs of insulin were not restricted to specific cells. In contrast, DAF-16 appeared to have a greater effect on lifespan when expressed in a combination of tissues. These results suggest that insulin signaling may regulate DAF-16 through cell-intrinsic and endocrine pathways. We also found that an insulin-dependent response to fasting in intestinal cells was preferentially regulated by intestinal insulin signaling and was less responsive to insulin signaling from non-intestinal cells. Together, these results show that C. elegans insulin signaling has endocrine as well as tissue-specific outputs which could influence lifespan in a combinatorial fashion.

Direct effect of gonadal and contraceptive steroids on insulin release from mouse pancreatic islets in organ culture

DEFF Research Database (Denmark)

Nielsen, Jens Høiriis

1984-01-01

Sex steroids are supposed to contribute to the normal glucose homeostasis and to the altered glucose and insulin metabolism in pregnancy and during contraception. In the present study isolated mouse pancreatic islets were maintained in tissue culture medium RPMI 1640 supplemented with 0.5% newborn...... calf serum and 100 ng/ml of one of the following steroids: oestradiol, progesterone, testosterone, megestrol acetate, medroxyprogesterone, chlormadinone acetate, norethynodrel, norethindrone acetate, and ethynyloestradiol. Release of insulin to the culture medium was measured during a 2 week culture...... in the presence of oestradiol, progesterone, or testosterone were subjected to 30 min stimulation with 5.5, 11, 22 mmol/l glucose, only the progesterone-treated islets released more insulin in response to glucose than the control islets. It is concluded that progesterone and its derivatives have a direct effect...

Gliclazide directly inhibits arginine-induced glucagon release

DEFF Research Database (Denmark)

Cejvan, Kenan; Coy, David H; Holst, Jens Juul

2002-01-01

Arginine-stimulated insulin and somatostatin release is enhanced by the sulfonylurea gliclazide. In contrast, gliclazide inhibits the glucagon response. The aim of the present study was to investigate whether this inhibition of glucagon release was mediated by a direct suppressive effect of glicl......Arginine-stimulated insulin and somatostatin release is enhanced by the sulfonylurea gliclazide. In contrast, gliclazide inhibits the glucagon response. The aim of the present study was to investigate whether this inhibition of glucagon release was mediated by a direct suppressive effect....... In islet perifusions with DC-41-33, arginine-induced glucagon release was inhibited by 66%. We therefore concluded that gliclazide inhibits glucagon release by a direct action on the pancreatic A cell....

Insulin receptor degradation is accelerated in cultured lymphocytes from patients with genetic syndromes of extreme insulin resistance

International Nuclear Information System (INIS)

McElduff, A.; Hedo, J.A.; Taylor, S.I.; Roth, J.; Gorden, P.

1984-01-01

The insulin receptor degradation rate was examined in B lymphocytes that were obtained from peripheral blood of normal subjects and patients with several syndromes of extreme insulin resistance. The insulin receptors were surface labeled using Na 125 I/lactoperoxidase and the cells were returned to incubate in growth media. After varying periods of incubation, aliquots of cells were solubilized and the cell content of labeled receptor subunits were measured by immunoprecipitation with anti-receptor antibodies and NaDodSO4/polyacrylamide gel electrophoresis. In cell lines from four patients in whom the number of insulin receptors was reduced by greater than 90%, the rate of receptor loss was greater than normal (t1/2 equals 3.8 +/- 0.9 h vs. 6.5 +/- 1.2 h; mean +/- SD, P less than 0.01). However, a similar acceleration in receptor degradation was seen in cells from five patients with extreme insulin resistance but low-normal insulin receptor concentration (t1/2 equals 4.4 +/- 0.9 h). Thus, all the patients with genetic syndromes of insulin resistance had accelerated receptor degradation, regardless of their receptor concentration. By contrast, insulin receptors on cultured lymphocytes that were obtained from patients with extreme insulin resistance secondary to autoantibodies to the insulin receptor had normal receptor degradation (t1/2 equals 6.1 +/- 1.9 h). We conclude that (a) accelerated insulin receptor degradation is an additional feature of cells from patients with genetic forms of insulin resistance; (b) that accelerated insulin receptor degradation may explain the low-normal receptor concentrations that were seen in some patients with extreme insulin resistance; and (c) that accelerated degradation does not explain the decreased receptor concentration in patients with very low insulin receptor binding and, therefore, by inference, a defect in receptor synthesis must be present in this subgroup

Exosomes released from breast cancer carcinomas stimulate cell movement.

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Dinari A Harris

Full Text Available For metastasis to occur cells must communicate with to their local environment to initiate growth and invasion. Exosomes have emerged as an important mediator of cell-to-cell signalling through the transfer of molecules such as mRNAs, microRNAs, and proteins between cells. Exosomes have been proposed to act as regulators of cancer progression. Here, we study the effect of exosomes on cell migration, an important step in metastasis. We performed cell migration assays, endocytosis assays, and exosome proteomic profiling on exosomes released from three breast cancer cell lines that model progressive stages of metastasis. Results from these experiments suggest: (1 exosomes promote cell migration and (2 the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3 exosomes are endocytosed at the same rate regardless of the cell type; (4 exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance. We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis.

Insulin-induced translocation of IR to the nucleus in insulin responsive cells requires a nuclear translocation sequence.

Science.gov (United States)

Kesten, Dov; Horovitz-Fried, Miriam; Brutman-Barazani, Tamar; Sampson, Sanford R

2018-04-01

Insulin binding to its cell surface receptor (IR) activates a cascade of events leading to its biological effects. The Insulin-IR complex is rapidly internalized and then is either recycled back to the plasma membrane or sent to lysosomes for degradation. Although most of the receptor is recycled or degraded, a small amount may escape this pathway and migrate to the nucleus of the cell where it might be important in promulgation of receptor signals. In this study we explored the mechanism by which insulin induces IR translocation to the cell nucleus. Experiments were performed cultured L6 myoblasts, AML liver cells and 3T3-L1 adipocytes. Insulin treatment induced a rapid increase in nuclear IR protein levels within 2 to 5 min. Treatment with WGA, an inhibitor of nuclear import, reduced insulin-induced increases nuclear IR protein; IR was, however, translocated to a perinuclear location. Bioinformatics tools predicted a potential nuclear localization sequence (NLS) on IR. Immunofluorescence staining showed that a point mutation on the predicted NLS blocked insulin-induced IR nuclear translocation. In addition, blockade of nuclear IR activation in isolated nuclei by an IR blocking antibody abrogated insulin-induced increases in IR tyrosine phosphorylation and nuclear PKCδ levels. Furthermore, over expression of mutated IR reduced insulin-induced glucose uptake and PKB phosphorylation. When added to isolated nuclei, insulin induced IR phosphorylation but had no effect on nuclear IR protein levels. These results raise questions regarding the possible role of nuclear IR in IR signaling and insulin resistance. Copyright © 2018 Elsevier B.V. All rights reserved.

Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

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Wang, Feng [Department of Gastroenterology, The Tenth People’s Hospital of Shanghai, Tongji University, Shanghai 200072 (China); Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Yang, Yong, E-mail: yyang@houstonmethodist.org [Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Department of Medicine, Weill Cornell Medical College, New York, NY 10065 (United States)

2014-10-03

Graphical abstract: - Highlights: • Quercetin inhibits insulin ligand–receptor interactions. • Quercetin reduces downstream insulin receptor signaling. • Quercetin blocks insulin induced glucose uptake. • Quercetin suppresses insulin stimulated cancer cell proliferation and tumor growth. - Abstract: Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers.

Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

International Nuclear Information System (INIS)

Wang, Feng; Yang, Yong

2014-01-01

Graphical abstract: - Highlights: • Quercetin inhibits insulin ligand–receptor interactions. • Quercetin reduces downstream insulin receptor signaling. • Quercetin blocks insulin induced glucose uptake. • Quercetin suppresses insulin stimulated cancer cell proliferation and tumor growth. - Abstract: Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers

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.

BAG3 regulates formation of the SNARE complex and insulin secretion

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Iorio, V; Festa, M; Rosati, A; Hahne, M; Tiberti, C; Capunzo, M; De Laurenzi, V; Turco, M C

2015-01-01

Insulin release in response to glucose stimulation requires exocytosis of insulin-containing granules. Glucose stimulation of beta cells leads to focal adhesion kinase (FAK) phosphorylation, which acts on the Rho family proteins (Rho, Rac and Cdc42) that direct F-actin remodeling. This process requires docking and fusion of secretory vesicles to the release sites at the plasma membrane and is a complex mechanism that is mediated by SNAREs. This transiently disrupts the F-actin barrier and allows the redistribution of the insulin-containing granules to more peripheral regions of the β cell, hence facilitating insulin secretion. In this manuscript, we show for the first time that BAG3 plays an important role in this process. We show that BAG3 downregulation results in increased insulin secretion in response to glucose stimulation and in disruption of the F-actin network. Moreover, we show that BAG3 binds to SNAP-25 and syntaxin-1, two components of the t-SNARE complex preventing the interaction between SNAP-25 and syntaxin-1. Upon glucose stimulation BAG3 is phosphorylated by FAK and dissociates from SNAP-25 allowing the formation of the SNARE complex, destabilization of the F-actin network and insulin release. PMID:25766323

The H+/K+ ATPase Inhibitor SCH-28080 Inhibits Insulin Secretion and Induces Cell Death in INS-1E Rat Insulinoma Cells

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Martin Jakab

2017-10-01

Full Text Available Background/Aims: Glucose-stimulated insulin secretion (GSIS of pancreatic β-cells involves glucose uptake and metabolism, closure of KATP channels and depolarization of the cell membrane potential (Vmem, activation of voltage-activated Ca2+ currents (ICav and influx of Ca2+, which eventually triggers hormone exocytosis. Beside this classical pathway, KATP-independent mechanisms such as changes in intracellular pH (pHi or cell volume, which also affect β-cell viability, can elicit or modify insulin release. In β-cells the regulation of pHi is mainly accomplished by Na+/H+ exchangers (NHEs. To investigate if other proton extrusion mechanisms than NHEs are involved in pH regulation, we tested for the presence of the non-gastric H+/K+ ATPase in rat insulinoma cells and assessed effects of the H+/K+ ATPase inhibitor SCH-28080 on insulin secretion, cell viability and apoptosis. Methods: In INS-1E cell cultures, H+/K+ ATPase gene and protein expression was analyzed by reverse transcription PCR and Western blotting. Intracellular pH (pHi recovery after acute acidic load was measured by NH4Cl prepulsing using BCECF. Insulin secretion was determined by ELISA from the cell culture supernatant. Vmem, K+ and Ca2+ currents were recorded using patch clamp. Overall cell responses were determined using resazurin (viability and cytotoxicity assays. The mean cell volume (MCV, cell granularity (side-scatter; SSC, phosphatidylserine (PS exposure, cell membrane integrity, caspase activity and the mitochondrial membrane potential (ΔΨm were measured by flow cytometry. Results: We found that the α-subunit of the non-gastric H+/K+ ATPase (HKα2 is expressed on mRNA and protein level. However, compared to rat colon tissue, in INS-1E cells mRNA abundance was very low. In NH4Cl prepulsing experiments no K+-dependent pHi recovery was observed under Na+-free extracellular conditions. Nonetheless within 1 h, 20 µM SCH-28080 inhibited GSIS by ∼50%, while basal release

Induced Pluripotent Stem Cell-Derived Endothelial Cells in Insulin Resistance and Metabolic Syndrome.

Science.gov (United States)

Carcamo-Orive, Ivan; Huang, Ngan F; Quertermous, Thomas; Knowles, Joshua W

2017-11-01

Insulin resistance leads to a number of metabolic and cellular abnormalities including endothelial dysfunction that increase the risk of vascular disease. Although it has been particularly challenging to study the genetic determinants that predispose to abnormal function of the endothelium in insulin-resistant states, the possibility of deriving endothelial cells from induced pluripotent stem cells generated from individuals with detailed clinical phenotyping, including accurate measurements of insulin resistance accompanied by multilevel omic data (eg, genetic and genomic characterization), has opened new avenues to study this relationship. Unfortunately, several technical barriers have hampered these efforts. In the present review, we summarize the current status of induced pluripotent stem cell-derived endothelial cells for modeling endothelial dysfunction associated with insulin resistance and discuss the challenges to overcoming these limitations. © 2017 American Heart Association, Inc.

Conjugated Linoleic Acids Mediate Insulin Release through Islet G Protein-coupled Receptor FFA1/GPR40

DEFF Research Database (Denmark)

Schmidt, Johannes; Liebscher, Kathrin; Merten, Nicole

2011-01-01

of insulin resistance and the risk of developing diabetes. However, the mechanisms accounting for the effects of CLAs on glucose homeostasis are incompletely understood. Herein we provide evidence that CLAs specifically activate the cell surface receptor FFA1, an emerging therapeutic target to treat type 2...... found to activate FFA1 in vitro at concentrations sufficient to also account for FFA1 activation in vivo. Each CLA isomer markedly increased glucose-stimulated insulin secretion in insulin-producing INS-1E cells that endogenously express FFA1 and in primary pancreatic β-cells of wild type but not FFA1......(-/-) knock-out mice. Our findings establish a clear mechanistic link between CLAs and insulin production and identify the cell surface receptor FFA1 as a molecular target for CLAs, explaining their acute stimulatory effects on insulin secretion in vivo. CLAs are also revealed as insulinotropic components...

[Molecular mechanism for ET-1-induced insulin resistance in skeletal muscle cells].

Science.gov (United States)

Horinouchi, Takahiro; Mazaki, Yuichi; Terada, Koji; Miwa, Soichi

2018-01-01

Insulin resistance is a condition where the sensitivity to insulin of the tissues expressing insulin receptor (InsR) is decreased due to a functional disturbance of InsR-mediated intracellular signaling. Insulin promotes the entry of glucose into the tissues and skeletal muscle is the most important tissue responsible for the insulin's action of decreasing blood glucose levels. Endothelin-1 (ET-1), a potent vasoconstrictor and pro-inflammatory peptide, induces insulin resistance through a direct action on skeletal muscle. However, the signaling pathways of ET-1-induced insulin resistance in skeletal muscle remain unclear. Here we show molecular mechanism underlying the inhibitory effect of ET-1 on insulin-stimulated Akt phosphorylation and glucose uptake in myotubes of rat L6 skeletal muscle cell line. mRNA expression levels of differentiation marker genes, MyoD and myogenin, were increased during L6 myoblasts differentiation into myotubes. Some of myotubes possessed the ability to spontaneously contract. In myotubes, insulin promoted Akt phosphorylation at Thr 308 and Ser 473 , and [ 3 H]-labelled 2-deoxy-D-glucose ([ 3 H]2-DG) uptake. The insulin-facilitated Akt phosphorylation and [ 3 H]2-DG uptake were inhibited by ET-1. The inhibitory effect of ET-1 was counteracted by blockade of ET type A receptor (ET A R), inhibition of G q/11 protein, and siRNA knockdown of G protein-coupled receptor kinase 2 (GRK2). The exogenously overexpressed GRK2 directly bound to endogenous Akt and their association was facilitated by ET-1. In summary, activation of ET A R with ET-1 inhibits insulin-induced Akt phosphorylation and [ 3 H]2-DG uptake in a G q/11 protein- and GRK2-dependent manner in skeletal muscle. These findings indicate that ET A R and GRK2 are potential targets for insulin resistance.

Insulin resistance in vascular endothelial cells promotes intestinal tumour formation

DEFF Research Database (Denmark)

Wang, X; Häring, M-F; Rathjen, Thomas

2017-01-01

in vascular endothelial cells. Strikingly, these mice had 42% more intestinal tumours than controls, no change in tumour angiogenesis, but increased expression of vascular cell adhesion molecule-1 (VCAM-1) in primary culture of tumour endothelial cells. Insulin decreased VCAM-1 expression and leukocyte...... adhesion in quiescent tumour endothelial cells with intact insulin receptors and partly prevented increases in VCAM-1 and leukocyte adhesion after treatment with tumour necrosis factor-α. Knockout of insulin receptors in endothelial cells also increased leukocyte adhesion in mesenteric venules...

Effect on Insulin-Stimulated Release of D-Chiro-Inositol-Containing Inositolphosphoglycan Mediator during Weight Loss in Obese Women with and without Polycystic Ovary Syndrome

OpenAIRE

Cheang, Kai I.; Sistrun, Sakita N.; Morel, Kelley S.; Nestler, John E.

2016-01-01

Background. A deficiency of D-chiro-inositol-inositolphosphoglycan mediator (DCI-IPG) may contribute to insulin resistance in polycystic ovary syndrome (PCOS). Whether the relationship between impaired DCI-IPG release and insulin resistance is specific to PCOS rather than obesity is unknown. We assessed insulin-released DCI-IPG and its relationship to insulin sensitivity at baseline and after weight loss in obese women with and without PCOS. Methods. Obese PCOS (n = 16) and normal (n = 15) wo...

Generation of insulin-producing cells from gnotobiotic porcine skin-derived stem cells

International Nuclear Information System (INIS)

Yang, Ji Hoon; Lee, Sung Ho; Heo, Young Tae; Uhm, Sang Jun; Lee, Hoon Taek

2010-01-01

A major problem in the treatment of type 1 diabetes mellitus is the limited availability of alternative sources of insulin-producing cells for islet transplantation. In this study, we investigated the effect of bone morphogenetic protein 4 (BMP-4) treatments of gnotobiotic porcine skin-derived stem cells (gSDSCs) on their reprogramming and subsequent differentiation into insulin-producing cells (IPCs). We isolated SDSCs from the ear skin of a gnotobiotic pig. During the proliferation period, the cells expressed stem-cell markers Oct-4, Sox-2, and CD90; nestin expression also increased significantly. The cells could differentiate into IPCs after treatments with activin-A, glucagon-like peptide-1 (GLP-1), and nicotinamide. After 15 days in the differentiation medium, controlled gSDSCs began expressing endocrine progenitor genes and proteins (Ngn3, Neuro-D, PDX-1, NKX2.2, NKX6.1, and insulin). The IPCs showed increased insulin synthesis after glucose stimulation. The results indicate that stem cells derived from the skin of gnotobiotic pigs can differentiate into IPCs under the appropriate conditions in vitro. Our three-stage induction protocol could be applied without genetic modification to source IPCs from stem cells in the skin of patients with diabetes for autologous transplantation.

Generation of insulin-producing cells from gnotobiotic porcine skin-derived stem cells

Energy Technology Data Exchange (ETDEWEB)

Yang, Ji Hoon; Lee, Sung Ho; Heo, Young Tae [Department of Bioscience and Biotechnology, Bio-Organ Research Center, Konkuk University, Seoul 143-701 (Korea, Republic of); Uhm, Sang Jun [Department of Animal Biotechnology, Bio-Organ Research Center, Konkuk University, Seoul 143-701 (Korea, Republic of); Lee, Hoon Taek, E-mail: htl3675@konkuk.ac.kr [Department of Animal Biotechnology, Bio-Organ Research Center, Konkuk University, Seoul 143-701 (Korea, Republic of)

2010-07-09

A major problem in the treatment of type 1 diabetes mellitus is the limited availability of alternative sources of insulin-producing cells for islet transplantation. In this study, we investigated the effect of bone morphogenetic protein 4 (BMP-4) treatments of gnotobiotic porcine skin-derived stem cells (gSDSCs) on their reprogramming and subsequent differentiation into insulin-producing cells (IPCs). We isolated SDSCs from the ear skin of a gnotobiotic pig. During the proliferation period, the cells expressed stem-cell markers Oct-4, Sox-2, and CD90; nestin expression also increased significantly. The cells could differentiate into IPCs after treatments with activin-A, glucagon-like peptide-1 (GLP-1), and nicotinamide. After 15 days in the differentiation medium, controlled gSDSCs began expressing endocrine progenitor genes and proteins (Ngn3, Neuro-D, PDX-1, NKX2.2, NKX6.1, and insulin). The IPCs showed increased insulin synthesis after glucose stimulation. The results indicate that stem cells derived from the skin of gnotobiotic pigs can differentiate into IPCs under the appropriate conditions in vitro. Our three-stage induction protocol could be applied without genetic modification to source IPCs from stem cells in the skin of patients with diabetes for autologous transplantation.

ATP release, generation and hydrolysis in exocrine pancreatic duct cells

DEFF Research Database (Denmark)

Kowal, Justyna Magdalena; Yegutkin, G.G.; Novak, Ivana

2015-01-01

Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, ou...... may be important in pancreas physiology and potentially in pancreas pathophysiology....... aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan...

Changes of insulin resistance and β-cell function in women with gestational diabetes mellitus and normal pregnant women during mid- and late pregnant period: a case-control study.

Science.gov (United States)

Wang, Yun-Hui; Wu, Hui-Hua; Ding, Hong; Li, Yan; Wang, Zhen-Hua; Li, Feng; Zhang, Jian-Ping

2013-03-01

The aim of this study was to observe insulin resistance and β-cell function changes among women diagnosed with gestational impaired glucose tolerance or gestational diabetes mellitus (GDM) in mid-pregnancy. Sixty-four pregnant women receiving prenatal care underwent an oral glucose tolerance test at 20-24 weeks of gestation and an insulin release test. The GDM group included 34 pregnant women diagnosed with gestational impaired glucose tolerance or GDM, and the subjects with normal blood glucose were the control group. Insulin resistance and islet β-cell function changes were observed with the oral glucose tolerance test and insulin release test. The homeostatic model assessment-β levels in late pregnancy were higher than those in mid-pregnancy for both groups, and the primary time effect was statistically significant. The early insulin secretion index (ΔI(30)/ΔG(30)) values in mid- and late pregnancy were lower in the GDM group. The values of the area under the curve of blood glucose in mid- and late pregnancy were higher in the GDM group than those in the control group. Insulin resistance was higher in GDM patients than in normal pregnant women. Insulin resistance was aggravated, and β-cell's ability to compensate for the increased insulin resistance by modulating insulin secretion was aggravated, as gestational week increased in women with gestational diabetes and normal pregnant women. Insulin resistance in women with GDM is higher than in pregnant women with normal metabolism of glucose. © 2012 The Authors. Journal of Obstetrics and Gynaecology Research © 2012 Japan Society of Obstetrics and Gynecology.

Novel Zn2+ Modulated GPR39 Receptor Agonists Do Not Drive Acute Insulin Secretion in Rodents.

Directory of Open Access Journals (Sweden)

Ola Fjellström

Full Text Available Type 2 diabetes (T2D occurs when there is insufficient insulin release to control blood glucose, due to insulin resistance and impaired β-cell function. The GPR39 receptor is expressed in metabolic tissues including pancreatic β-cells and has been proposed as a T2D target. Specifically, GPR39 agonists might improve β-cell function leading to more adequate and sustained insulin release and glucose control. The present study aimed to test the hypothesis that GPR39 agonism would improve glucose stimulated insulin secretion in vivo. A high throughput screen, followed by a medicinal chemistry program, identified three novel potent Zn2+ modulated GPR39 agonists. These agonists were evaluated in acute rodent glucose tolerance tests. The results showed a lack of glucose lowering and insulinotropic effects not only in lean mice, but also in diet-induced obese (DIO mice and Zucker fatty rats. It is concluded that Zn2+ modulated GPR39 agonists do not acutely stimulate insulin release in rodents.

Introduction of exogenous growth hormone receptors augments growth hormone-responsive insulin biosynthesis in rat insulinoma cells

DEFF Research Database (Denmark)

Billestrup, N; Møldrup, A; Serup, P

1990-01-01

The stimulation of insulin biosynthesis in the pancreatic insulinoma cell line RIN5-AH by growth hormone (GH) is initiated by GH binding to specific receptors. To determine whether the recently cloned rat hepatic GH receptor is able to mediate the insulinotropic effect of GH, we have transfected ...

Fucosterol activates the insulin signaling pathway in insulin resistant HepG2 cells via inhibiting PTP1B.

Science.gov (United States)

Jung, Hyun Ah; Bhakta, Himanshu Kumar; Min, Byung-Sun; Choi, Jae Sue

2016-10-01

Insulin resistance is a characteristic feature of type 2 diabetes mellitus (T2DM) and is characterized by defects in insulin signaling. This study investigated the modulatory effects of fucosterol on the insulin signaling pathway in insulin-resistant HepG2 cells by inhibiting protein tyrosine phosphatase 1B (PTP1B). In addition, molecular docking simulation studies were performed to predict binding energies, the specific binding site of fucosterol to PTP1B, and to identify interacting residues using Autodock 4.2 software. Glucose uptake was determined using a fluorescent D-glucose analogue and the glucose tracer 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose, and the signaling pathway was detected by Western blot analysis. We found that fucosterol enhanced insulin-provoked glucose uptake and conjointly decreased PTP1B expression level in insulin-resistant HepG2 cells. Moreover, fucosterol significantly reduced insulin-stimulated serine (Ser307) phosphorylation of insulin receptor substrate 1 (IRS1) and increased phosphorylation of Akt, phosphatidylinositol-3-kinase, and extracellular signal- regulated kinase 1 at concentrations of 12.5, 25, and 50 µM in insulin-resistant HepG2 cells. Fucosterol inhibited caspase-3 activation and nuclear factor kappa B in insulin-resistant hepatocytes. These results suggest that fucosterol stimulates glucose uptake and improves insulin resistance by downregulating expression of PTP1B and activating the insulin signaling pathway. Thus, fucosterol has potential for development as an anti-diabetic agent.

Essential roles of aspartate aminotransferase 1 and vesicular glutamate transporters in β-cell glutamate signaling for incretin-induced insulin secretion.

Directory of Open Access Journals (Sweden)

Naoya Murao

Full Text Available Incretins (GLP-1 and GIP potentiate insulin secretion through cAMP signaling in pancreatic β-cells in a glucose-dependent manner. We recently proposed a mechanistic model of incretin-induced insulin secretion (IIIS that requires two critical processes: 1 generation of cytosolic glutamate through the malate-aspartate (MA shuttle in glucose metabolism and 2 glutamate transport into insulin granules by cAMP signaling to promote insulin granule exocytosis. To directly prove the model, we have established and characterized CRISPR/Cas9-engineered clonal mouse β-cell lines deficient for the genes critical in these two processes: aspartate aminotransferase 1 (AST1, gene symbol Got1, a key enzyme in the MA shuttle, which generates cytosolic glutamate, and the vesicular glutamate transporters (VGLUT1, VGLUT2, and VGLUT3, gene symbol Slc17a7, Slc17a6, and Slc17a8, respectively, which participate in glutamate transport into secretory vesicles. Got1 knockout (KO β-cell lines were defective in cytosolic glutamate production from glucose and showed impaired IIIS. Unexpectedly, different from the previous finding that global Slc17a7 KO mice exhibited impaired IIIS from pancreatic islets, β-cell specific Slc17a7 KO mice showed no significant impairment in IIIS, as assessed by pancreas perfusion experiment. Single Slc17a7 KO β-cell lines also retained IIIS, probably due to compensatory upregulation of Slc17a6. Interestingly, triple KO of Slc17a7, Slc17a6, and Slc17a8 diminished IIIS, which was rescued by exogenously introduced wild-type Slc17a7 or Slc17a6 genes. The present study provides direct evidence for the essential roles of AST1 and VGLUTs in β-cell glutamate signaling for IIIS and also shows the usefulness of the CRISPR/Cas9 system for studying β-cells by simultaneous disruption of multiple genes.

Adiponectin release and insulin receptor targeting share trans-Golgi-dependent endosomal trafficking routes

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Maria Rödiger

2018-02-01

Full Text Available Objective: Intracellular vesicle trafficking maintains cellular structures and functions. The assembly of cargo-laden vesicles at the trans-Golgi network is initiated by the ARF family of small GTPases. Here, we demonstrate the role of the trans-Golgi localized monomeric GTPase ARFRP1 in endosomal-mediated vesicle trafficking of mature adipocytes. Methods: Control (Arfrp1flox/flox and inducible fat-specific Arfrp1 knockout (Arfrp1iAT−/− mice were metabolically characterized. In vitro experiments on mature 3T3-L1 cells and primary mouse adipocytes were conducted to validate the impact of ARFRP1 on localization of adiponectin and the insulin receptor. Finally, secretion and transferrin-based uptake and recycling assays were performed with HeLa and HeLa M-C1 cells. Results: We identified the ARFRP1-based sorting machinery to be involved in vesicle trafficking relying on the endosomal compartment for cell surface delivery. Secretion of adiponectin from fat depots was selectively reduced in Arfrp1iAT−/− mice, and Arfrp1-depleted 3T3-L1 adipocytes revealed an accumulation of adiponectin in Rab11-positive endosomes. Plasma adiponectin deficiency of Arfrp1iAT−/− mice resulted in deteriorated hepatic insulin sensitivity, increased gluconeogenesis and elevated fasting blood glucose levels. Additionally, the insulin receptor, undergoing endocytic recycling after ligand binding, was less abundant at the plasma membrane of adipocytes lacking Arfrp1. This had detrimental effects on adipose insulin signaling, followed by insufficient suppression of basal lipolytic activity and impaired adipose tissue expansion. Conclusions: Our findings suggest that adiponectin secretion and insulin receptor surface targeting utilize the same post-Golgi trafficking pathways that are essential for an appropriate systemic insulin sensitivity and glucose homeostasis. Keywords: Adiponectin, ARFRP1, Exocytosis, Insulin receptor, trans-Golgi

The zinc transporter ZNT3 co-localizes with insulin in INS-1E pancreatic beta cells and influences cell survival, insulin secretion capacity, and ZNT8 expression

DEFF Research Database (Denmark)

Smidt, Kamille; Larsen, Agnete; Brønden, Andreas

2016-01-01

Zinc trafficking in pancreatic beta cells is tightly regulated by zinc transporting (ZNTs) proteins. The role of different ZNTs in the beta cells is currently being clarified. ZNT8 transports zinc into insulin granules and is critical for a correct insulin crystallization and storage in the granu......Zinc trafficking in pancreatic beta cells is tightly regulated by zinc transporting (ZNTs) proteins. The role of different ZNTs in the beta cells is currently being clarified. ZNT8 transports zinc into insulin granules and is critical for a correct insulin crystallization and storage...

Clinical study on insulin receptors of mononuclear cells in diabetes

Energy Technology Data Exchange (ETDEWEB)

Dalimunthe, D [Hiroshima Univ. (Japan). School of Medicine

1980-12-01

/sup 125/I-insulin binding activity to mononuclear cells was studied in 75 noninsulin-dependent diabetic subjects and 31 normal subjects and the following results were obtained. 1. /sup 125/I-insulin binding is directly proportional to the mononuclear cell concentrations. There is a linear increase of specific /sup 125/I-insulin binding. 2. The binding of /sup 125/I-insulin to mononuclear cells is displaced by the increasing concentration of native insulin. 3. The /sup 125/I-insulin degradation in the incubation medium after incubation of mononuclear cells for 24 hours at 4/sup 0/C was almost 5% in this study. 4. The insulin binding activity in diabetic subjects was lower than that in normal subjects (P < 0.001) without any significant difference in affinity constant. 5. The relationship of binding activity to age of diabetics (r = 0.06, N.S), relative body weitht (r = 0.06, N.S) and duration of diabetes from onset was not significant. 6. In untreated noninsulin-dependent diabetics the insulin binding activity was inversely correlated to fasting blood glucose level (r = 0.78, P < 0.001) and slightly inversely correlated to serum insulin level (r = 0.47, P < 0.01). A slight inverse correlation was also observed in serum triglyceride level (r = 0.53, P < 0.01) and in total cholesterol level (r = 0.29, P < 0.05). 7. No significant difference between the binding activity was observed by grade of diabetic retinopathy. 8. After treatment with diet and/or sulfonylurea, the diabetics exhibited a significant increase in insulin binding activity (P < 0.005) but no significant difference in plasma insulin level, body weight and plasma lipid levels was observed.

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

Development of the insulin secretion mechanism in fetal and neonatal rat pancreatic B-cells: response to glucose, K+, theophylline, and carbamylcholine

Directory of Open Access Journals (Sweden)

A.C. Mendonça

1998-06-01

Full Text Available We studied the development of the insulin secretion mechanism in the pancreas of fetal (19- and 21-day-old, neonatal (3-day-old, and adult (90-day-old rats in response to stimulation with 8.3 or 16.7 mM glucose, 30 mM K+, 5 mM theophylline (Theo and 200 µM carbamylcholine (Cch. No effect of glucose or high K+ was observed on the pancreas from 19-day-old fetuses, whereas Theo and Cch significantly increased insulin secretion at this age (82 and 127% above basal levels, respectively. High K+ also failed to alter the insulin secretion in the pancreas from 21-day-old fetuses, whereas 8.3 mM and 16.7 mM glucose significantly stimulated insulin release by 41 and 54% above basal levels, respectively. Similar results were obtained with Theo and Cch. A more marked effect of glucose on insulin secretion was observed in the pancreas of 3-day-old rats, reaching 84 and 179% above basal levels with 8.3 mM and 16.7 mM glucose, respectively. At this age, both Theo and Cch increased insulin secretion to close to two-times basal levels. In islets from adult rats, 8.3 mM and 16.7 mM glucose, Theo, and Cch increased the insulin release by 104, 193, 318 and 396% above basal levels, respectively. These data indicate that pancreatic B-cells from 19-day-old fetuses were already sensitive to stimuli that use either cAMP or IP3 and DAG as second messengers, but insensitive to stimuli such as glucose and high K+ that induce membrane depolarization. The greater effect of glucose on insulin secretion during the neonatal period indicates that this period is crucial for the maturation of the glucose-sensing mechanism in B-cells.

Generation, isolation, and maintenance of rodent mast cells and mast cell lines

DEFF Research Database (Denmark)

Jensen, Bettina M; Swindle, Emily J; Iwaki, Shoko

2006-01-01

Antigen-mediated mast cell activation, with subsequent mediator release, is a major initiator of the inflammatory allergic response associated with such conditions as asthma. A comprehensive understanding of the principles involved in this process therefore is key to the development of novel...... therapies for the treatment of these disease states. In vitro models of mast cell function have allowed significant progress to be made in the recognition of the fundamental principles of mast cell activation via the high-affinity IgE receptor (FcvarepsilonRI) and, more recently, other receptors expressed...... on mast cells. In addition to human mast cells, the major cell culture systems employed to investigate these responses are rat and mouse peritoneal mast cells, mouse bone-marrow-derived mast cells, the rat basophilic leukemia cell line RBL-2H3, and the mouse MC/9 mast cell line. In this unit, we describe...

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...

Impaired insulin secretion and glucose intolerance in synaptotagmin-7 null mutant mice

DEFF Research Database (Denmark)

Gustavsson, Natalia; Lao, Ye; Maximov, Anton

2008-01-01

and insulin release. Here, we show that synaptotagmin-7 is required for the maintenance of systemic glucose tolerance and glucose-stimulated insulin secretion. Mutant mice have normal insulin sensitivity, insulin production, islet architecture and ultrastructural organization, and metabolic and calcium...... secretion in pancreatic beta-cells. Of these other synaptotagmins, synaptotagmin-7 is one of the most abundant and is present in pancreatic beta-cells. To determine whether synaptotagmin-7 regulates Ca(2+)-dependent insulin secretion, we analyzed synaptotagmin-7 null mutant mice for glucose tolerance...... responses but exhibit impaired glucose-induced insulin secretion, indicating a calcium-sensing defect during insulin-containing secretory granule exocytosis. Taken together, our findings show that synaptotagmin-7 functions as a positive regulator of insulin secretion and may serve as a calcium sensor...

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.

Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

Energy Technology Data Exchange (ETDEWEB)

Kelly, Catriona, E-mail: catriona.kelly@qub.ac.uk [SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine (United Kingdom); Flatt, Peter R.; McClenaghan, Neville H. [SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine (United Kingdom)

2010-08-20

Research highlights: {yields} TGP52 cells display enhanced functionality in pseudoislet form. {yields} Somatostatin content was reduced, but secretion increased in high glucose conditions. {yields} Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mM glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.

Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

International Nuclear Information System (INIS)

Kelly, Catriona; Flatt, Peter R.; McClenaghan, Neville H.

2010-01-01

Research highlights: → TGP52 cells display enhanced functionality in pseudoislet form. → Somatostatin content was reduced, but secretion increased in high glucose conditions. → Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mM glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.

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.

CNC-bZIP protein Nrf1-dependent regulation of glucose-stimulated insulin secretion.

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Zheng, Hongzhi; Fu, Jingqi; Xue, Peng; Zhao, Rui; Dong, Jian; Liu, Dianxin; Yamamoto, Masayuki; Tong, Qingchun; Teng, Weiping; Qu, Weidong; Zhang, Qiang; Andersen, Melvin E; Pi, Jingbo

2015-04-01

The inability of pancreatic β-cells to secrete sufficient insulin in response to glucose stimulation is a major contributing factor to the development of type 2 diabetes (T2D). We investigated both the in vitro and in vivo effects of deficiency of nuclear factor-erythroid 2-related factor 1 (Nrf1) in β-cells on β-cell function and glucose homeostasis. Silencing of Nrf1 in β-cells leads to a pre-T2D phenotype with disrupted glucose metabolism and impaired insulin secretion. Specifically, MIN6 β-cells with stable knockdown of Nrf1 (Nrf1-KD) and isolated islets from β-cell-specific Nrf1-knockout [Nrf1(b)-KO] mice displayed impaired glucose responsiveness, including elevated basal insulin release and decreased glucose-stimulated insulin secretion (GSIS). Nrf1(b)-KO mice exhibited severe fasting hyperinsulinemia, reduced GSIS, and glucose intolerance. Silencing of Nrf1 in MIN6 cells resulted in oxidative stress and altered glucose metabolism, with increases in both glucose uptake and aerobic glycolysis, which is associated with the elevated basal insulin release and reduced glucose responsiveness. The elevated glycolysis and reduced glucose responsiveness due to Nrf1 silencing likely result from altered expression of glucose metabolic enzymes, with induction of high-affinity hexokinase 1 and suppression of low-affinity glucokinase. Our study demonstrated a novel role of Nrf1 in regulating glucose metabolism and insulin secretion in β-cells and characterized Nrf1 as a key transcription factor that regulates the coupling of glycolysis and mitochondrial metabolism and GSIS. Nrf1 plays critical roles in regulating glucose metabolism, mitochondrial function, and insulin secretion, suggesting that Nrf1 may be a novel target to improve the function of insulin-secreting β-cells.

Cardiac regeneration by pharmacologically active microcarriers releasing growth factors and/or transporting adipose-derived stem cells

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Monia Savi

2014-01-01

Full Text Available We tested the hypothesis that cardiac regeneration through local delivery of adipose-derived stem cells (ASCs, activation of resident cardiac stem cells via growth factors (GFs [hepatocyte growth factor (HGF and insulin-like growth factor 1 (IGF-1:GFs] or both, are improved by pharmacologically active microcarriers (PAMs interacting with cells/molecules conveyed on their surface. Rats with one-month old myocardial infarction were treated with ASCs, ASCs+PAMs, GF-releasing PAMs, ASCs+GF-releasing PAMs or vehicle. Two weeks later, hemodynamic function and inducibility of ventricular arrhythmias (VAs were assessed. Eventually, the hearts were subjected to anatomical and immunohistochemical analyses. A significant ASCs engraftment and the largest improvement in cardiac mechanics occurred in ASC+GF-releasing PAM rats which by contrast were more vulnerable to VAs. Thus, PAMs may improve cell/GF-based cardiac regeneration although caution should be paid on the electrophysiological impact of their physical interaction with the myocardium.

NKX6.1 induced pluripotent stem cell reporter lines for isolation and analysis of functionally relevant neuronal and pancreas populations

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Shailesh Kumar Gupta

2018-05-01

Full Text Available Recent studies have reported significant advances in the differentiation of human pluripotent stem cells to clinically relevant cell types such as the insulin producing beta-like cells and motor neurons. However, many of the current differentiation protocols lead to heterogeneous cell cultures containing cell types other than the targeted cell fate. Genetically modified human pluripotent stem cells reporting the expression of specific genes are of great value for differentiation protocol optimization and for the purification of relevant cell populations from heterogeneous cell cultures. Here we present the generation of human induced pluripotent stem cell (iPSC lines with a GFP reporter inserted in the endogenous NKX6.1 locus. Characterization of the reporter lines demonstrated faithful GFP labelling of NKX6.1 expression during pancreas and motor neuron differentiation. Cell sorting and gene expression profiling by RNA sequencing revealed that NKX6.1-positive cells from pancreatic differentiations closely resemble human beta cells. Furthermore, functional characterization of the isolated cells demonstrated that glucose-stimulated insulin secretion is mainly confined to the NKX6.1-positive cells. We expect that the NKX6.1-GFP iPSC lines and the results presented here will contribute to the further refinement of differentiation protocols and characterization of hPSC-derived beta cells and motor neurons for disease modelling and cell replacement therapies. Keywords: Human induced pluripotent stem cells, NKX6.1, Reporter cell line, Directed differentiation, hiPSC-derived beta cells

THP-1 cell line: an in vitro cell model for immune modulation approach.

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Chanput, Wasaporn; Mes, Jurriaan J; Wichers, Harry J

2014-11-01

THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review attempts to summarize and discuss recent publications related to the THP-1 cell model. An overview on the biological similarities and dissimilarities between the THP-1 cell line and human peripheral blood mononuclear cell (PBMC) derived-monocytes and macrophages, as well as the advantages and disadvantages of the use of THP-1 cell line, is included. The review summarizes different published co-cultivation studies of THP-1 cells with other cell types, for instance, intestinal cells, adipocytes, T-lymphocytes, platelets, and vascular smooth muscle cells, which can be an option to study cell-cell interaction in vitro and can be an approach to better mimic in vivo conditions. Macrophage polarization is a relatively new topic which gains interest for which the THP-1 cell line also may be relevant. Besides that an overview of newly released commercial THP-1 engineered-reporter cells and THP-1 inflammasome test-cells is also given. Evaluation of recent papers leads to the conclusion that the THP-1 cell line has unique characteristics as a model to investigate/estimate immune-modulating effects of compounds in both activated and resting conditions of the cells. Although the THP-1 response can hint to potential responses that might occur ex vivo or in vivo, these should be, however, validated by in vivo studies to draw more definite conclusions. Copyright © 2013. Published by Elsevier B.V.

Mitochondrial Dynamics Mediated by Mitofusin 1 Is Required for POMC Neuron Glucose-Sensing and Insulin Release Control.

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Ramírez, Sara; Gómez-Valadés, Alicia G; Schneeberger, Marc; Varela, Luis; Haddad-Tóvolli, Roberta; Altirriba, Jordi; Noguera, Eduard; Drougard, Anne; Flores-Martínez, Álvaro; Imbernón, Mónica; Chivite, Iñigo; Pozo, Macarena; Vidal-Itriago, Andrés; Garcia, Ainhoa; Cervantes, Sara; Gasa, Rosa; Nogueiras, Ruben; Gama-Pérez, Pau; Garcia-Roves, Pablo M; Cano, David A; Knauf, Claude; Servitja, Joan-Marc; Horvath, Tamas L; Gomis, Ramon; Zorzano, Antonio; Claret, Marc

2017-06-06

Proopiomelanocortin (POMC) neurons are critical sensors of nutrient availability implicated in energy balance and glucose metabolism control. However, the precise mechanisms underlying nutrient sensing in POMC neurons remain incompletely understood. We show that mitochondrial dynamics mediated by Mitofusin 1 (MFN1) in POMC neurons couple nutrient sensing with systemic glucose metabolism. Mice lacking MFN1 in POMC neurons exhibited defective mitochondrial architecture remodeling and attenuated hypothalamic gene expression programs during the fast-to-fed transition. This loss of mitochondrial flexibility in POMC neurons bidirectionally altered glucose sensing, causing abnormal glucose homeostasis due to defective insulin secretion by pancreatic β cells. Fed mice lacking MFN1 in POMC neurons displayed enhanced hypothalamic mitochondrial oxygen flux and reactive oxygen species generation. Central delivery of antioxidants was able to normalize the phenotype. Collectively, our data posit MFN1-mediated mitochondrial dynamics in POMC neurons as an intrinsic nutrient-sensing mechanism and unveil an unrecognized link between this subset of neurons and insulin release. Copyright © 2017 Elsevier Inc. All rights reserved.

Immune responses to an encapsulated allogeneic islet β-cell line in diabetic NOD mice

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Black, Sasha P.; Constantinidis, Ioannis; Cui, Hong; Tucker-Burden, Carol; Weber, Collin J.; Safley, Susan A.

2006-01-01

Our goal is to develop effective islet grafts for treating type 1 diabetes. Since human islets are scarce, we evaluated the efficacy of a microencapsulated insulin-secreting conditionally transformed allogeneic β-cell line (βTC-tet) in non-obese diabetic mice treated with tetracycline to inhibit cell growth. Relatively low serum levels of tetracycline controlled proliferation of βTC-tet cells without inhibiting effective control of hyperglycemia in recipients. There was no significant host cellular reaction to the allografts or host cell adherence to microcapsules, and host cytokine levels were similar to those of sham-operated controls. We conclude that encapsulated allogeneic β-cell lines may be clinically relevant, because they effectively restore euglycemia and do not elicit a strong cellular immune response following transplantation. To our knowledge, this is First extensive characterization of the kinetics of host cellular and cytokine responses to an encapsulated islet cell line in an animal model of type 1 diabetes

Insulin sensitizers prevent fine particulate matter-induced vascular insulin resistance and changes in endothelial progenitor cell homeostasis.

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Haberzettl, Petra; McCracken, James P; Bhatnagar, Aruni; Conklin, Daniel J

2016-06-01

Exposure to fine particular matter (PM2.5) increases the risk of developing cardiovascular disease and Type 2 diabetes. Because blood vessels are sensitive targets of air pollutant exposure, we examined the effects of concentrated ambient PM2.5 (CAP) on vascular insulin sensitivity and circulating levels of endothelial progenitor cells (EPCs), which reflect cardiovascular health. We found that CAP exposure for 9 days decreased insulin-stimulated Akt phosphorylation in the aorta of mice maintained on control diet. This change was accompanied by the induction of IL-1β and increases in the abundance of cleaved IL-18 and p10 subunit of Casp-1, consistent with the activation of the inflammasome pathway. CAP exposure also suppressed circulating levels of EPCs (Flk-1(+)/Sca-1(+) cells), while enhancing the bone marrow abundance of these cells. Although similar changes in vascular insulin signaling and EPC levels were observed in mice fed high-fat diet, CAP exposure did not exacerbate diet-induced changes in vascular insulin resistance or EPC homeostasis. Treatment with an insulin sensitizer, metformin or rosiglitazone, prevented CAP-induced vascular insulin resistance and NF-κB and inflammasome activation and restored peripheral blood and bone marrow EPC levels. These findings suggest that PM2.5 exposure induces diet-independent vascular insulin resistance and inflammation and prevents EPC mobilization, and that this EPC mobilization defect could be mediated by vascular insulin resistance. Impaired vascular insulin sensitivity may be an important mechanism underlying PM2.5-induced vascular injury, and pharmacological sensitization to insulin action could potentially prevent deficits in vascular repair and mitigate vascular inflammation due to exposure to elevated levels of ambient air pollution. Copyright © 2016 the American Physiological Society.

Biological assessment of self-assembled polymeric micelles for pulmonary administration of insulin.

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Andrade, Fernanda; das Neves, José; Gener, Petra; Schwartz, Simó; Ferreira, Domingos; Oliva, Mireia; Sarmento, Bruno

2015-10-01

Pulmonary delivery of drugs for both local and systemic action has gained new attention over the last decades. In this work, different amphiphilic polymers (Soluplus®, Pluronic® F68, Pluronic® F108 and Pluronic® F127) were used to produce lyophilized formulations for inhalation of insulin. Development of stimuli-responsive, namely glucose-sensitive, formulations was also attempted with the addition of phenylboronic acid (PBA). Despite influencing the in vitro release of insulin from micelles, PBA did not confer glucose-sensitive properties to formulations. Lyophilized powders with aerodynamic diameter (<6 μm) compatible with good deposition in the lungs did not present significant in vitro toxicity for respiratory cell lines. Additionally, some formulations, in particular Pluronic® F127-based formulations, enhanced the permeation of insulin through pulmonary epithelial models and underwent minimal internalization by macrophages in vitro. Overall, formulations based on polymeric micelles presenting promising characteristics were developed for the delivery of insulin by inhalation. The ability to deliver other systemic drugs via inhalation has received renewed interests in the clinical setting. This is especially true for drugs which usually require injections for delivery, like insulin. In this article, the authors investigated their previously developed amphiphilic polymers for inhalation of insulin in an in vitro model. The results should provide basis for future in vivo studies. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Novel nuclear localization and potential function of insulin-like growth factor-1 receptor/insulin receptor hybrid in corneal epithelial cells.

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Yu-Chieh Wu

Full Text Available BACKGROUND: Type I insulin-like growth factor receptor (IGF-1R and insulin receptor (INSR are highly homologous molecules, which can heterodimerize to form an IGF-1R/INSR hybrid (Hybrid-R. The presence and biological significance of the Hybrid-R in human corneal epithelium has not yet been established. In addition, while nuclear localization of IGF-1R was recently reported in cancer cells and human corneal epithelial cells, the function and profile of nuclear IGF-1R is unknown. In this study, we characterized the nuclear localization and function of the Hybrid-R and the role of IGF-1/IGF-1R and Hybrid-R signaling in the human corneal epithelium. METHODOLOGY/PRINCIPLE FINDINGS: IGF-1-mediated signaling and cell growth were examined in a human telomerized corneal epithelial (hTCEpi cell line using co-immunoprecipitation, immunoblotting and cell proliferation assays. The presence of Hybrid-R in hTCEpi and primary cultured human corneal epithelial cells was confirmed by immunofluorescence and reciprocal immunoprecipitation of whole cell lysates. We found that IGF-1 stimulated Akt and promoted cell growth through IGF-1R activation, which was independent of the Hybrid-R. The presence of Hybrid-R, but not IGF-1R/IGF-1R, was detected in nuclear extracts. Knockdown of INSR by small interfering RNA resulted in depletion of the INSR/INSR and preferential formation of Hybrid-R. Chromatin-immunoprecipitation sequencing assay with anti-IGF-1R or anti-INSR was subsequently performed to identify potential genomic targets responsible for critical homeostatic regulatory pathways. CONCLUSION/SIGNIFICANCE: In contrast to previous reports on nuclear localized IGF-1R, this is the first report identifying the nuclear localization of Hybrid-R in an epithelial cell line. The identification of a nuclear Hybrid-R and novel genomic targets suggests that IGF-1R traffics to the nucleus as an IGF-1R/INSR heterotetrameric complex to regulate corneal epithelial homeostatic

A clinical study on insulin receptors of mononuclear cells in diabetes

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Dalimunthe, D.

1980-01-01

125 I-insulin binding activity to mononuclear cells was studied in 75 noninsulin-dependent diabetic subjects and 31 normal subjects and the following results were obtained. 1. 125 I-insulin binding is directly proportional to the mononuclear cell concentrations. There is a linear increase of specific 125 I-insulin binding. 2. The binding of 125 I-insulin to mononuclear cells is displaced by the increasing concentration of native insulin. 3. The 125 I-insulin degradation in the incubation medium after incubation of mononuclear cells for 24 hours at 4 0 C was almost 5% in this study. 4. The insulin binding activity in diabetic subjects was lower than that in normal subjects (P < 0.001) without any significant difference in affinity constant. 5. The relationship of binding activity to age of diabetics (r = 0.06, N.S), relative body weitht (r = 0.06, N.S) and duration of diabetes from onset was not significant. 6. In untreated noninsulin-dependent diabetics the insulin binding activity was inversely correlated to fasting blood glucose level (r = 0.78, P < 0.001) and slightly inversely correlated to serum insulin level (r = 0.47, P < 0.01). A slight inverse correlation was also observed in serum triglyceride level (r = 0.53, P < 0.01) and in total cholesterol level (r = 0.29, P < 0.05). 7. No significant difference between the binding activity was observed by grade of diabetic retinopathy. 8. After treatment with diet and/or sulfonylurea, the diabetics exhibited a significant increase in insulin binding activity (P < 0.005) but no significant difference in plasma insulin level, body weight and plasma lipid levels was observed. (author)

Secretome Analysis of Lipid-Induced Insulin Resistance in Skeletal Muscle Cells by a Combined Experimental and Bioinformatics Workflow

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Deshmukh, Atul S; Cox, Juergen; Jensen, Lars Juhl

2015-01-01

, in principle, allows an unbiased and comprehensive analysis of cellular secretomes; however, the distinction of bona fide secreted proteins from proteins released upon lysis of a small fraction of dying cells remains challenging. Here we applied highly sensitive MS and streamlined bioinformatics to analyze......-resistant conditions. Our study demonstrates an efficient combined experimental and bioinformatics workflow to identify putative secreted proteins from insulin-resistant skeletal muscle cells, which could easily be adapted to other cellular models....

Indian culinary plants enhance glucose-induced insulin secretion and glucose consumption in INS-1 β-cells and 3T3-L1 adipocytes.

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Kaur, Lovedeep; Han, Kyoung-Sik; Bains, Kiran; Singh, Harjinder

2011-12-01

Six Indian plants, commonly used as culinary plants, herbs or spices (kikar; jamun; neem; harad; fenugreek; bitter gourd), were screened and compared for their antidiabetic potential in vitro. Aqueous plant extracts were prepared and assessed for their effect on the insulin secretion activity of rat pancreatic INS-1 β-cells and glucose consumption in mouse 3T3-L1 adipocytes in order to study their specific mechanisms of action. The effect of the plant extract concentration (25-1000μg/ml) on insulin release and glucose consumption was also studied. All the extracts had a significant stimulatory effect on the insulin secretion of INS-1 cells. In the presence of kikar extract (100μg/ml), an increase of 228% in insulin release was recorded compared to the control (5.6mM glucose) whereas that was 270% and 367% in the presence of kikar and jamun extracts (500μg/ml), respectively. 3T3-L1 cells treated with jamun extract (100μg/ml) exhibited the highest increase in glucose consumption by the cells (94%, compared with the control) followed by harad (53%) and fenugreek (50%) extracts. A significant inhibitory effect of the fenugreek, kikar and jamun extracts on glucose diffusion across a dialysis membrane suggested that these extracts could partly act by decreasing glucose absorption in the small intestine. The results showed that a combination of these plants in diet could help in the management of both type 1 and type 2 diabetes. Copyright © 2011 Elsevier Ltd. All rights reserved.

Insulin-induced inhibition of gluconeogenesis genes, including glutamic pyruvic transaminase 2, is associated with reduced histone acetylation in a human liver cell line.

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Honma, Kazue; Kamikubo, Michiko; Mochizuki, Kazuki; Goda, Toshinao

2017-06-01

Hepatic glutamic pyruvic transaminase (GPT; also known as alanine aminotransferase) is a gluconeogenesis enzyme that catalyzes conversions between alanine and pyruvic acid. It is also used as a blood biomarker for hepatic damage. In this study, we investigated whether insulin regulates GPT expression, as it does for other gluconeogenesis genes, and if this involves the epigenetic modification of histone acetylation. Human liver-derived HepG2 cells were cultured with 0.5-100nM insulin for 8h, and the mRNA expression of GPT, glutamic-oxaloacetic transaminase (GOT), γ-glutamyltransferase (GGT), PCK1, G6PC and FBP1 was measured. We also investigated the extent of histone acetylation around these genes. Insulin suppressed the mRNA expression of gluconeogenesis genes (GPT2, GOT1, GOT2, GGT1, GGT2, G6PC, and PCK1) in HepG2 cells in a dose-dependent manner. mRNA levels of GPT2, but not GPT1, were decreased by insulin. Histone acetylation was also reduced around GPT2, G6PC, and PCK1 in response to insulin. The expression of GPT2 and other gluconeogenesis genes such as G6PC and PCK1 was suppressed by insulin, in association with decreases in histone H3 and H4 acetylation surrounding these genes. Copyright © 2017 Elsevier Inc. All rights reserved.

Sodium arsenite impairs insulin secretion and transcription in pancreatic β-cells

International Nuclear Information System (INIS)

Diaz-Villasenor, Andrea; Sanchez-Soto, M. Carmen; Cebrian, Mariano E.; Ostrosky-Wegman, Patricia; Hiriart, Marcia

2006-01-01

Human studies have shown that chronic inorganic arsenic (iAs) exposure is associated with a high prevalence and incidence of type 2 diabetes. However, the mechanism(s) underlying this effect are not well understood, and practically, there is no information available on the effects of arsenic on pancreatic β-cells functions. Thus, since insulin secreted by the pancreas plays a crucial role in maintaining glucose homeostasis, our aim was to determine if sodium arsenite impairs insulin secretion and mRNA expression in single adult rat pancreatic β-cells. Cells were treated with 0.5, 1, 2, 5 and 10 μM sodium arsenite and incubated for 72 and 144 h. The highest dose tested (10 μM) decreased β-cell viability, by 33% and 83%, respectively. Insulin secretion and mRNA expression were evaluated in the presence of 1 and 5 μM sodium arsenite. Basal insulin secretion, in 5.6 mM glucose, was not significantly affected by 1 or 5 μM treatment for 72 h, but basal secretion was reduced when cells were exposed to 5 μM sodium arsenite for 144 h. On the other hand, insulin secretion in response to 15.6 mM glucose decreased with sodium arsenite in a dose-dependent manner in such a way that cells were no longer able to distinguish between different glucose concentrations. We also showed a significant decrease in insulin mRNA expression of cells exposed to 5 μM sodium arsenite during 72 h. Our data suggest that arsenic may contribute to the development of diabetes mellitus by impairing pancreatic β-cell functions, particularly insulin synthesis and secretion

TSA-induced cell death in prostate cancer cell lines is caspase-2 dependent and involves the PIDDosome.

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Taghiyev, Agshin F; Guseva, Natalya V; Glover, Rebecca A; Rokhlin, Oskar W; Cohen, Michael B

2006-09-01

The histone deacetylase inhibitor Trichostatin A (TSA) has previously been found to induce caspase activity in the human prostate cancer cell lines DU145 and LNCaP. TSA treatment resulted in the release of cytochrome c and Smac/DIABLO from mitochondria in DU145, and activation of caspase-9 in both cell lines. We concluded that TSA mediated its effect via the mitochondrial pathway. The aim of the current study was to determine how TSA initiated the caspase cascade. The results revealed that caspase-2 plays an important role in TSA-induced apoptosis. Inhibition of caspase-2 by siRNA or expression of caspase-2dn substantially decreased caspase activity after TSA treatment in both cell lines, siRNA caspase-2 also inhibited TSA-induced cell death. Caspase-2 acts upstream of caspase-8 and -9 and mediates mitochondrial cytochrome c release. Coimmunoprecipitation experiments show that caspase-2 formed protein complexes with RADD/RAIDD and PIDD. Together, these data indicate that caspase-2 initiates caspase cascade after TSA treatment and involves the formation of the PIDDosome.

Streptozotocin Aggravated Osteopathology and Insulin Induced Osteogenesis Through Co-treatment with Fluoride.

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Yang, Chen; Zhang, Mengmeng; Li, Yagang; Wang, Yan; Mao, Weixian; Gao, Yuan; Xu, Hui

2015-12-01

The role of insulin in the mechanism underlying the excessive fluoride that causes skeletal lesion was studied. The in vitro bone marrow stem cells (BMSC) collected from Kunming mice were exposed to varying concentrations of fluoride with or without insulin. The cell viability and early differentiation of BMSC co-treated with fluoride and insulin were measured by using cell counting kit-8 and Gomori modified calcium-cobalt method, respectively. We further investigated the in vivo effects of varying dose of fluoride on rats co-treated with streptozotocin (STZ). Wistar rats were divided into six groups which included normal control, 10 mg fluoride/kg day group, 20 mg fluoride/kg day group, STZ control, STZ+10 mg fluoride/kg day group, and STZ+20 mg fluoride/kg day group. The rats were administered with sodium fluoride (NaF) by gavage with water at doses 10 and 20 mg fluoride/kg day for 2 months. In a period of one month, half of rats in every group were treated with streptozotocin (STZ) once through intraperitoneal injection at 52 mg/kg body weight. The serum glucose, HbA1c, and insulin were determined. Bone mineral content and insulin release were assessed. The results showed insulin combined with fluoride stimulated BMSC cell viability in vitro. The bone mineral content reduced in rats treated with higher dose of fluoride and decreased immensely in rat co-treated with fluoride and STZ. Similarly, a combination treatment of a high dose of fluoride and STZ decreased insulin sensitivity and activity. To sum up, these data indicated fluoride influenced insulin release, activity, and sensitivity. Furthermore, the insulin state in vivo interfered in the osteogenesis in turn and implied there was a close relation between insulin and bone pathogenesis in the mechanism of fluoride toxicity.

Neuronal calcium sensor synaptotagmin-9 is not involved in the regulation of glucose homeostasis or insulin secretion

DEFF Research Database (Denmark)

Gustavsson, Natalia; Wang, Xiaorui; Wang, Yue

2010-01-01

the identities of proteins that are responsible for sensing calcium changes and for transmitting the calcium signal to release machineries. Synaptotagmins are primarily expressed in brain and endocrine cells and exhibit diverse calcium binding properties. Synaptotagmin-1, -2 and -9 are calcium sensors for fast......BACKGROUND: Insulin secretion is a complex and highly regulated process. It is well established that cytoplasmic calcium is a key regulator of insulin secretion, but how elevated intracellular calcium triggers insulin granule exocytosis remains unclear, and we have only begun to define...... neurotransmitter release in respective brain regions, while synaptotagmin-7 is a positive regulator of calcium-dependent insulin release. Unlike the three neuronal calcium sensors, whose deletion abolished fast neurotransmitter release, synaptotagmin-7 deletion resulted in only partial loss of calcium...

Evaluation of insulin expression and secretion in genetically engineered gut K and L-cells

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Ahmad Zalinah

2012-09-01

Full Text Available Abstract Background Gene therapy could provide an effective treatment of diabetes. Previous studies have investigated the potential for several cell and tissue types to produce mature and active insulin. Gut K and L-cells could be potential candidate hosts for gene therapy because of their special features. Results In this study, we isolated gut K and L-cells to compare the potential of both cell types to produce insulin when exposed to similar conditions. The isolated pure K and L-cells were transfected with recombinant plasmids encoding insulin and with specific promoters for K or L-cells. Insulin expression was studied in response to glucose or meat hydrolysate. We found that glucose and meat hydrolysate efficiently induced insulin secretion from K and L-cells. However, the effects of meat hydrolysate on insulin secretion were more potent in both cells compared with glucose. Results of enzyme-linked immunosorbent assays showed that L-cells secreted more insulin compared with K-cells regardless of the stimulator, although this difference was not statistically significant. Conclusion The responses of K and L-cells to stimulation with glucose or meat hydrolysate were generally comparable. Therefore, both K and L-cells show similar potential to be used as surrogate cells for insulin gene expression in vitro. The potential use of these cells for diabetic gene therapy warrants further investigation.

Potent Insulin Secretagogue from Scoparia dulcis Linn of Nepalese Origin.

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Sharma, Khaga Raj; Adhikari, Achyut; Hafizur, Rahman M; Hameed, Abdul; Raza, Sayed Ali; Kalauni, Surya Kant; Miyazaki, Jun-Ichi; Choudhary, M Iqbal

2015-10-01

Ethno-botanical inspired isolation from plant Scoparia dulcis Linn. (Sweet Broomweed) yielded six compounds, coixol (1), glutinol (2), glutinone (3), friedelin (4), betulinic acid (5), and tetratriacontan-1-ol (6). There structures were identified using mass and 1D- and 2D-NMR spectroscopy techniques. Compounds 1-6 were evaluated for their insulin secretory activity on isolated mice islets and MIN-6 pancreatic β-cell line, and compounds 1 and 2 were found to be potent and mildly active, respectively. Compound 1 was further evaluated for insulin secretory activity on MIN-6 cells. Compound 1 was subjected to in vitro cytotoxicity assay against MIN-6, 3T3 cell lines, and islet cells, and in vivo acute toxicity test in mice that was found to be non-toxic. The insulin secretory activity of compounds 1 and 2 supported the ethno-botanic uses of S. dulcis as an anti-diabetic agent. Copyright © 2015 John Wiley & Sons, Ltd.

Human interleukin 1. beta. stimulates islet insulin release by a mechanism not dependent on changes in phospholipase C and protein kinase C activities or Ca sup 2+ handling

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Welsh, N.; Nilsson, T.; Hallberg, A.; Arkhammar, P.; Berggren, P.-O.; Sandler, S.

1989-01-01

Isolated islets from adult rats or obese hyperglycemic (ob/ob) mice were incubated with human recombinant interleukin 1{beta} in order to study whether the acute effects of the cytokine on islet insulin release are associated with changes in islet phospholipase C activity, Ca{sup 2+} handling or protein phosphorylation. The cytokine stimulated insulin release both at low and high glucose concentrations during one hour incubations. In shortterm incubations (<1 min) interleukin 1{beta} did not affect the production of inositoltrisphosphate. Addition of interleukin 1{beta} affected neither the cytoplasmic free Ca{sup 2+} concentration at rest nor that observed subsequent to stimulation with a high concentration of glucose. Furthermore, the endogenous protein kinase C activity, as visualized by immunoprecipitation of a {sup 32}P-labelled substrate for this enzyme, was not altered by interleukin 1{beta}. Separation of {sup 32}P-labelled proteins by means of 2-dimensional gel electrophoresis failed to reveal any specific effects of the cytokine on the total protein phosphorylation activity. These results suggest that the stimulatory effects on insulin release exerted by interleukin 1{beta} are not caused by acute activation of phospholipase C and protein kinase C or by an alternation of islet Ca{sup 2+} handling of the B-cells. (author).

Insulin sensitivity and β-cell function in normoglycemic offspring of individuals with type 2 diabetes mellitus: Impact of line of inheritance

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Edavan P Praveen

2012-01-01

Full Text Available Aims: The aim was to study the effect of family history of type 2 diabetes mellitus (T2DM on insulin sensitivity and b-cell function in normoglycemic offspring. Material and Methods: Offspring of T2DM patients (cases and individuals without family history of T2DM (controls were the subjects for this cross-sectional study. All participants underwent 75 g OGTT and samples were collected for plasma insulin, C-peptide, and proinsulin at 0, 30, 60, and 120 minutes. Results: A total of 271 cases (age 22 ± 10 years; 53% males and 259 controls (28 ± 10 years, 66% males were enrolled for the study. BMI, plasma insulin, C-peptide, proinsulin, HOMA-IR, and insulinogenic index (0-120 were significantly higher and whole-body insulin sensitivity (WBISI and disposition index (0-120 [DI 120] were lower in cases compared to controls. After adjusting for BMI, proinsulin at 120 minutes, area under the curve (AUC of proinsulin (during OGTT and AUC proinsulin/AUC C-peptide were significantly higher in cases. Cases were subdivided into four groups according to inheritance pattern; paternal DM (PDM, maternal DM (MDM, grandparental DM (GPDM, and both parents DM (BPDM. The magnitude of differences varied with relationship (greater when both parents and grandparents were affected. Mean HOMA-IR was higher by 127% and 50% and DI 120 was lower by 33% and 18% (adjusted for age and gender in the BPDM and GPDM groups respectively compared to controls. Conclusions: We observed higher BMI, plasma insulin, C-peptide, and proinsulin and lower insulin sensitivity and b-cell compensation in normoglycemic offspring of T2DM subjects compared to controls. Differences were greater when both parents and grandparents had T2DM.

Similar weight-adjusted insulin secretion and insulin sensitivity in short-duration late autoimmune diabetes of adulthood (LADA) and Type 2 diabetes

DEFF Research Database (Denmark)

Juhl, C B; Bradley, U; Holst, Jens Juul

2014-01-01

AIMS: To explore insulin sensitivity and insulin secretion in people with latent autoimmune diabetes in adulthood (LADA) compared with that in people with Type 2 diabetes. METHODS: A total of 12 people with LADA, defined as glutamic acid decarboxylase (GAD) antibody positivity and > 1 year...... of insulin independency (group A) were age-matched pairwise to people with Type 2 diabetes (group B) and to six people with Type 2 diabetes of similar age and BMI (group C). β-cell function (first-phase insulin secretion and assessment of insulin pulsatility), insulin sensitivity (hyperinsulinemic......-euglycemic clamp) and metabolic response during a mixed meal were studied. RESULTS: Both first-phase insulin secretion and insulin release during the meal were greater (P = 0.05 and P = 0.009, respectively) in Type 2 diabetes as compared with LADA; these differences were lost on adjustment for BMI (group C...

Release behavior and toxicity profiles towards A549 cell lines of ciprofloxacin from its layered zinc hydroxide intercalation compound.

Science.gov (United States)

Abdul Latip, Ahmad Faiz; Hussein, Mohd Zobir; Stanslas, Johnson; Wong, Charng Choon; Adnan, Rohana

2013-01-01

Layered hydroxides salts (LHS), a layered inorganic compound is gaining attention in a wide range of applications, particularly due to its unique anion exchange properties. In this work, layered zinc hydroxide nitrate (LZH), a family member of LHS was intercalated with anionic ciprofloxacin (CFX), a broad spectrum antibiotic via ion exchange in a mixture solution of water:ethanol. Powder x-ray diffraction (XRD), Fourier transform infrared (FTIR) and thermogravimetric analysis (TGA) confirmed the drug anions were successfully intercalated in the interlayer space of LZH. Specific surface area of the obtained compound was increased compared to that of the host due to the different pore textures between the two materials. CFX anions were slowly released over 80 hours in phosphate-buffered saline (PBS) solution due to strong interactions that occurred between the intercalated anions and the host lattices. The intercalation compound demonstrated enhanced antiproliferative effects towards A549 cancer cells compared to the toxicity of CFX alone. Strong host-guest interactions between the LZH lattice and the CFX anion give rise to a new intercalation compound that demonstrates sustained release mode and enhanced toxicity effects towards A549 cell lines. These findings should serve as foundations towards further developments of the brucite-like host material in drug delivery systems.

Bioresponsive release of insulin-like growth factor-I from its PEGylated conjugate.

Science.gov (United States)

Braun, Alexandra C; Gutmann, Marcus; Mueller, Thomas D; Lühmann, Tessa; Meinel, Lorenz

2018-06-10

PEGylation of protein ligands, the attachment of polyethylene glycol (PEG) polymers to a therapeutic protein, increases therapeutics' half-life but frequently comes at the cost of reduced bioactivity. We are now presenting a bioinspired strategy leading out of this dilemma. To this end, we selected a position within insulin-like growth factor I (IGF-I) for decoration with a PEG 30kDa -modified protease-sensitive peptide linker (PSL) using a combination of enzymatic and chemical bioorthogonal coupling strategies. The PSL sequence responded to matrix metalloproteinases (MMP) to provide a targeted release in diseased tissue. The IGF-PSL-PEG conjugate had different binding protein affinity, cell proliferation, and endocytosis patterns as compared to the wild type. Exposure of the conjugate to elevated levels of activated MMPs, as present in inflamed tissues, fully reestablished the wild type properties through effective PSL cleavage. In conclusion, this bioinspired approach provided a blueprint for PEGylated therapeutics combining the pharmacokinetic advantages of PEGylation, while locally restoring the full suite of biological potential of therapeutics. Copyright © 2018 Elsevier B.V. All rights reserved.

Nutrient sensing and insulin signaling in neuropeptide-expressing immortalized, hypothalamic neurons: A cellular model of insulin resistance.

Science.gov (United States)

Fick, Laura J; Belsham, Denise D

2010-08-15

Obesity and type 2 diabetes mellitus represent a significant global health crisis. These two interrelated diseases are typified by perturbed insulin signaling in the hypothalamus. Using novel hypothalamic cell lines, we have begun to elucidate the molecular and intracellular mechanisms involved in the hypothalamic control of energy homeostasis and insulin resistance. In this review, we present evidence of insulin and glucose signaling pathways that lead to changes in neuropeptide gene expression. We have identified some of the molecular mechanisms involved in the control of de novo hypothalamic insulin mRNA expression. And finally, we have defined key mechanisms involved in the etiology of cellular insulin resistance in hypothalamic neurons that may play a fundamental role in cases of high levels of insulin or saturated fatty acids, often linked to the exacerbation of obesity and diabetes.

[Anti-tumor effect of 5-FU-PLLA-CNTs on human gastric carcinoma cell lines in vitro].

Science.gov (United States)

Gu, Jun; Li, Maolan; Wu, Xiangsong; Wu, Wenguang; Zhang, Lin; Ding, Qichen; Yang, Jiahua; Weng, Hao; Ding, Qian; Bao, Runfa; Shu, Yijun; Liu, Yingbin

2014-04-01

To prepare cisPLLAtin-loaded polylactic acid/cnts, and to study the anti-tumor effect of 5-FU-PLLA-CNTs on human gastric carcinoma cell lines(MGC803 and MNK45). 5-FU-PLLA-CNTs were prepared with ultrasound emulsification. The morphology of 5-FU-PLLA-CNTs was determined by scanning electron microscope(SEM), and its drug loading and drug release curve in vitro were detected by UV-Vis-NIR spectrophotometer. Cells were divided into experiment, positive control and negative control groups. CCK8 method was used to test the cytotoxic effect of 5-FU-PLLA-CNTs in different concentrations on MGC803 and MNK45 cell proliferation. Flow cytometry was employed to measure the apoptotic rate of MGC803 and MNK45 cells before and after the intervention of 5-FU-PLLA-CNTs. Deep layer film of 5-FU-PLLA-CNTs was successfully established, whose drug-load rate was(4.54±0.43)%, entrapment rate was(21.56±2.36)%. In vitro release test showed release rate within 24 h of 5-FU-PLLA-CNTs was 23.9% in a as lowly increasing manner, and accumulating release rate was 85.3% at day 31. CCk8 experiment revealed, as compared to control group, 5-FU-PLLA-CNTs significantly inhibited the proliferation of two cell lines in dose-dependent and time-dependent manner. The best 5-FU-PLLA-CNTs concentration of inhibition for human gastric cancer cell lines was 1 mg/well. Flow cytometry indicated the apoptotic rate of MGC803 and MNK45 cells in experiment group treated by 1 mg/well 5-FU-PLLA-CNTs significantly increased as compared to negative control group (P0.05). The 5-FU-PLLA-CNTs has good drug sustained-release capacity, and can significantly kill and inhibit the proliferation of MGC803 and MNK45 cell lines.

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans.

Science.gov (United States)

MacDonald, Patrick E; De Marinis, Yang Zhang; Ramracheya, Reshma; Salehi, Albert; Ma, Xiaosong; Johnson, Paul R V; Cox, Roger; Eliasson, Lena; Rorsman, Patrik

2007-06-01

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

The establishment of insulin resistance model in FL83B and L6 cell

Science.gov (United States)

Liu, Lanlan; Han, Jizhong; Li, Haoran; Liu, Mengmeng; Zeng, Bin

2017-10-01

The insulin resistance models of mouse liver epithelial and rat myoblasts cells were induced by three kinds of inducers: dexamethasone, high insulin and high glucose. The purpose is to select the optimal insulin resistance model, to provide a simple and reliable TR cell model for the study of the pathogenesis of TR and the improvement of TR drugs and functional foods. The MTT method is used for toxicity screening of three compounds, selecting security and suitable concentration. We performed a Glucose oxidase peroxidase (GOD-POD) method involving FL83B and L6 cell with dexamethasone, high insulin and high glucose-induced insulin resistance. Results suggested that FL83B cells with dexamethasone-induced (0.25uM) were established insulin resistance and L6 cells with high-glucose (30mM) and dexamethasone-induced (0.25uM) were established insulin resistance.

Phosphorylation events implicating p38 and PI3K mediate tungstate-effects in MIN6 beta cells

International Nuclear Information System (INIS)

Piquer, Sandra; Barcelo-Batllori, Silvia; Julia, Marta; Marzo, Nuria; Nadal, Belen; Guinovart, Joan J.; Gomis, Ramon

2007-01-01

Oral administration of sodium tungstate is an effective treatment for diabetes in animal models. Several lines of evidence indicate the pancreatic beta cell as one of the targets of tungstate action. Here, we examined the molecular mechanism by which this compound exerts its effects on the beta cell line MIN6. Tungstate treatment induced phosphorylation and subsequent activation of p38 and PI3K which in turn are implicated in tungstate PDX-1 nuclear localization and activation. Although no effect was observed in glucose-induced insulin secretion we found that tungstate activates basal insulin release, a process driven, at least in part, by activation of p38. These results show a direct involvement of p38 and PI3K phosphorylation in the mechanism of action of tungstate in the beta cell

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.

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.

Insulin Secretagogues

Science.gov (United States)

... than sulfonylureas. What are the side effects and disadvantages of insulin secretagogues? Both types of insulin-releasing ... help find the cause. Questions to ask your doctor What else can I do to keep my ...

Gonadotropin-releasing hormone receptor activates GTPase RhoA and inhibits cell invasion in the breast cancer cell line MDA-MB-231

International Nuclear Information System (INIS)

Aguilar-Rojas, Arturo; Huerta-Reyes, Maira; Maya-Núñez, Guadalupe; Arechavaleta-Velásco, Fabián; Conn, P Michael; Ulloa-Aguirre, Alfredo; Valdés, Jesús

2012-01-01

Gonadotropin-releasing hormone (GnRH) and its receptor (GnRHR) are both expressed by a number of malignant tumors, including those of the breast. In the latter, both behave as potent inhibitors of invasion. Nevertheless, the signaling pathways whereby the activated GnRH/GnRHR system exerts this effect have not been clearly established. In this study, we provide experimental evidence that describes components of the mechanism(s) whereby GnRH inhibits breast cancer cell invasion. Actin polymerization and substrate adhesion was measured in the highly invasive cell line, MDA-MB-231 transiently expressing the wild-type or mutant DesK191 GnRHR by fluorometry, flow cytometric analysis, and confocal microscopy, in the absence or presence of GnRH agonist. The effect of RhoA-GTP on stress fiber formation and focal adhesion assembly was measured in MDA-MB-231 cells co-expressing the GnRHRs and the GAP domain of human p190Rho GAP-A or the dominant negative mutant GAP-Y1284D. Cell invasion was determined by the transwell migration assay. Agonist-stimulated activation of the wild-type GnRHR and the highly plasma membrane expressed mutant GnRHR-DesK191 transiently transfected to MDA-MB-231 cells, favored F-actin polymerization and substrate adhesion. Confocal imaging allowed detection of an association between F-actin levels and the increase in stress fibers promoted by exposure to GnRH. Pull-down assays showed that the effects observed on actin cytoskeleton resulted from GnRH-stimulated activation of RhoA GTPase. Activation of this small G protein favored the marked increase in both cell adhesion to Collagen-I and number of focal adhesion complexes leading to inhibition of the invasion capacity of MDA-MB-231 cells as disclosed by assays in Transwell Chambers. We here show that GnRH inhibits invasion of highly invasive breast cancer-derived MDA-MB-231 cells. This effect is mediated through an increase in substrate adhesion promoted by activation of RhoA GTPase and formation of

Border cell release

DEFF Research Database (Denmark)

Mravec, Jozef

2017-01-01

Plant border cells are specialised cells derived from the root cap with roles in the biomechanics of root growth and in forming a barrier against pathogens. The mechanism of highly localised cell separation which is essential for their release to the environment is little understood. Here I present...... in situ analysis of Brachypodium distachyon, a model organism for grasses which possess type II primary cell walls poor in pectin content. Results suggest similarity in spatial dynamics of pectic homogalacturonan during dicot and monocot border cell release. Integration of observations from different...... species leads to the hypothesis that this process most likely does not involve degradation of cell wall material but rather employs unique cell wall structural and compositional means enabling both the rigidity of the root cap as well as detachability of given cells on its surface....

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.

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.

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.

Endogenous pyrogen production by Hodgkin's disease and human histiocytic lymphoma cell lines in vitro.

OpenAIRE

Bodel, P; Ralph, P; Wenc, K; Long, J C

1980-01-01

Fever not explained by infection may occur in patients with malignant lymphoma presumably caused by a release of endogenous pyrogen. Although pyrogen has been found in some tumors with a mixed cell population, production of endogenous pyrogen by the neoplastic cells has not been demonstrated. This report documents the apparently spontaneous synthesis and release of such pyrogen by two human tumor cell lines derived from patients with Hodgkin's disease and histiocytic lymphoma. The endogenous ...

Relation between the insulin receptor number in cells, autophosphorylation and insulin-stimulated Ras.GTP formation

NARCIS (Netherlands)

Osterop, A.P.R.M.; Medema, R.H.; Bos, J.L.; Zon, G.C.M. van der; Moller, D.E.; Flier, J.S.; Möller, W.; Maassen, J.A.

1992-01-01

We showed previously that upon insulin stimulation of an insulin receptor overexpressing cell linme,o st of the p2lras warsa pidly converted into the GTP bound state (Burgering, B. M. T., Medema, R. H., Maassen, J. A., Van de Wetering, M. L., Van der Eb, A. J., McCormick, F., and Bos, J. L.

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.

Insulin resistance enhances the mitogen-activated protein kinase signaling pathway in ovarian granulosa cells.

Directory of Open Access Journals (Sweden)

Linghui Kong

Full Text Available The ovary is the main regulator of female fertility. Granulosa cell dysfunction may be involved in various reproductive endocrine disorders. Here we investigated the effect of insulin resistance on the metabolism and function of ovarian granulosa cells, and dissected the functional status of the mitogen-activated protein kinase signaling pathway in these cells. Our data showed that dexamethasone-induced insulin resistance in mouse granulosa cells reduced insulin sensitivity, accompanied with an increase in phosphorylation of p44/42 mitogen-activated protein kinase. Furthermore, up-regulation of cytochrome P450 subfamily 17 and testosterone and down-regulation of progesterone were observed in insulin-resistant mouse granulosa cells. Inhibition of p44/42 mitogen-activated protein kinase after induction of insulin resistance in mouse granulosa cells decreased phosphorylation of p44/42 mitogen-activated protein kinase, downregulated cytochrome P450 subfamily 17 and lowered progesterone production. This insulin resistance cell model can successfully demonstrate certain mechanisms such as hyperandrogenism, which may inspire a new strategy for treating reproductive endocrine disorders by regulating cell signaling pathways.

Lipofection of insulin-producing RINm5F cells: methodological improvements.

Science.gov (United States)

Barbu, Andreea; Welsh, Nils

2007-01-01

Cationic lipid/DNA-complexes have been widely used as gene transfer vectors because they are less toxic and immunogenic than viral vectors. The aim of the present study was to improve and characterize lipofection of an insulin-producing cell line. We compared the transfection efficiency of seven commercially available lipid formulations (Lipotaxi, SuperFect, Fugene, TransFast, Dosper, GenePORTER and LipofectAMINE) by flow cytometry analysis of GFP-expression. In addition, we have determined the influences of centrifugation, serum and a nuclear localization signal peptide on the lipofection efficiency. We observed that two lipid formulations, GenePORTER and LipofectAMINE, were able to promote efficient gene transfer in RINm5F cells. However, GenePORTER exhibited the important advantage of being able to transfect cells in the presence of serum and with less cytotoxicity than LipofectAMINE. LipofectAMINE-induced RINm5F cell death could partially be counteracted by TPA, forskolin or fumonisin beta(1). Finally, both centrifugation and a nuclear localization signal peptide increased transfection efficiency.

Liver-derived systemic factors drive β-cell hyperplasia in insulin resistant states

Energy Technology Data Exchange (ETDEWEB)

El Ouaamari, Abdelfattah; Kawamori, Dan; Dirice, Ercument; Liew, Chong Wee; Shadrach, Jennifer L.; Hu, Jiang; Katsuta, Hitoshi; Hollister-Lock, Jennifer; Qian, Weijun; Wagers, Amy J.; Kulkarni, Rohit N.

2013-02-21

Integrative organ cross-talk regulates key aspects of energy homeostasis and its dysregulation may underlie metabolic disorders such as obesity and diabetes. To test the hypothesis that cross-talk between the liver and pancreatic islets modulates β-cell growth in response to insulin resistance, we used the Liver-specific Insulin Receptor Knockout (LIRKO) mouse, a unique model that exhibits dramatic islet hyperplasia. Using complementary in vivo parabiosis and transplantation assays, and in vitro islet culture approaches, we demonstrate that humoral, non-neural, non-cell autonomous factor(s) induce β-cell proliferation in LIRKO mice. Furthermore, we report that a hepatocyte-derived factor(s) stimulates mouse and human β-cell proliferation in ex vivo assays, independent of ambient glucose and insulin levels. These data implicate the liver as a critical source of β-cell growth factors in insulin resistant states.

Chronic Insulin Exposure Induces ER Stress and Lipid Body Accumulation in Mast Cells at the Expense of Their Secretory Degranulation Response.

Directory of Open Access Journals (Sweden)

William E Greineisen

Full Text Available Lipid bodies (LB are reservoirs of precursors to inflammatory lipid mediators in immunocytes, including mast cells. LB numbers are dynamic, increasing dramatically under conditions of immunological challenge. We have previously shown in vitro that insulin-influenced lipogenic pathways induce LB biogenesis in mast cells, with their numbers attaining steatosis-like levels. Here, we demonstrate that in vivo hyperinsulinemia resulting from high fat diet is associated with LB accumulation in murine mast cells and basophils. We characterize the lipidome of purified insulin-induced LB, and the shifts in the whole cell lipid landscape in LB that are associated with their accumulation, in both model (RBL2H3 and primary mast cells. Lipidomic analysis suggests a gain of function associated with LB accumulation, in terms of elevated levels of eicosanoid precursors that translate to enhanced antigen-induced LTC4 release. Loss-of-function in terms of a suppressed degranulation response was also associated with LB accumulation, as were ER reprogramming and ER stress, analogous to observations in the obese hepatocyte and adipocyte. Taken together, these data suggest that chronic insulin elevation drives mast cell LB enrichment in vitro and in vivo, with associated effects on the cellular lipidome, ER status and pro-inflammatory responses.

Pancreatic β-Cell Electrical Activity and Insulin Secretion: of Mice and Men

Science.gov (United States)

Rorsman, Patrik; Ashcroft, Frances M

2018-01-01

The pancreatic β-cell plays a key role in glucose homeostasis by secreting insulin, the only hormone capable of lowering the blood glucose concentration. Impaired insulin secretion results in the chronic hyperglycaemia that characterizes type 2 diabetes (T2DM), which currently afflicts >450 million people worldwide. The healthy β-cell acts as a glucose sensor matching its output to the circulating glucose concentration. It does so via metabolically induced changes in electrical activity, which culminate in an increase in the cytoplasmic Ca2+ concentration and initiation of Ca2+-dependent exocytosis of insulin-containing secretory granules. Here, we review recent advances in our understanding of the β-cell transcriptome, electrical activity and insulin exocytosis. We highlight salient differences between mouse and human β-cells, provide models of how the different ion channels contribute to their electrical activity and insulin secretion, and conclude by discussing how these processes become perturbed in T2DM. PMID:29212789

Nanovesicles released by Dictyostelium cells: a potential carrier for drug delivery.

Science.gov (United States)

Lavialle, Françoise; Deshayes, Sophie; Gonnet, Florence; Larquet, Eric; Kruglik, Sergei G; Boisset, Nicolas; Daniel, Régis; Alfsen, Annette; Tatischeff, Irène

2009-10-01

Nanovesicles released by Dictyostelium discoideum cells grown in the presence of the DNA-specific dye Hoechst 33342 have been previously shown to mediate the transfer of the dye into the nuclei of Hoechst-resistant cells. The present investigation extends this work by conducting experiments in the presence of hypericin, a fluorescent therapeutic photosensitizer assayed for antitumoral photodynamic therapy. Nanovesicles released by Dictyostelium cells exhibit an averaged diameter between 50 and 150 nm, as measured by transmission cryoelectron microscopy. A proteomic analysis reveals a predominance of actin and actin-related proteins. The detection of a lysosomal membrane protein (LIMP II) indicates that these vesicles are likely generated in the late endosomal compartment. The use of the hypericin-containing nanovesicles as nanodevices for in vitro drug delivery was investigated by fluorescence microscopy. The observed signal was almost exclusively located in the perinuclear area of two human cell lines, skin fibroblasts (HS68) and cervix carcinoma (HeLa) cells. Studies by confocal microscopy with specific markers of cell organelles, provided evidence that hypericin was accumulated in the Golgi apparatus. All these data shed a new light on in vitro drug delivery by using cell-released vesicles as carriers.

Insulin regulation of Na/K pump activity in rat hepatoma cells

International Nuclear Information System (INIS)

Gelehrter, T.D.; Shreve, P.D.; Dilworth, V.M.

1984-01-01

Insulin rapidly increases Na/K pump activity in HTC rat hepatoma cells in tissue culture, as measured by the ouabain-sensitive influx of the potassium analogue 86Rb+. Increased influx is observed within minutes and is maximal (70% above control) within 1-2 h. The effect appears to be mediated by the insulin receptors, as: the concentration dependence on insulin is identical to that for insulin induction of tyrosine aminotransferase and stimulation of 2-aminoisobutyric acid transport, proinsulin is 6% as potent as insulin, and the effect is blocked by anti-receptor antibodies. The early stimulation of potassium influx is not blocked by cycloheximide and is not associated with an increased number of pump sites as measured by 3 H-ouabain binding. The insulin effect is blocked by amiloride, which blocks sodium influx, and is mimicked by the sodium ionophore monensin, which increases sodium influx and intracellular accumulation. Insulin also rapidly increases the initial rate of 22 Na+ influx, suggesting that insulin may enhance Na/K pump activity, in part, by increasing intracellular sodium concentration. Incubation of HTC cells with insulin for 24 h causes complete unresponsiveness to the insulin induction of transaminase and stimulation of amino acid transport, a phenomenon mediated by postbinding mechanisms. In contrast, similar incubation with insulin does not cause unresponsiveness to the insulin stimulation of Na/K pump activity. Therefore, the site of regulation of responsiveness to insulin must be distal to, or separate from, those events causing stimulation of ion fluxes

An aqueous extract of Curcuma longa (turmeric) rhizomes stimulates insulin release and mimics insulin action on tissues involved in glucose homeostasis in vitro.

Science.gov (United States)

Mohankumar, Sureshkumar; McFarlane, James R

2011-03-01

Curcuma longa (turmeric) has been used widely as a spice, particularly in Asian countries. It is also used in the Ayurvedic system of medicine as an antiinflammatory and antimicrobial agent and for numerous other curative properties. The aim of this study was to investigate the effects of an aqueous extract of Curcuma longa (AEC) on tissues involved in glucose homeostasis. The extract was prepared by soaking 100 g of ground turmeric in 1 L of water, which was filtered and stored at -20°C prior to use. Pancreas and muscle tissues of adult mice were cultured in DMEM with 5 or 12 mmol/L glucose and varying doses of extract. The AEC stimulated insulin secretion from mouse pancreatic tissues under both basal and hyperglycaemic conditions, although the maximum effect was only 68% of that of tolbutamide. The AEC induced stepwise stimulation of glucose uptake from abdominal muscle tissues in the presence and absence of insulin, and the combination of AEC and insulin significantly potentiated the glucose uptake into abdominal muscle tissue. However, this effect was attenuated by wortmannin, suggesting that AEC possibly acts via the insulin-mediated glucose uptake pathway. In summary, water soluble compounds of turmeric exhibit insulin releasing and mimicking actions within in vitro tissue culture conditions. Copyright © 2010 John Wiley & Sons, Ltd.

Suppression of the Nuclear Factor Eny2 Increases Insulin Secretion in Poorly Functioning INS-1E Insulinoma Cells

Directory of Open Access Journals (Sweden)

P. Dames

2012-01-01

Full Text Available Eny2, the mammalian ortholog of yeast Sus1 and drosophila E(y2, is a nuclear factor that participates in several steps of gene transcription and in mRNA export. We had previously found that Eny2 expression changes in mouse pancreatic islets during the metabolic adaptation to pregnancy. We therefore hypothesized that the protein contributes to the regulation of islet endocrine cell function and tested this hypothesis in rat INS-1E insulinoma cells. Overexpression of Eny2 had no effect but siRNA-mediated knockdown of Eny2 resulted in markedly increased glucose and exendin-4-induced insulin secretion from otherwise poorly glucose-responsive INS-1E cells. Insulin content, cellular viability, and the expression levels of several key components of glucose sensing remained unchanged; however glucose-dependent cellular metabolism was higher after Eny2 knockdown. Suppression of Eny2 enhanced the intracellular incretin signal downstream of cAMP. The use of specific cAMP analogues and pathway inhibitors primarily implicated the PKA and to a lesser extent the EPAC pathway. In summary, we identified a potential link between the nuclear protein Eny2 and insulin secretion. Suppression of Eny2 resulted in increased glucose and incretin-induced insulin release from a poorly glucose-responsive INS-1E subline. Whether these findings extend to other experimental conditions or to in vivo physiology needs to be determined in further studies.

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans.

Directory of Open Access Journals (Sweden)

Patrick E MacDonald

2007-06-01

Full Text Available Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+ responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+ signalling was blocked, but was reversed by low concentrations (1-20 muM of the ATP-sensitive K(+ (KATP channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM. Higher diazoxide concentrations (>/=30 muM decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (10 muM were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM, glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+ (TTX and N-type Ca(2+ channels (omega-conotoxin, but not L-type Ca(2+ channels (nifedipine, prevented glucagon secretion. Both the N-type Ca(2+ channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

Effects of insulin on the survival of irradiated chinese hamster lung cells

Energy Technology Data Exchange (ETDEWEB)

Lin, P S; Kwock, L; Hefter, K; Wallach, D F.H.; Brotman, R [Tufts-New England Medical Center, Boston, Mass. (USA)

1977-01-01

Insulin treatment (10/sup -7/-10/sup -9/ M) before ..gamma.. irradiation (50 to 500 rads) increases the long term survival of Chinese hamster lung cells (DON). Our data indicates that the radioprotective effect of insulin is not due to a modulation of cyclic-adenosine-3',5'-monophosphate levels within these cells. The results suggest that the radiosensitive plasma membrane component postulated to be involved in the interphase death of thymocytes and protected by insulin may have a counterpart in DON cells.

Effect on Insulin-Stimulated Release of D-Chiro-Inositol-Containing Inositolphosphoglycan Mediator during Weight Loss in Obese Women with and without Polycystic Ovary Syndrome.

Science.gov (United States)

Cheang, Kai I; Sistrun, Sakita N; Morel, Kelley S; Nestler, John E

2016-01-01

Background. A deficiency of D-chiro-inositol-inositolphosphoglycan mediator (DCI-IPG) may contribute to insulin resistance in polycystic ovary syndrome (PCOS). Whether the relationship between impaired DCI-IPG release and insulin resistance is specific to PCOS rather than obesity is unknown. We assessed insulin-released DCI-IPG and its relationship to insulin sensitivity at baseline and after weight loss in obese women with and without PCOS. Methods. Obese PCOS ( n = 16) and normal ( n = 15) women underwent 8 weeks of a hypocaloric diet. The Matsuda index, area under the curve DCI-IPG (AUC DCI-IPG ), AUC insulin , and AUC DCI-IPG /AUC insulin were measured during a 2 hr OGTT at baseline and 8 weeks. Results. PCOS women had lower AUC DCI-IPG /AUC insulin at baseline and a significant relationship between AUC DCI-IPG /AUC insulin and Matsuda index ( p = 0.0003), which was not present in controls. Weight loss was similar between PCOS (-4.08 kg) and normal women (-4.29 kg, p = 0.6281). Weight loss in PCOS women did not change the relationship between AUC DCI-IPG /AUC insulin and Matsuda index ( p = 0.0100), and this relationship remained absent in control women. Conclusion. The association between AUC DCI-IPG /AUC insulin and insulin sensitivity was only found in PCOS but not in normal women, and this relationship was unaffected by weight loss. DCI and its messenger may contribute to insulin resistance in PCOS independent of obesity.

Hypoxia induces adipogenic differentitation of myoblastic cell lines

Energy Technology Data Exchange (ETDEWEB)

Itoigawa, Yoshiaki [Tohoku University School of Medicine, Sendai (Japan); Juntendo University School of Medicine, Tokyo (Japan); Kishimoto, Koshi N., E-mail: kishimoto@med.tohoku.ac.jp [Tohoku University School of Medicine, Sendai (Japan); Okuno, Hiroshi; Sano, Hirotaka [Tohoku University School of Medicine, Sendai (Japan); Kaneko, Kazuo [Juntendo University School of Medicine, Tokyo (Japan); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan)

2010-09-03

Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

Difference in membrane repair capacity between cancer cell lines and a normal cell line

DEFF Research Database (Denmark)

Frandsen, Stine Krog; McNeil, Anna K.; Novak, Ivana

2016-01-01

repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique...... cancer cell lines (p immortalized cell line (p

Nitric oxide-releasing nanoparticles: synthesis, characterization, and cytotoxicity to tumorigenic cells

Energy Technology Data Exchange (ETDEWEB)

Pelegrino, Milena T. [Universidade Federal de São Paulo, Exact and Earth Sciences Department (Brazil); Silva, Letícia C.; Watashi, Carolina M. [Universidade Federal do ABC, UFABC, Center of Natural and Human Sciences (Brazil); Haddad, Paula S. [Universidade Federal de São Paulo, Exact and Earth Sciences Department (Brazil); Rodrigues, Tiago; Seabra, Amedea B., E-mail: amedea.seabra@ufabc.edu.br [Universidade Federal do ABC, UFABC, Center of Natural and Human Sciences (Brazil)

2017-02-15

Nitric oxide (NO) is involved in several biological processes, including toxicity against tumor cells. The aim of this study was to synthesize, characterize, and evaluate the cytotoxicity of NO-releasing chitosan nanoparticles. A thiol-containing molecule, mercaptosuccinic acid (MSA), was encapsulated (encapsulation efficiency of 99%) in chitosan/sodium tripolyphosphate nanoparticles (CS NPs). The obtained nanoparticles showed an average hydrodynamic size of 108.40 ± 0.96 nm and polydispersity index of 0.26 ± 0.01. MSA-CS NPs were nitrosated leading to S-nitroso-MSA-CS NPs, which act as NO donor. The cytotoxicity of CS NPs, MSA-CS NPs, and S-nitroso-MSA-CS NPs were evaluated in several tumor cells, including human hepatocellular carcinoma (HepG2), mouse melanoma (B16F10), and human chronic myeloid leukemia (K562) cell lines and Lucena-1, a vincristine-resistant K562 cell line. Both CS NPs and MSA-CS NPs did not cause toxic effects in these cells, whereas S-nitroso-MSA-CS NPs caused potent cytotoxic effects in all the tested tumor cell lines. The half-maximal inhibitory concentration values of S-nitroso-MSA-CS NPs were 19.7, 10.5, 22.8, and 27.8 μg·mL{sup −1} for HepG2, B16F10, K562, and Lucena-1 cells, respectively. In contrast, S-nitroso-MSA-CS NPs exhibited lower cytotoxic to non-tumorigenic melanocytes (Melan-A) when compared with melanoma B16F10. Therefore, the results highlight the potential use of NO-releasing CS NPs in antitumor chemotherapy.

[Inhibitory effect of exogenous insulin-like growth factor binding protein 7 on proliferation of human breast cancer cell line MDA-MB-453 and its mechanism].

Science.gov (United States)

Yuan, Lei; Fan, Wen-Juan; Yang, Xu-Guang; Rao, Shu-Mei; Song, Jin-Ling; Song, Guo-Hua

2013-10-25

The present study was to investigate the effects of exogenous insulin-like growth factor binding protein 7 (IGFBP7) on the proliferation of human breast cancer cell line MDA-MB-453 and its possible mechanism. By means of MTT method in vitro, the results showed exogenous IGFBP7 inhibited the growth of MDA-MB-453 cells (IC50 of IGFBP7 = 8.49 μg/mL) in time- and concentration-dependent manner. SB203580, p38(MAPK) inhibitor, blocked the anti-proliferative effect of exogenous IGFBP7. The flow cytometry assay showed that exogenous IGFBP7 remarkably induced G0/G1 arrest in MDA-MB-453 cells. The Western blot showed that exogenous IGFBP7 promoted phosphorylation of p38(MAPK), up-regulated expression of p21(CIP1/WAF1), and inhibited phosphorylation of Rb. SB203580 restrained exogenous IGFBP7-induced regulation of p21(CIP1/WAF1) and p-Rb in MDA-MB-453 cells. In conclusion, the present study suggests that exogenous IGFBP7 could activate the p38(MAPK) signaling pathway, upregulate p21(CIP1/WAF1) expression, inhibit phosphorylation of Rb, and finally induce G0/G1 arrest in MDA-MB-453 cells.

Stimuli sensitive polymethacrylic acid microparticles (PMAA)--oral insulin delivery.

Science.gov (United States)

Victor, Sunita Prem; Sharma, Chandra P

2002-10-01

This study investigated polymethacrylic acid (PMAA) microparticles for controlled release of Insulin in oral administration. The microparticles were characterised by scanning electron microscopy (SEM) for morphological studies. The swelling behaviour and drug release profile in various pH media were studied. The % swelling of gels was found to be inversely related to the amount of crosslinker added. Inclusion complex of betaCD and Insulin was studied using polyacrylamide gel electrophoresis (PAGE). Optimum complexation was obtained in the ratio 100 mg betaCD: 200 IU Insulin. The release pattern of Insulin from Insulin-betaCD complex encapsulated PMAA microparticles showed release of Insulin for more than seven hours.

Combined contributions of over-secreted glucagon-like peptide 1 and suppressed insulin secretion to hyperglycemia induced by gatifloxacin in rats

Energy Technology Data Exchange (ETDEWEB)

Yu, Yunli, E-mail: chrisyu1255@yahoo.com.cn [Department of Pharmaceutics, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009 (China); Wang, Xinting, E-mail: wxinting1986@yahoo.com.cn [Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009 (China); Liu, Can, E-mail: ltsan@163.com [Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009 (China); Yao, Dan, E-mail: erinyao@126.com [Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009 (China); Shanghai Institute of Materia Medica, Shanghai 201203 (China); Hu, Mengyue, E-mail: juliahmy@126.com [Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009 (China); Li, Jia, E-mail: ljbzd@163.com [Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009 (China); Hu, Nan, E-mail: hn_324@163.com [Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009 (China); Liu, Li, E-mail: liulee@cpu.edu.cn [Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009 (China); Liu, Xiaodong, E-mail: xdliu@cpu.edu.cn [Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009 (China)

2013-02-01

Accumulating evidences have showed that gatifloxacin causes dysglycemia in both diabetic and non-diabetic patients. Our preliminary study demonstrated that gatifloxacin stimulated glucagon-like peptide 1 (GLP-1) secretion from intestinal cells. The aim of the study was to investigate the association between gatifloxacin-stimulated GLP-1 release and dysglycemia in both normal and streptozotocin-induced diabetic rats and explore the possible mechanisms. Oral administration of gatifloxacin (100 mg/kg/day and 200 mg/kg/day) for 3 and 12 days led to marked elevation of GLP-1 levels, accompanied by significant decrease in insulin levels and increase in plasma glucose. Similar results were found in normal rats treated with 3-day gatifloxacin. Gatifloxacin-stimulated GLP-1 release was further confirmed in NCI-H716 cells, which was abolished by diazoxide, a K{sub ATP} channel opener. QT-PCR analysis showed that gatifloxacin also upregulated expression of proglucagon and prohormone convertase 3 mRNA. To clarify the contradiction on elevated GLP-1 without insulinotropic effect, effects of GLP-1 and gatifloxacin on insulin release were investigated using INS-1 cells. We found that short exposure (2 h) to GLP-1 stimulated insulin secretion and biosynthesis, whereas long exposure (24 h and 48 h) to high level of GLP-1 inhibited insulin secretion and biosynthesis. Moreover, we also confirmed gatifloxacin acutely stimulated insulin secretion while chronically inhibited insulin biosynthesis. All the results gave an inference that gatifloxacin stimulated over-secretion of GLP-1, in turn, high levels of GLP-1 and gatifloxacin synergistically impaired insulin release, worsening hyperglycemia. -- Highlights: ► Gatifloxacin induced hyperglycemia both in diabetic rats and normal rats. ► Gatifloxacin enhanced GLP-1 secretion but inhibited insulin secretion in rats. ► Long-term exposure to high GLP-1 inhibited insulin secretion and biosynthesis. ► GLP-1 over-secretion may be