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�

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.

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

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Mohamad Hafizi Abu Bakar

2015-05-01

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

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

DEFF Research Database (Denmark)

Bouzakri, Karim; Plomgaard, Peter; Berney, Thierry

2011-01-01

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

Insulin release by glucagon and secretin

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Kofod, Hans; Andreu, D; Thams, P

1988-01-01

Secretin and glucagon potentiate glucose-induced insulin release. We have compared the effects of secretin and glucagon with that of four hybrid molecules of the two hormones on insulin release and formation of cyclic AMP (cAMP) in isolated mouse pancreatic islets. All six peptides potentiated...... the release of insulin at 10 mM D-glucose, and their effects were indistinguishable with respect to the dynamics of release, dose-response relationship, and glucose dependency. However, measurements of cAMP accumulation in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (10(-4) M...... potentiating effects of secretin and glucagon on glucose-induced insulin release, their modes of action may be different....

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

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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.

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

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

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

2012-01-01

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

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

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Irit Meivar-Levy

2011-01-01

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

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.

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

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

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Mohamed I Husseiny

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

Induction of insulin secretion in engineered liver cells by nitric oxide

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Özcan Sabire

2007-10-01

Full Text Available Abstract Background Type 1 Diabetes Mellitus results from an autoimmune destruction of the pancreatic beta cells, which produce insulin. The lack of insulin leads to chronic hyperglycemia and secondary complications, such as cardiovascular disease. The currently approved clinical treatments for diabetes mellitus often fail to achieve sustained and optimal glycemic control. Therefore, there is a great interest in the development of surrogate beta cells as a treatment for type 1 diabetes. Normally, pancreatic beta cells produce and secrete insulin only in response to increased blood glucose levels. However in many cases, insulin secretion from non-beta cells engineered to produce insulin occurs in a glucose-independent manner. In the present study we engineered liver cells to produce and secrete insulin and insulin secretion can be stimulated via the nitric oxide pathway. Results Expression of either human insulin or the beta cell specific transcription factors PDX-1, NeuroD1 and MafA in the Hepa1-6 cell line or primary liver cells via adenoviral gene transfer, results in production and secretion of insulin. Although, the secretion of insulin is not significantly increased in response to high glucose, treatment of these engineered liver cells with L-arginine stimulates insulin secretion up to three-fold. This L-arginine-mediated insulin release is dependent on the production of nitric oxide. Conclusion Liver cells can be engineered to produce insulin and insulin secretion can be induced by treatment with L-arginine via the production of nitric oxide.

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

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

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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.

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

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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.

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

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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.

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

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

2005-05-01

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

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.

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

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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.

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

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Darwin V Lee

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

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.

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...

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.

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.

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

International Nuclear Information System (INIS)

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

1988-01-01

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

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)

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

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

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...

Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells

International Nuclear Information System (INIS)

Timper, Katharina; Seboek, Dalma; Eberhardt, Michael; Linscheid, Philippe; Christ-Crain, Mirjam; Keller, Ulrich; Mueller, Beat; Zulewski, Henryk

2006-01-01

Mesenchymal stem cells (MSC) from mouse bone marrow were shown to adopt a pancreatic endocrine phenotype in vitro and to reverse diabetes in an animal model. MSC from human bone marrow and adipose tissue represent very similar cell populations with comparable phenotypes. Adipose tissue is abundant and easily accessible and could thus also harbor cells with the potential to differentiate in insulin producing cells. We isolated human adipose tissue-derived MSC from four healthy donors. During the proliferation period, the cells expressed the stem cell markers nestin, ABCG2, SCF, Thy-1 as well as the pancreatic endocrine transcription factor Isl-1. The cells were induced to differentiate into a pancreatic endocrine phenotype by defined culture conditions within 3 days. Using quantitative PCR a down-regulation of ABCG2 and up-regulation of pancreatic developmental transcription factors Isl-1, Ipf-1, and Ngn3 were observed together with induction of the islet hormones insulin, glucagon, and somatostatin

Protein Crystal Recombinant Human Insulin

Science.gov (United States)

1994-01-01

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

Expression of an insulin/interleukin-1 receptor antagonist hybrid gene in insulin-producing cell lines (HIT-T15 and NIT-1) confers resistance against interleukin-1-induced nitric oxide production.

Science.gov (United States)

Welsh, N; Bendtzen, K; Welsh, M

1995-01-01

A hybrid gene consisting of the insulin gene enhancer/promoter region, the signal sequence, the insulin B- and C-chains, and the human interleukin-1 receptor antagonist (IL-1ra) gene was constructed. This hybrid gene was transfected together with the pSV2-neo construct into the insulin-producing cell lines HIT-T15 and NIT-1. One of the geneticin-selected clones, HITra2, expressed a 1.4-kb mRNA, which hybridized both to insulin and IL-1ra-cDNA in Northern blot analysis. Three proteins, with the mol wt 23, 17, and 14 kD, were immunoprecipitated with anti-IL-1ra antibodies from [35S]methionine-labeled HITra2 cells. Both at a low and at a high glucose concentration, 4-5 ng of IL-1ra/10(6) cells (ELISA) was released from these cells. On the other hand, a high glucose concentration evoked a three-fold increase in the release of insulin, suggesting that IL-1ra was released constitutively. Measured by nitrite production, transfected HIT, and NIT-1 cells exhibited a more than 10-fold decrease in IL-1 beta sensitivity. Since the conditioned culture media from the HITra2 cells exhibited an anti-IL-1 beta activity of only 0.5 U/ml, and mixed culture of HITra2 cells and isolated rat islets prevented IL-1 beta induced inhibition of insulin release, it is likely that IL-1ra acts locally at the cell surface. It is concluded that expression of a hybrid insulin/IL-1ra gene confers resistance to IL-1 and that this technique may be used to elucidate the role of IL-1 in autoimmune disorders such as insulin-dependent diabetes mellitus. Images PMID:7706480

Differentiation of PDX1 gene-modified human umbilical cord mesenchymal stem cells into insulin-producing cells in vitro.

Science.gov (United States)

He, Dongmei; Wang, Juan; Gao, Yangjun; Zhang, Yuan

2011-12-01

Mesenchymal stem cells (MSCs) have significant advantages over other stem cell types, and greater potential for immediate clinical application. MSCs would be an interesting cellular source for treatment of type 1 diabetes. In this study, MSCs from human umbilical cord were differentiated into functional insulin-producing cells in vitro by introduction of the pancreatic and duodenal homeobox factor 1 (PDX1) and in the presence of induction factors. The expressions of cell surface antigens were detected by flow cytometry. After induction in an adipogenic medium or an osteogenic medium, the cells were observed by Oil Red O staining and alkaline phosphatase staining. Recombinant adenovirus carrying the PDX1 gene was constructed and MSCs were infected by the recombinant adenovirus, then treated with several inducing factors for differentiation into islet β-like cells. The expression of the genes and protein related to islet β-cells was detected by immunocytochemistry, RT-PCR and Western blot analysis. Insulin and C-peptide secretion were assayed. Our results show that the morphology and immunophenotype of MSCs from human umbilical cord were similar to those present in human bone marrow. The MSCs could be induced to differentiate into osteocytes and adipocytes. After induction by recombined adenovirus vector with induction factors, MSCs were aggregated and presented islet-like bodies. Dithizone staining of these cells was positive. The genes' expression related to islet β-cells was found. After induction, insulin and C-peptide secretion in the supernatant were significantly increased. In conclusion, our results demonstrated that PDX1 gene-modified human umbilical cord mesenchymal stem cells could be differentiated into insulin-producing cells in vitro.

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.

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

Science.gov (United States)

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.

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

International Nuclear Information System (INIS)

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

2005-01-01

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

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

Science.gov (United States)

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

2014-01-01

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

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.

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

Directory of Open Access Journals (Sweden)

Xiaoya Sun

2017-11-01

Full Text Available Abstract Background Insulin resistance is one of the most common and important pathological features of type 2 diabetes (T2D. Recently, insulin resistance is increasingly considered to be associated with systemic chronic inflammation. Elevated levels of tumor necrosis factor (TNF-α and interleukin (IL-1β in blood are predictive indicators of the development of T2D. Mesenchymal stem cell (MSC-based therapies have been proven to have potential immunomodulation and anti-inflammatory properties through their paracrine effects; however, the mechanism for the anti-inflammatory effect of MSCs in enhancing insulin sensitivity is still uncertain. Methods In the present experiment, we used HepG2 cells, a human hepatoma cell line, and a MSC-HepG2 transwell culturing system to investigate the anti-inflammatory mechanism of human umbilical cord-derived MSCs (UC-MSCs under palmitic acid (PA and lipopolysaccharide (LPS-induced insulin resistance in vitro. Insulin resistance was confirmed by glycogen assay kit and glucose assay kit. Inflammatory factor release was detected by ELISA, gene expression was tested by quantitative real-time PCR, and insulin signaling activation was determined by western blotting analysis. The changes of inflammatory factors and insulin signaling protein were also tested in T2D rats injected with UC-MSCs. Results Treating HepG2 cells with PA–LPS caused NLRP3 inflammation activation, including overexpression of NLRP3 and caspase-1, and overproduction of IL-1β and IL-18 as well as TNF-α from HepG2 cells. The elevated levels of these inflammatory cytokines impaired insulin receptor action and thereby prevented downstream signaling pathways, exacerbating insulin resistance in HepG2 cells. Importantly, UC-MSCs cocultured with HepG2 could effectively alleviate PA and LPS-induced insulin resistance by blocking the NLRP3 inflammasome activation and inflammatory agents. Furthermore, knockdown of NLRP3 or IL-1β partially improved PA and

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

Science.gov (United States)

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

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

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Maria L. Mizgier

2017-01-01

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

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

Differential responsiveness of luteinized human granulosa cells to gonadotropins and insulin-like growth factor I for induction of aromatase activity

International Nuclear Information System (INIS)

Christman, G.M.; Randolph, J.F. Jr.; Peegel, H.; Menon, K.M.

1991-01-01

The objective of this study was to examine the in vitro responsiveness of cultured luteinized human granulosa cells over time to insulin-like growth factor 1 (IGF-1), human follicle-stimulating hormone (FSH), and human chorionic gonadotropin (hCG) for the induction of aromatase activity. Granulosa cells were retrieved from preovulatory follicles in patients undergoing in vitro fertilization. Cells were cultured for a period of 72 hours or 10 days. The ability of hCG, human FSH, and/or IGF-I to induce aromatase activity was assayed by the stereospecific release of tritium from [1B-3H]androstenedione. Short-term cultures (72 hours) demonstrated a marked rise in aromatase activity in response to human FSH and IGF-I, whereas a smaller response to hCG was observed. In contrast, 10-day cultures demonstrated responsiveness predominantly to hCG rather than human FSH for the induction of aromatase activity with no remarkable effect of IGF-I. Luteinized human granulosa cells undergo a transformation from an initial human FSH and IGF-I responsive state to an hCG responsive state in long-term cultures

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

Differentiation of Human Mesenchymal Stem Cells into Insulin Producing Cells by Using A Lentiviral Vector Carrying PDX1.

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Allahverdi, Amir; Abroun, Saied; Jafarian, Arefeh; Soleimani, Masoud; Taghikhani, Mohammad; Eskandari, Fatemeh

2015-01-01

Type I diabetes is an immunologically-mediated devastation of insulin producing cells (IPCs) in the pancreatic islet. Stem cells that produce β-cells are a new promising tool. Adult stem cells such as mesenchymal stem cells (MSCs) are self renewing multi potent cells showing capabilities to differentiate into ectodermal, mesodermal and endodermal tissues. Pancreatic and duodenal homeobox factor 1 (PDX1) is a master regulator gene required for embryonic development of the pancreas and is crucial for normal pancreatic islets activities in adults. We induced the over-expression of the PDX1 gene in human bone marrow MSCs (BM-MSCs) by Lenti-PDX1 in order to generate IPCs. Next, we examine the ability of the cells by measuring insulin/c-peptide production and INSULIN and PDX1 gene expressions. After transduction, MSCs changed their morphology at day 5 and gradually differentiated into IPCs. INSULIN and PDX1 expressions were confirmed by real time polymerase chain reaction (RT-PCR) and immunostaining. IPC secreted insulin and C-peptide in the media that contained different glucose concentrations. MSCs differentiated into IPCs by genetic manipulation. Our result showed that lentiviral vectors could deliver PDX1 gene to MSCs and induce pancreatic differentiation.

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

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Binhai Ren

2016-04-01

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

High oxygen condition facilitates the differentiation of mouse and human pluripotent stem cells into pancreatic progenitors and insulin-producing cells.

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Hakim, Farzana; Kaitsuka, Taku; Raeed, Jamiruddin Mohd; Wei, Fan-Yan; Shiraki, Nobuaki; Akagi, Tadayuki; Yokota, Takashi; Kume, Shoen; Tomizawa, Kazuhito

2014-04-04

Pluripotent stem cells have potential applications in regenerative medicine for diabetes. Differentiation of stem cells into insulin-producing cells has been achieved using various protocols. However, both the efficiency of the method and potency of differentiated cells are insufficient. Oxygen tension, the partial pressure of oxygen, has been shown to regulate the embryonic development of several organs, including pancreatic β-cells. In this study, we tried to establish an effective method for the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells by culturing under high oxygen (O2) conditions. Treatment with a high O2 condition in the early stage of differentiation increased insulin-positive cells at the terminus of differentiation. We found that a high O2 condition repressed Notch-dependent gene Hes1 expression and increased Ngn3 expression at the stage of pancreatic progenitors. This effect was caused by inhibition of hypoxia-inducible factor-1α protein level. Moreover, a high O2 condition activated Wnt signaling. Optimal stage-specific treatment with a high O2 condition resulted in a significant increase in insulin production in both mouse embryonic stem cells and human iPSCs and yielded populations containing up to 10% C-peptide-positive cells in human iPSCs. These results suggest that culturing in a high O2 condition at a specific stage is useful for the efficient generation of insulin-producing cells.

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

Insulin-Producing Cells Differentiated from Human Bone Marrow Mesenchymal Stem Cells In Vitro Ameliorate Streptozotocin-Induced Diabetic Hyperglycemia.

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Ying Xin

Full Text Available The two major obstacles in the successful transplantation of islets for diabetes treatment are inadequate supply of insulin-producing tissue and immune rejection. Induction of the differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs into insulin-producing cells (IPCs for autologous transplantation may alleviate those limitations.hMSCs were isolated and induced to differentiate into IPCs through a three-stage differentiation protocol in a defined media with high glucose, nicotinamide, and exendin-4. The physiological characteristics and functions of IPCs were then evaluated. Next, about 3 × 10(6 differentiated cells were transplanted into the renal sub-capsular space of streptozotocin (STZ-induced diabetic nude mice. Graft survival and function were assessed by immunohistochemistry, TUNEL staining and measurements of blood glucose levels in the mice.The differentiated IPCs were characterized by Dithizone (DTZ positive staining, expression of pancreatic β-cell markers, and human insulin secretion in response to glucose stimulation. Moreover, 43% of the IPCs showed L-type Ca2+ channel activity and similar changes in intracellular Ca2+ in response to glucose stimulation as that seen in pancreatic β-cells in the process of glucose-stimulated insulin secretion. Transplantation of functional IPCs into the renal subcapsular space of STZ-induced diabetic nude mice ameliorated the hyperglycemia. Immunofluorescence staining revealed that transplanted IPCs sustainably expressed insulin, c-peptide, and PDX-1 without apparent apoptosis in vivo.IPCs derived from hMSCs in vitro can ameliorate STZ-induced diabetic hyperglycemia, which indicates that these hMSCs may be a promising approach to overcome the limitations of islet transplantation.

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

Energy Technology Data Exchange (ETDEWEB)

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-producing Cells from Adult Human Bone Marrow Mesenchymal Stromal Cells Could Control Chemically Induced Diabetes in Dogs: A Preliminary Study.

Science.gov (United States)

Gabr, Mahmoud M; Zakaria, Mahmoud M; Refaie, Ayman F; Ismail, Amani M; Khater, Sherry M; Ashamallah, Sylvia A; Azzam, Maha M; Ghoneim, Mohamed A

2018-01-01

Ten mongrel dogs were used in this study. Diabetes was chemically induced in 7 dogs, and 3 dogs served as normal controls. For each diabetic dog, 5 million human bone marrow-derived mesenchymal stem cells/kg were differentiated to form insulin-producing cells using a trichostatin-based protocol. Cells were then loaded in 2 TheraCyte capsules which were transplanted under the rectus sheath. One dog died 4 d postoperatively from pneumonia. Six dogs were followed up with for 6 to 18 mo. Euglycemia was achieved in 4 dogs. Their glucose tolerance curves exhibited a normal pattern demonstrating that the encapsulated cells were glucose sensitive and insulin responsive. In the remaining 2 dogs, the fasting blood sugar levels were reduced but did not reach normal values. The sera of all transplanted dogs contained human insulin and C-peptide with a negligible amount of canine insulin. Removal of the transplanted capsules was followed by prompt return of diabetes. Intracytoplasmic insulin granules were seen by immunofluorescence in cells from the harvested capsules. Furthermore, all pancreatic endocrine genes were expressed. This study demonstrated that the TheraCyte capsule or a similar device can provide adequate immunoisolation, an important issue when stem cells are considered for the treatment of type 1 diabetes mellitus.

Differentiation of human multipotent dermal fibroblasts into islet-like cell clusters

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Liu Wei

2010-06-01

Full Text Available Abstract Background We have previously obtained a clonal population of cells from human foreskin that is able to differentiate into mesodermal, ectodermal and endodermal progenies. It is of great interest to know whether these cells could be further differentiated into functional insulin-producing cells. Results Sixty-one single-cell-derived dermal fibroblast clones were established from human foreskin by limiting dilution culture. Of these, two clones could be differentiated into neuron-, adipocyte- or hepatocyte-like cells under certain culture conditions. In addition, those two clones were able to differentiate into islet-like clusters under pancreatic induction. Insulin, glucagon and somatostatin were detectable at the mRNA and protein levels after induction. Moreover, the islet-like clusters could release insulin in response to glucose in vitro. Conclusions This is the first study to demonstrate that dermal fibroblasts can differentiate into insulin-producing cells without genetic manipulation. This may offer a safer cell source for future stem cell-based therapies.

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.

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.

Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.

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D'Amour, Kevin A; Bang, Anne G; Eliazer, Susan; Kelly, Olivia G; Agulnick, Alan D; Smart, Nora G; Moorman, Mark A; Kroon, Evert; Carpenter, Melissa K; Baetge, Emmanuel E

2006-11-01

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor--en route to cells that express endocrine hormones. The hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal beta-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell-derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.

Internalization and localization of basal insulin peglispro in cells.

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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

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

DEFF Research Database (Denmark)

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

2007-01-01

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

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats.

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Matveyenko, Aleksey V; Georgia, Senta; Bhushan, Anil; Butler, Peter C

2010-11-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in development of viable glucose-responsive pancreatic endocrine tissue. Athymic nude rats were implanted with PE derived from hESC either via implantation into the epididymal fat pads or by subcutaneous implantation into TheraCyte encapsulation devices for 20 wk. Blood glucose, weight, and human insulin/C-peptide secretion were monitored by weekly blood draws. Graft β-cell function was assessed by a glucose tolerance test, and graft morphology was assessed by immunohistochemistry and immunofluorescence. At 20 wk postimplantation, epididymal fat-implanted PE progressed to develop islet-like structures in 50% of implants, with a mean β-cell fractional area of 0.8 ± 0.3%. Human C-peptide and insulin were detectable, but at very low levels (C-peptide = 50 ± 26 pmol/l and insulin = 15 ± 7 pmol/l); however, there was no increase in human C-peptide/insulin levels after glucose challenge. There was no development of viable pancreatic tissue or meaningful secretory function when human PE was implanted in the TheraCyte encapsulation devices. These data confirm that islet-like structures develop from hESC differentiated to PE by the protocol developed by NovoCell. However, the extent of endocrine cell formation and secretory function is not yet sufficient to be clinically relevant.

Characterization of stimulus-secretion coupling in the human pancreatic EndoC-βH1 beta cell line.

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Lotta E Andersson

Full Text Available Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay, gene expression (Gene Chip array, metabolite levels (GC/MS, respiration (Seahorse XF24 Extracellular Flux Analyzer, glucose utilization (radiometric, lactate release (enzymatic colorimetric, ATP levels (enzymatic bioluminescence and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the

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

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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.

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.

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

DEFF Research Database (Denmark)

Øzbay, Aygen; Møller, Niels; Juhl, Claus

2012-01-01

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

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

International Nuclear Information System (INIS)

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

1983-01-01

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

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

Dual effect of insulin resistance and cadmium on human granulosa cells - In vitro study

Energy Technology Data Exchange (ETDEWEB)

Belani, Muskaan, E-mail: muskaanbelani@gmail.com [Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390 002, Gujarat, India. (India); Shah, Preeti, E-mail: preeti.shah@novaivifertility.com [Nova IVI Fertility, Behind Xavier' s Ladies Hostel, 108, Swastik Society Rd., Navrangpura, Ahmedabad 390009, Gujarat, India. (India); Banker, Manish, E-mail: manish.banker@novaivifertility.com [Nova IVI Fertility, Behind Xavier' s Ladies Hostel, 108, Swastik Society Rd., Navrangpura, Ahmedabad 390009, Gujarat, India. (India); Gupta, Sarita, E-mail: saritagupta9@gmail.com [Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390 002, Gujarat, India. (India)

2016-12-15

Combined exposure of cadmium (Cd) and insulin resistance (IR) might be responsible for subfertility. In the present study, we investigated the effects of Cd in vitro in IR human granulosa cells. Isolated human granulosa cells from control and polycystic ovary syndrome (PCOS) follicular fluid samples were confirmed for IR by decrease in protein expression of insulin receptor-β. Control and IR human granulosa cells were then incubated with or without 32 μM Cd. The combined effect of IR with 32 μM Cd in granulosa cells demonstrated significant decrease in expression of StAR, CYP11A1, CYP19A1, 17β-HSD, 3β-HSD, FSH-R and LH-R. Decrease was also observed in progesterone and estradiol concentrations as compared to control. Additionally, increase in protein expression of cleaved PARP-F2, active caspase-3 and a positive staining for Annexin V and PI indicated apoptosis as the mode of increased cell death ultimately leading to decreased steroidogenesis, as observed through the combined exposure. Taken together the results suggest decrease in steroidogenesis ultimately leading to abnormal development of the follicle thus compromising fertility at the level of preconception. - Highlights: • Protein expression of INSR-β in granulosa cells to differentiate PCOS-IR and NIR • Cd and IR together decrease steroidogenesis in human granulosa cells in vitro. • Cd and IR increase human granulosa cell death by increase in apoptosis. • Environment and life style are set to hamper pregnancies at preconception level.

Dual effect of insulin resistance and cadmium on human granulosa cells - In vitro study

International Nuclear Information System (INIS)

Belani, Muskaan; Shah, Preeti; Banker, Manish; Gupta, Sarita

2016-01-01

Combined exposure of cadmium (Cd) and insulin resistance (IR) might be responsible for subfertility. In the present study, we investigated the effects of Cd in vitro in IR human granulosa cells. Isolated human granulosa cells from control and polycystic ovary syndrome (PCOS) follicular fluid samples were confirmed for IR by decrease in protein expression of insulin receptor-β. Control and IR human granulosa cells were then incubated with or without 32 μM Cd. The combined effect of IR with 32 μM Cd in granulosa cells demonstrated significant decrease in expression of StAR, CYP11A1, CYP19A1, 17β-HSD, 3β-HSD, FSH-R and LH-R. Decrease was also observed in progesterone and estradiol concentrations as compared to control. Additionally, increase in protein expression of cleaved PARP-F2, active caspase-3 and a positive staining for Annexin V and PI indicated apoptosis as the mode of increased cell death ultimately leading to decreased steroidogenesis, as observed through the combined exposure. Taken together the results suggest decrease in steroidogenesis ultimately leading to abnormal development of the follicle thus compromising fertility at the level of preconception. - Highlights: • Protein expression of INSR-β in granulosa cells to differentiate PCOS-IR and NIR • Cd and IR together decrease steroidogenesis in human granulosa cells in vitro. • Cd and IR increase human granulosa cell death by increase in apoptosis. • Environment and life style are set to hamper pregnancies at preconception level.

Insulin-Producing Endocrine Cells Differentiated In Vitro From Human Embryonic Stem Cells Function in Macroencapsulation Devices In Vivo.

Science.gov (United States)

Agulnick, Alan D; Ambruzs, Dana M; Moorman, Mark A; Bhoumik, Anindita; Cesario, Rosemary M; Payne, Janice K; Kelly, Jonathan R; Haakmeester, Carl; Srijemac, Robert; Wilson, Alistair Z; Kerr, Justin; Frazier, Mauro A; Kroon, Evert J; D'Amour, Kevin A

2015-10-01

The PEC-01 cell population, differentiated from human embryonic stem cells (hESCs), contains pancreatic progenitors (PPs) that, when loaded into macroencapsulation devices (to produce the VC-01 candidate product) and transplanted into mice, can mature into glucose-responsive insulin-secreting cells and other pancreatic endocrine cells involved in glucose metabolism. We modified the protocol for making PEC-01 cells such that 73%-80% of the cell population consisted of PDX1-positive (PDX1+) and NKX6.1+ PPs. The PPs were further differentiated to islet-like cells (ICs) that reproducibly contained 73%-89% endocrine cells, of which approximately 40%-50% expressed insulin. A large fraction of these insulin-positive cells were single hormone-positive and expressed the transcription factors PDX1 and NKX6.1. To preclude a significant contribution of progenitors to the in vivo function of ICs, we used a simple enrichment process to remove remaining PPs, yielding aggregates that contained 93%-98% endocrine cells and 1%-3% progenitors. Enriched ICs, when encapsulated and implanted into mice, functioned similarly to the VC-01 candidate product, demonstrating conclusively that in vitro-produced hESC-derived insulin-producing cells can mature and function in vivo in devices. A scaled version of our suspension culture was used, and the endocrine aggregates could be cryopreserved and retain functionality. Although ICs expressed multiple important β cell genes, the cells contained relatively low levels of several maturity-associated markers. Correlating with this, the time to function of ICs was similar to PEC-01 cells, indicating that ICs required cell-autonomous maturation after delivery in vivo, which would occur concurrently with graft integration into the host. Type 1 diabetes (T1D) affects approximately 1.25 million people in the U.S. alone and is deadly if not managed with insulin injections. This paper describes the production of insulin-producing cells in vitro and a new

Characterization of Microvesicles Released from Human Red Blood Cells

Directory of Open Access Journals (Sweden)

Duc Bach Nguyen

2016-03-01

Full Text Available Background/Aims: Extracellular vesicles (EVs are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. Methods: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS, scanning electron microscopy (SEM, atomic force microscopy (AFM and dynamic light scattering (DLS. The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Results: Treatment of RBCs with 4-bromo-A23187 (positive control, lysophosphatidic acid (LPA, or phorbol-12 myristate-13 acetate (PMA in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 m

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.

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

Science.gov (United States)

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

2003-12-15

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

An ibuprofen-antagonized plasmin inhibitor released by human endothelial cells.

Science.gov (United States)

Rockwell, W B; Ehrlich, H P

1991-02-01

Serum-free culture medium harvested from endothelial cell monolayer cultures derived from human scars and dermis was examined for inhibition of fibrinolysis using a fibrin plate assay. Human cultured fibroblasts and smooth muscle cells did not produce any detectable inhibitory activity. The inhibitor is spontaneously released from the cultured endothelial cells over time. In the fibrin plate assay of plasmin-induced fibrinolysis, one nonsteroidal antiinflammatory (NSAI) drug, ibuprofen, was demonstrated to antagonize the inhibition of fibrinolysis. The antagonistic activity of ibuprofen appears unrelated to its NSAI drug activity because other NSAI drugs such as indomethacin and tolmetin have minimal antagonistic activity. Heating the cultured endothelial cells to 42 degrees C stimulates greater release of the inhibitor in a shorter period of time. This plasmin inhibitor, which is produced by endothelial cells, may contribute to postburn vascular occlusion, leading to secondary progressive necrosis in burn-traumatized patients.

[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.

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

Science.gov (United States)

Wubetu, Gizachew Yismaw; Utsunomiya, Tohru; Ishikawa, Daichi; Ikemoto, Tetsuya; Yamada, Shinichiro; Morine, Yuji; Iwahashi, Shuichi; Saito, Yu; Arakawa, Yusuke; Imura, Satoru; Arimochi, Hideki; Shimada, Mitsuo

2014-09-01

Branched chain amino acid (BCAA) dietary supplementation inhibits activation of the insulin-like growth factor (IGF)/IGF-I receptor (IGF-IR) axis in diabetic animal models. However, the in vitro effect of BCAA on human cancer cell lines under hyper-insulinemic conditions remains unclear. Colon (HCT-116) and hepatic (HepG2) tumor cells were treated with varying concentrations of BCAA with or without fluorouracil (5-FU). The effect of BCAA on insulin-initiated proliferation was determined. Gene and protein expression was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. BCAA supplementation had no significant effect on cell proliferation and did not show significant synergistic or antagonistic effects with 5-FU. However, BCAA significantly decreased insulin-initiated proliferation of human colon and hepatic cancer cell lines in vitro. BCAA supplementation caused a marked decrease in activated IGF-IR expression and significantly enhanced both mRNA and protein expression of LC3-II and BECN1 (BECLIN-1). BCAA could be a useful chemopreventive modality for cancer in hyperinsulinemic conditions. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

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.

Acetylcholine release by human colon cancer cells mediates autocrine stimulation of cell proliferation.

Science.gov (United States)

Cheng, Kunrong; Samimi, Roxana; Xie, Guofeng; Shant, Jasleen; Drachenberg, Cinthia; Wade, Mark; Davis, Richard J; Nomikos, George; Raufman, Jean-Pierre

2008-09-01

Most colon cancers overexpress M3 muscarinic receptors (M3R), and post-M3R signaling stimulates human colon cancer cell proliferation. Acetylcholine (ACh), a muscarinic receptor ligand traditionally regarded as a neurotransmitter, may be produced by nonneuronal cells. We hypothesized that ACh release by human colon cancer cells results in autocrine stimulation of proliferation. H508 human colon cancer cells, which have robust M3R expression, were used to examine effects of muscarinic receptor antagonists, acetylcholinesterase inhibitors, and choline transport inhibitors on cell proliferation. A nonselective muscarinic receptor antagonist (atropine), a selective M3R antagonist (p-fluorohexahydro-sila-difenidol hydrochloride), and a choline transport inhibitor (hemicholinum-3) all inhibited unstimulated H508 colon cancer cell proliferation by approximately 40% (P<0.005). In contrast, two acetylcholinesterase inhibitors (eserine-hemisulfate and bis-9-amino-1,2,3,4-tetrahydroacridine) increased proliferation by 2.5- and 2-fold, respectively (P<0.005). By using quantitative real-time PCR, expression of choline acetyltransferase (ChAT), a critical enzyme for ACh synthesis, was identified in H508, WiDr, and Caco-2 colon cancer cells. By using high-performance liquid chromatography-electrochemical detection, released ACh was detected in H508 and Caco-2 cell culture media. Immunohistochemistry in surgical specimens revealed weak or no cytoplasmic staining for ChAT in normal colon enterocytes (n=25) whereas half of colon cancer specimens (n=24) exhibited moderate to strong staining (P<0.005). We conclude that ACh is an autocrine growth factor in colon cancer. Mechanisms that regulate colon epithelial cell production and release of ACh warrant further investigation.

In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

International Nuclear Information System (INIS)

Rowe, Alexander M.; Brundage, Kathleen M.; Barnett, John B.

2007-01-01

The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells

Human interleukin for DA cells or leukemia inhibitory factor is released by Vero cells in human embryo coculture.

Science.gov (United States)

Papaxanthos-Roche, A; Taupin, J L; Mayer, G; Daniel, J Y; Moreau, J F

1994-09-01

In the light of the newly discovered implications of human interleukin for DA cells and leukemia inhibitory factor in embryology, we searched for the presence of this soluble cytokine in the supernatant of Vero cell coculture systems. Using a bioassay as well as a specific ELISA, we demonstrated that Vero cells are able to release large quantities of human interleukin for DA cells and leukemia inhibitory factor in the embryo-growing medium of such cocultures.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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

2011-06-01

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

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.

Derivation of Insulin Producing Cells From Human Endometrial Stromal Stem Cells and Use in the Treatment of Murine Diabetes

OpenAIRE

Santamaria, Xavier; Massasa, Efi E; Feng, Yuzhe; Wolff, Erin; Taylor, Hugh S

2011-01-01

Pancreatic islet cell transplantation is an effective approach to treat type 1 diabetes, however the shortage of cadaveric donors and limitations due to rejection require alternative solutions. Multipotent cells derived from the uterine endometrium have the ability to differentiate into mesodermal and ectodermal cellular lineages, suggesting the existence of mesenchymal stem cells in this tissue. We differentiated human endometrial stromal stem cells (ESSC) into insulin secreting cells using ...

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+

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

Science.gov (United States)

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

1991-11-01

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

Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo.

Science.gov (United States)

Rezania, Alireza; Bruin, Jennifer E; Xu, Jean; Narayan, Kavitha; Fox, Jessica K; O'Neil, John J; Kieffer, Timothy J

2013-11-01

Human embryonic stem cells (hESCs) are considered a potential alternative to cadaveric islets as a source of transplantable cells for treating patients with diabetes. We previously described a differentiation protocol to generate pancreatic progenitor cells from hESCs, composed of mainly pancreatic endoderm (PDX1/NKX6.1-positive), endocrine precursors (NKX2.2/synaptophysin-positive, hormone/NKX6.1-negative), and polyhormonal cells (insulin/glucagon-positive, NKX6.1-negative). However, the relative contributions of NKX6.1-negative versus NKX6.1-positive cell fractions to the maturation of functional β-cells remained unclear. To address this question, we generated two distinct pancreatic progenitor cell populations using modified differentiation protocols. Prior to transplant, both populations contained a high proportion of PDX1-expressing cells (~85%-90%) but were distinguished by their relatively high (~80%) or low (~25%) expression of NKX6.1. NKX6.1-high and NKX6.1-low progenitor populations were transplanted subcutaneously within macroencapsulation devices into diabetic mice. Mice transplanted with NKX6.1-low cells remained hyperglycemic throughout the 5-month post-transplant period whereas diabetes was reversed in NKX6.1-high recipients within 3 months. Fasting human C-peptide levels were similar between groups throughout the study, but only NKX6.1-high grafts displayed robust meal-, glucose- and arginine-responsive insulin secretion as early as 3 months post-transplant. NKX6.1-low recipients displayed elevated fasting glucagon levels. Theracyte devices from both groups contained almost exclusively pancreatic endocrine tissue, but NKX6.1-high grafts contained a greater proportion of insulin-positive and somatostatin-positive cells, whereas NKX6.1-low grafts contained mainly glucagon-expressing cells. Insulin-positive cells in NKX6.1-high, but not NKX6.1-low grafts expressed nuclear MAFA. Collectively, this study demonstrates that a pancreatic endoderm

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)

Mesenchymal stromal cells improve human islet function through released products and extracellular matrix.

Science.gov (United States)

Arzouni, Ahmed A; Vargas-Seymour, Andreia; Rackham, Chloe L; Dhadda, Paramjeet; Huang, Guo-Cai; Choudhary, Pratik; Nardi, Nance; King, Aileen J F; Jones, Peter M

2017-12-01

The aims of the present study were (i) to determine whether the reported beneficial effects of mesenchymal stromal cells (MSCs) on mouse islet function extend to clinically relevant human tissues (islets and MSCs), enabling translation into improved protocols for clinical human islet transplantation; and (ii) to identify possible mechanisms through which human MSCs influence human islet function. Human islets were co-cultured with human adipose tissue-derived MSCs (hASCs) or pre-treated with its products - extracellular matrix (ECM) and annexin A1 (ANXA1). Mouse islets were pre-treated with mouse MSC-derived ECM. Islet insulin secretory function was assessed in vitro by radioimmunoassay. Quantitative RT-PCR was used to screen human adipMSCs for potential ligands of human islet G-protein-coupled receptors. We show that co-culture with hASCs improves human islet secretory function in vitro , as measured by glucose-stimulated insulin secretion, confirming previous reports using rodent tissues. Furthermore, we demonstrate that these beneficial effects on islet function can be partly attributed to the MSC-derived products ECM and ANXA1. Our results suggest that hASCs have the potential to improve the quality of human islets isolated for transplantation therapy of Type 1 diabetes. Furthermore, it may be possible to achieve improvements in human islet quality in a cell-free culture system by using the MSC-derived products ANXA1 and ECM. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Co-culture of clonal beta cells with GLP-1 and glucagon-secreting cell line impacts on beta cell insulin secretion, proliferation and susceptibility to cytotoxins.

Science.gov (United States)

Green, Alastair D; Vasu, Srividya; Moffett, R Charlotte; Flatt, Peter R

2016-06-01

We investigated the direct effects on insulin releasing MIN6 cells of chronic exposure to GLP-1, glucagon or a combination of both peptides secreted from GLUTag L-cell and αTC1.9 alpha-cell lines in co-culture. MIN6, GLUTag and αTC1.9 cell lines exhibited high cellular hormone content and release of insulin, GLP-1 and glucagon, respectively. Co-culture of MIN6 cells with GLUTag cells significantly increased cellular insulin content, beta-cell proliferation, insulin secretory responses to a range of established secretogogues and afforded protection against exposure cytotoxic concentrations of glucose, lipid, streptozotocin or cytokines. Benefits of co-culture of MIN6 cells with αTC1.9 alphacells were limited to enhanced beta-cell proliferation with marginal positive actions on both insulin secretion and cellular protection. In contrast, co-culture of MIN6 with GLUTag cells plus αTC1.9 cells, markedly enhanced both insulin secretory responses and protection against beta-cell toxins compared with co-culture with GLUTag cells alone. These data indicate important long-term effects of conjoint GLP-1 and glucagon exposure on beta-cell function. This illustrates the possible functional significance of alpha-cell GLP-1 production as well as direct beneficial effects of dual agonism at beta-cell GLP-1 and glucagon receptors. Copyright © 2016 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

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

Science.gov (United States)

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

2013-01-01

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

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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...

Protein kinase C is differentially regulated by thrombin, insulin, and epidermal growth factor in human mammary tumor cells

Energy Technology Data Exchange (ETDEWEB)

Gomez, M.L.; Tellez-Inon, M.T. (Instituto de Ingenieria Genetica y Biologia Molecular, Buenos Aires (Argentina)); Medrano, E.E.; Cafferatta, E.G.A. (Instituto de Investigaciones Bioquimicas Fundacion Campomar, Buenos Aires (Argentina))

1988-03-01

The exposure of serum-deprived mammary tumor cells MCF-7 and T-47D to insulin, thrombin, and epidermal growth factor (EGF) resulted in dramatic modifications in the activity and in the translocation capacity of protein kinase C from cytosol to membrane fractions. Insulin induces a 600% activation of the enzyme after 5 h of exposure to the hormone in MCF-7 cells; thrombin either activates (200% in MCF-7) or down-regulates (in T-47D), and EGF exerts only a moderate effect. Thus, the growth factors studied modulate differentially the protein kinase C activity in human mammary tumor cells. The physiological significance of the results obtained are discussed in terms of the growth response elicited by insulin, thrombin, and EGF.

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

Human beta-cell precursors mature into functional insulin-producing cells in an immunoisolation device: implications for diabetes cell therapies.

Science.gov (United States)

Lee, Seung-Hee; Hao, Ergeng; Savinov, Alexei Y; Geron, Ifat; Strongin, Alex Y; Itkin-Ansari, Pamela

2009-04-15

Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human beta-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human beta-cells and their progenitors and (2) the engraftment of encapsulated murine beta-cells in allo- and autoimmune settings. Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets. Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary beta-cells ameliorated diabetes without stimulating a detectable T-cell response. We demonstrate for the first time that human beta-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of beta-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells.

Human β-cell Precursors Mature Into Functional Insulin-producing Cells in an Immunoisolation Device: Implications for Diabetes Cell Therapies

Science.gov (United States)

Lee, Seung-Hee; Hao, Ergeng; Savinov, Alexei Y.; Geron, Ifat; Strongin, Alex Y.; Itkin-Ansari, Pamela

2009-01-01

Background Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human β-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human β-cells and their progenitors and (2) the engraftment of encapsulated murine β-cells in allo- and autoimmune settings. Methods Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets. Results Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary β-cells ameliorated diabetes without stimulating a detectable T-cell response. Conclusions We demonstrate for the first time that human β-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of β-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells. PMID:19352116

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

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

DEFF Research Database (Denmark)

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

1992-01-01

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

Insulin receptor internalization defect in an insulin-resistant mouse melanoma cell line

International Nuclear Information System (INIS)

Androlewicz, M.J.; Straus, D.S.; Brandenburg, D.F.

1989-01-01

Previous studies from this laboratory demonstrated that the PG19 mouse melanoma cell line does not exhibit a biological response to insulin, whereas melanoma x mouse embryo fibroblast hybrids do respond to insulin. To investigate the molecular basis of the insulin resistance of the PG19 melanoma cells, insulin receptors from the insulin-resistant melanoma cells and insulin-sensitive fibroblast x melanoma hybrid cells were analyzed by the technique of photoaffinity labeling using the photoprobe 125 I-NAPA-DP-insulin. Photolabeled insulin receptors from the two cell types have identical molecular weights as determined by SDS gel electrophoresis under reducing and nonreducing conditions, indicating that the receptors on the two cell lines are structurally similar. Insulin receptor internalization studies revealed that the hybrid cells internalize receptors to a high degree at 37 degree C, whereas the melanoma cells internalize receptors to a very low degree or not at all. The correlation between ability to internalize insulin receptors and sensitivity to insulin action in this system suggests that uptake of the insulin-receptor complex may be required for insulin action in these cells. Insulin receptors from the two cell lines autophosphorylate in a similar insulin-dependent manner both in vitro and in intact cells, indicating that insulin receptors on the melanoma and hybrid cells have functional tyrosine protein kinase activity. Therefore, the block in insulin action in the PG19 melanoma cells appears to reside at a step beyond insulin-stimulated receptor autophosphorylation

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

The Generation of Insulin Producing Cells from Human Mesenchymal Stem Cells by MiR-375 and Anti-MiR-9.

Science.gov (United States)

Jafarian, Arefeh; Taghikani, Mohammad; Abroun, Saeid; Allahverdi, Amir; Lamei, Maryam; Lakpour, Niknam; Soleimani, Masoud

2015-01-01

MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs that regulate gene expression at the post-transcriptional level. A number of studies have led to the notion that some miRNAs have key roles in control of pancreatic islet development and insulin secretion. Based on some studies on miRNAs pattern, the researchers in this paper investigated the pancreatic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) by up-regulation of miR-375 and down-regulation of miR-9 by lentiviruses containing miR-375 and anti-miR-9. After 21 days of induction, islet-like clusters containing insulin producing cells (IPCs) were confirmed by dithizone (DTZ) staining. The IPCs and β cell specific related genes and proteins were detected using qRT-PCR and immunofluorescence on days 7, 14 and 21 of differentiation. Glucose challenge test was performed at different concentrations of glucose so extracellular and intracellular insulin and C-peptide were assayed using ELISA kit. Although derived IPCs by miR-375 alone were capable to express insulin and other endocrine specific transcription factors, the cells lacked the machinery to respond to glucose. It was found that over-expression of miR-375 led to a reduction in levels of Mtpn protein in derived IPCs, while treatment with anti-miR-9 following miR-375 over-expression had synergistic effects on MSCs differentiation and insulin secretion in a glucose-regulated manner. The researchers reported that silencing of miR-9 increased OC-2 protein in IPCs that may contribute to the observed glucose-regulated insulin secretion. Although the roles of miR-375 and miR-9 are well known in pancreatic development and insulin secretion, the use of these miRNAs in transdifferentiation was never demonstrated. These findings highlight miRNAs functions in stem cells differentiation and suggest that they could be used as therapeutic tools for gene-based therapy in diabetes mellitus.

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

Osteoinductive activity of insulin-functionalized cell culture surfaces obtained using diazonium chemistry

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Mikulska, Anna; Filipowska, Joanna; Osyczka, Anna; Nowakowska, Maria; Szczubiałka, Krzysztof

2014-12-01

Polymeric surfaces suitable for cell culture (DR/Pec) were constructed from diazoresin (DR) and pectin (Pec) in a form of ultrathin films using the layer-by-layer (LbL) technique. The surfaces were functionalized with insulin using diazonium chemistry. Such functionalized surfaces were used to culture human mesenchymal stem cells (hMSCs) to assess their suitability for bone tissue engineering and regeneration. The activity of insulin immobilized on the surfaces (DR/Pec/Ins) was compared to that of insulin dissolved in the culture medium. Human MSC grown on insulin-immobilized DR/Pec surfaces displayed increased proliferation and higher osteogenic activity. The latter was determined by means of alkaline phosphatase (ALP) activity, which increases at early stages of osteoblasts differentiation. Insulin dissolved in the culture medium did not stimulate cell proliferation and its osteogenic activity was significantly lower. Addition of recombinant human bone morphogenetic protein 2 (rhBMP-2) to the culture medium further increased ALP activity in hMSCs indicating additive osteogenic action of immobilized insulin and rhBMP-2

Osteoinductive activity of insulin-functionalized cell culture surfaces obtained using diazonium chemistry

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Anna eMikulska

2015-01-01

Full Text Available Polymeric surfaces suitable for cell culture (DR/Pec were constructed from diazoresin (DR and pectin (Pec in a form of ultrathin films using the layer-by-layer (LbL technique. The surfaces were functionalized with insulin using diazonium chemistry. Such functionalized surfaces were used to culture human mesenchymal stem cells (hMSCs to assess their suitability for bone tissue engineering and regeneration. The activity of insulin immobilized on the surfaces (DR/Pec/Ins was compared to that of insulin dissolved in the culture medium. Human MSC grown on insulin-immobilized DR/Pec surfaces displayed increased proliferation and higher osteogenic activity. The latter was determined by means of alkaline phosphatase (ALP activity, which increases at early stages of osteoblasts differentiation. Insulin dissolved in the culture medium did not stimulate cell proliferation and its osteogenic activity was significantly lower. Addition of recombinant human bone morphogenetic protein 2 (rhBMP-2 to the culture medium further increased ALP activity in hMSCs indicating additive osteogenic action of immobilized insulin and rhBMP-2

Palmitate-induced inflammatory pathways in human adipose microvascular endothelial cells promote monocyte adhesion and impair insulin transcytosis.

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Pillon, Nicolas J; Azizi, Paymon M; Li, Yujin E; Liu, Jun; Wang, Changsen; Chan, Kenny L; Hopperton, Kathryn E; Bazinet, Richard P; Heit, Bryan; Bilan, Philip J; Lee, Warren L; Klip, Amira

2015-07-01

Obesity is associated with inflammation and immune cell recruitment to adipose tissue, muscle and intima of atherosclerotic blood vessels. Obesity and hyperlipidemia are also associated with tissue insulin resistance and can compromise insulin delivery to muscle. The muscle/fat microvascular endothelium mediates insulin delivery and facilitates monocyte transmigration, yet its contribution to the consequences of hyperlipidemia is poorly understood. Using primary endothelial cells from human adipose tissue microvasculature (HAMEC), we investigated the effects of physiological levels of fatty acids on endothelial inflammation and function. Expression of cytokines and adhesion molecules was measured by RT-qPCR. Signaling pathways were evaluated by pharmacological manipulation and immunoblotting. Surface expression of adhesion molecules was determined by immunohistochemistry. THP1 monocyte interaction with HAMEC was measured by cell adhesion and migration across transwells. Insulin transcytosis was measured by total internal reflection fluorescence microscopy. Palmitate, but not palmitoleate, elevated the expression of IL-6, IL-8, TLR2 (Toll-like receptor 2), and intercellular adhesion molecule 1 (ICAM-1). HAMEC had markedly low fatty acid uptake and oxidation, and CD36 inhibition did not reverse the palmitate-induced expression of adhesion molecules, suggesting that inflammation did not arise from palmitate uptake/metabolism. Instead, inhibition of TLR4 to NF-κB signaling blunted palmitate-induced ICAM-1 expression. Importantly, palmitate-induced surface expression of ICAM-1 promoted monocyte binding and transmigration. Conversely, palmitate reduced insulin transcytosis, an effect reversed by TLR4 inhibition. In summary, palmitate activates inflammatory pathways in primary microvascular endothelial cells, impairing insulin transport and increasing monocyte transmigration. This behavior may contribute in vivo to reduced tissue insulin action and enhanced tissue

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

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Lorna W Harries

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

Differentiation of human induced pluripotent stem cells into insulin-like cell clusters with miR-186 and miR-375 by using chemical transfection.

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Shaer, Anahita; Azarpira, Negar; Karimi, Mohammad Hosein

2014-09-01

Diabetes mellitus is characterized by either the inability to produce insulin or insensitivity to insulin secreted by the body. Islet cell replacement is an effective approach for diabetes treatment; however, it is not sufficient for all the diabetic patients. MicroRNAs (miRNAs) are a class of small noncoding RNAs that play an important role in mediating a broad and expanding range of biological activities, such as pancreas development. The present study aimed to develop a protocol to efficiently differentiate human induced pluripotent stem (iPS) cells into islet-like cell clusters (ILCs) in vitro by using miR-186 and miR-375. The human iPS colonies were transfected with hsa-miR-186 and hsa-miR-375 by using siPORT™ NeoFX™ Transfection Agent, and the differentiation was compared to controls. Total RNA was extracted 24 and 48 h after transfection. The gene expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, Glucagon, and OCT4 were then evaluated through real-time qPCR. On the third day, the potency of the clusters was assessed in response to high glucose levels. Dithizone (DTZ) was used to identify the existence of the β-cells. Besides, the presence of insulin and NGN3 proteins was investigated by immunocytochemistry. Morphological changes were observed on the first day after the chemical transfection, and cell clusters were formed on the third day. The expression of pancreatic specific transcription factors was increased on the first day and significantly increased on the second day. The ILCs were positive for insulin and NGN3 proteins in the immunocytochemistry. Besides, the clusters were stained with DTZ and secreted insulin in glucose challenge test. Overexpression of miR-186 and miR-375 can be an alternative strategy for producing ILCs from the iPS cells in a short time. This work provides a new approach by using patient-specific iPSCs for β-cell replacement therapy in diabetic patients.

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.

Synthesis and release of fatty acids by human trophoblast cells in culture

International Nuclear Information System (INIS)

Coleman, R.A.; Haynes, E.B.

1987-01-01

In order to determine whether placental cells can synthesize and release fatty acids, trophoblast cells from term human placentas were established in monolayer culture. The cells continued to secrete placental lactogen and progesterone and maintained specific activities of critical enzymes of triacylglycerol and phosphatidylcholine biosynthesis for 24 to 72 hr in culture. Fatty acid was rapidly synthesized from [ 14 C]acetate and released by the cells. Palmitoleic, palmitic, and oleic acids were the major fatty acids synthesized from [ 14 C]acetate and released. Small amounts of lauric, myristic, and stearic acids were also identified. [ 14 C]acetate was also incorporated into cellular triacylglycerol, phospholipid, and cholesterol, but radiolabeled free fatty acid did not accumulate intracellularly. In a pulse-chase experiment, cellular glycerolipids were labeled with [1- 14 C]oleate; trophoblast cells then released 14 C-labeled fatty acid into the media as the cellular content of labeled phospholipid and triacylglycerol decreased without intracellular accumulation of free fatty acid. Twenty percent of the 14 C-label lost from cellular glycerolipid could not be recovered as a chloroform-extractable product, suggesting that some of the hydrolyzed fatty acid had been oxidized. These data indicate that cultured placenta trophoblast cells can release fatty acids that have either been synthesized de novo or that have been hydrolyzed from cellular glycerolipids. Trophoblast cells in monolayer culture should provide an excellent model for molecular studies of placental fatty acid metabolism and release

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.

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

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Peng Li

2017-01-01

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

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.

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, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells.

Science.gov (United States)

Kulikova, Veronika; Shabalin, Konstantin; Nerinovski, Kirill; Dölle, Christian; Niere, Marc; Yakimov, Alexander; Redpath, Philip; Khodorkovskiy, Mikhail; Migaud, Marie E; Ziegler, Mathias; Nikiforov, Andrey

2015-11-06

NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibition of histamine and eicosanoid release from dispersed human lung cells in vitro by quinotolast.

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Okayama, Y; Hiroi, J; Lau, L C; Church, M K

1995-12-01

We have examined the effects of a new anti-allergic drug, quinotolast [sodium 5-(4-oxo-1-phenoxy-4H-quinolizine-3-carboxamido) yetrazolate monohydrate], in inhibiting the release of histamine and the generation of leukotriene (LT) C4 and prostaglandin (PG) D2 from dispersed human lung cells and compared this with those of its active metabolite in the rat, hydroxy quinotolast, and reference drugs, tranilast and sodium cromoglycate (SCG). Quinotolast in the concentration range of 1-100 micrograms/ml inhibited histamine and LTC4 release in a concentration-dependent manner. The inhibitory effect of quinotolast on histamine release from dispersed lung cells was largely independent of the preincubation period, no tachyphylaxis being observed. Hydroxy quinotolast and tranilast showed a weak inhibition of histamine release only when the drugs were added to the cells simultaneously with anti-IgE challenge. Quinotolast, 100 micrograms/ml, and SCG, 1 mM, significantly inhibited PGD2 and LTC4 release. Quinotolast inhibited PGD2 release by 100% and LTC4 release by 54%, whereas SCG inhibited PDG2 release by 33% and LTC4 release by 100%. No cross-tachyphylaxis between quinotolast and SCG was observed. The results demonstrated that quinotolast showed a significant inhibition of inflammatory mediators from human dispersed lung cells, suggesting that quinotolast is a good candidate for a clinical anti-allergic drug.

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

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Ackerman, G A; Wolken, K W

1981-10-01

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

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

International Nuclear Information System (INIS)

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

1981-01-01

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

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

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...

Activated α2-macroglobulin binding to human prostate cancer cells triggers insulin-like responses.

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Misra, Uma Kant; Pizzo, Salvatore Vincent

2015-04-10

Ligation of cell surface GRP78 by activated α2-macroglobulin (α2M*) promotes cell proliferation and suppresses apoptosis. α2M*-treated human prostate cancer cells exhibit a 2-3-fold increase in glucose uptake and lactate secretion, an effect similar to insulin treatment. In both α2M* and insulin-treated cells, the mRNA levels of SREBP1-c, SREBP2, fatty-acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, and Glut-1 were significantly increased together with their protein levels, except for SREBP2. Pretreatment of cells with α2M* antagonist antibody directed against the carboxyl-terminal domain of GRP78 blocks these α2M*-mediated effects, and silencing GRP78 expression by RNAi inhibits up-regulation of ATP citrate lyase and fatty-acid synthase. α2M* induces a 2-3-fold increase in lipogenesis as determined by 6-[(14)C]glucose or 1-[(14)C]acetate incorporation into free cholesterol, cholesterol esters, triglycerides, free fatty acids, and phosphatidylcholine, which is blocked by inhibitors of fatty-acid synthase, PI 3-kinase, mTORC, or an antibody against the carboxyl-terminal domain of GRP78. We also assessed the incorporation of [(14)CH3]choline into phosphatidylcholine and observed similar effects. Lipogenesis is significantly affected by pretreatment of prostate cancer cells with fatostatin A, which blocks sterol regulatory element-binding protein proteolytic cleavage and activation. This study demonstrates that α2M* functions as a growth factor, leading to proliferation of prostate cancer cells by promoting insulin-like responses. An antibody against the carboxyl-terminal domain of GRP78 may have important applications in prostate cancer therapy. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

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.

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.

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

International Nuclear Information System (INIS)

Randazzo, P.A.; Jarett, L.

1990-01-01

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

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.

NK cell-released exosomes

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Fais, Stefano

2013-01-01

We have recently reported that human natural killer (NK) cells release exosomes that express both NK-cell markers and cytotoxic molecules. Similar results were obtained with circulating exosomes from human healthy donors. Both NK-cell derived and circulating exosomes exerted a full functional activity and killed both tumor and activated immune cells. These findings indicate that NK-cell derived exosomes might constitute a new promising therapeutic tool. PMID:23482694

Evidence that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of gene expression from human cytomegalovirus promoter in CHO cells

OpenAIRE

Zhang, Yingpei; Katakura, Yoshinori; Seto, Perry; Shirahata, Sanetaka

1997-01-01

The signal transduction from insulin to its receptors and Ras has been extensively studied, while little has been reported beyond these steps. We found that the expression of human interleukin 6 gene under the control of immediate early gene promoter of human cytomegalovirus was enhanced by insulin sitmulation in Chinese hamster ovary cells. The induction effect of insulin was not significantly affected by inhibitors or activators of conventional protein kinase C, cAMP dependent protein kinas...

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...

Baculovirus-mediated gene transfer and recombinant protein expression do not interfere with insulin dependent phosphorylation of PKB/Akt in human SHSY-5Y and C3A cells

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Selander Martin

2007-02-01

Full Text Available Abstract Background Recombinant adenovirus vectors and transfection agents comprising cationic lipids are widely used as gene delivery vehicles for functional expression in cultured cells. Consequently, these tools are utilized to investigate the effects of functional over-expression of proteins on insulin mediated events. However, we have previously reported that cationic lipid reagents cause a state of insulin unresponsiveness in cell cultures. In addition, we have found that cultured cells often do not respond to insulin stimulation following adenovirus treatment. Infection with adenovirus compromises vital functions of the host cell leading to the activation of protein kinases central to insulin signalling, such as protein kinase B/Akt. Therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of Akt activation in cultured cells. Moreover, we investigated the use of baculovirus as a heterologous viral gene delivery vehicle to circumvent these phenomena. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of this viral system in gene delivery has greatly expanded and one advantage is the virtual absence of cytotoxicity in mammalian cells. Results We show that infection of human neuroblastoma SHSY-5Y and liver C3A cells with recombinant adenovirus results in the activation of Akt in a dose dependent manner. In addition, this activation makes treated cells unresponsive to insulin stimulation as determined by an apparent lack of differential phosphorylation of Akt on serine-473. Our data further indicate that the use of recombinant baculovirus does not increase the phosphorylation of Akt in SHSY-5Y and C3A cells. Moreover, following infection with baculovirus, SHSY-5Y and C3A cells respond to insulin by means of phosphorylation of Akt on serine-473 in the same manner as uninfected cells. Conclusion Widely-used adenovirus vectors for gene delivery cause a state of

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

Pancreatic islet-like clusters from bone marrow mesenchymal stem cells of human first-trimester abortus can cure streptozocin-induced mouse diabetes.

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Zhang, Yihua; Shen, Wenzheng; Hua, Jinlian; Lei, Anmin; Lv, Changrong; Wang, Huayan; Yang, Chunrong; Gao, Zhimin; Dou, Zhongying

2010-12-01

Bone marrow mesenchymal stem cells (BMSCs) have been reported to possess low immunogenicity and cause immunosuppression of recipients when allografted. They can differentiate into insulin-producing cells and may be a valuable source for islet formation. However, the extremely low differentiating rate of adult BMSCs toward insulin-producing cells and the insufficient insulin secretion of the differentiated BMSCs in vitro prevent their clinical use in diabetes treatment. Little is known about the potential of cell replacement therapy with human BMSCs. Previously, we isolated and identified human first-trimester fetal BMSCs (hfBMSCs). Under a novel four-step induction procedure established in this study, the hfBMSCs effectively differentiated into functional pancreatic islet-like cell clusters that contained 62 ± 14% insulin-producing cells, expressed a broad gene profile related to pancreatic islet β-cell development, and released high levels of insulin (2.245 ± 0.222 pmol/100 clusters per 30 min) and C-peptide (2.200 ± 0.468 pmol/100 clusters per 30 min) in response to 25 mmol/L glucose stimulus in vitro. The pancreatic islet-like cell clusters normalized the blood glucose level of diabetic model mice for at least 9 weeks when xenografted; blood glucose levels in these mice rose abnormally again when the grafts were removed. Examination of the grafts indicated that the transplanted cells survived in recipients and produced human insulin and C-peptide in situ. These results demonstrate that hfBMSCs derived from a human first-trimester abortus can differentiate into pancreatic islet-like cell clusters following an established four-step induction. The insulin-producing clusters present advantages in cell replacement therapy of type 1 diabetic model mice.

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

Science.gov (United States)

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

Extrinsic Factors Involved in the Differentiation of Stem Cells into Insulin-Producing Cells: An Overview

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Rebecca S. Y. Wong

2011-01-01

Full Text Available Diabetes mellitus is a chronic disease with many debilitating complications. Treatment of diabetes mellitus mainly revolves around conventional oral hypoglycaemic agents and insulin replacement therapy. Recently, scientists have turned their attention to the generation of insulin-producing cells (IPCs from stem cells of various sources. To date, many types of stem cells of human and animal origins have been successfully turned into IPCs in vitro and have been shown to exert glucose-lowering effect in vivo. However, scientists are still faced with the challenge of producing a sufficient number of IPCs that can in turn produce sufficient insulin for clinical use. A careful choice of stem cells, methods, and extrinsic factors for induction may all be contributing factors to successful production of functional beta-islet like IPCs. It is also important that the mechanism of differentiation and mechanism by which IPCs correct hyperglycaemia are carefully studied before they are used in human subjects.

Stimulation of GPR30 increases release of EMMPRIN-containing microvesicles in human uterine epithelial cells.

Science.gov (United States)

Burnett, Lindsey A; Light, Mallory M; Mehrotra, Pavni; Nowak, Romana A

2012-12-01

Uterine remodeling is highly dependent on the glycosylated transmembrane protein extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN). Previous studies indicate estradiol can increase EMMPRIN expression in uterine cells and promote subsequent induction of MMP production. The aim of this study was to investigate the role of G protein-coupled receptor 30 (GPR30) stimulation on EMMPRIN microvesicle release in the human uterine epithelial cell line hTERT-EEC (EECs). We examined EMMPRIN release by human EECs in response to GPR30 stimulation by microvesicle isolation, Western blot, and immunocytochemistry. We employed a pharmacological approach using the GPR30-selective agonist G1 and the antagonist G15 to determine the receptor specificity of this response. We demonstrated GPR30 expression in EECs and release of EMMPRIN in microvesicles in response to stimulation of GPR30. G1, estradiol, and cholera toxin stimulated EMMPRIN release in microvesicles as detected by Western blot and immunocytochemistry, indicating that stimulation of GPR30 can induce EMMPRIN microvesicle release. These data indicate that EMMPRIN release in microvesicles can be mediated by stimulation of GPR30 in human EECs, suggesting that inappropriate stimulation or expression of this receptor may be significant in uterine pathology.

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....

Histamine release from rodent and human mast cells induced by protoporphyrin and ultraviolet light: studies of the mechanism of mast-cell activation in erythropoietic protoporphyria

International Nuclear Information System (INIS)

Glover, R.A.; Bailey, C.S.; Barrett, K.E.; Wasserman, S.I.; Gigli, I.

1990-01-01

We report that protoporphyrin (PP) and ultraviolet light (UVA) induces histamine release from rat peritoneal mast cells, mouse bone marrow mast cells and human cutaneous mast cells in a dose- and temperature-dependent manner. The mast-cell activation was associated with loss of membrane integrity and inhibited by the hydrogen peroxide scavenger, catalase. Histamine release was independent of extracellular calcium in the rodent mast cells, but was markedly reduced in the absence of calcium in human cells. These findings indicate that PP and UVA induce mast-cell-mediator release by a process that may involve hydrogen peroxide formation. There appear to be differences in response to PP and UVA between rodent and human mast cells. (author)

Histamine release from rodent and human mast cells induced by protoporphyrin and ultraviolet light: studies of the mechanism of mast-cell activation in erythropoietic protoporphyria

Energy Technology Data Exchange (ETDEWEB)

Glover, R.A.; Bailey, C.S.; Barrett, K.E.; Wasserman, S.I.; Gigli, I. (California Univ., San Diego, CA (USA). Dept. of Medicine)

1990-04-01

We report that protoporphyrin (PP) and ultraviolet light (UVA) induces histamine release from rat peritoneal mast cells, mouse bone marrow mast cells and human cutaneous mast cells in a dose- and temperature-dependent manner. The mast-cell activation was associated with loss of membrane integrity and inhibited by the hydrogen peroxide scavenger, catalase. Histamine release was independent of extracellular calcium in the rodent mast cells, but was markedly reduced in the absence of calcium in human cells. These findings indicate that PP and UVA induce mast-cell-mediator release by a process that may involve hydrogen peroxide formation. There appear to be differences in response to PP and UVA between rodent and human mast cells. (author).

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

Development of Gonadotropin-Releasing Hormone-Secreting Neurons from Human Pluripotent Stem Cells

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Carina Lund

2016-08-01

Full Text Available Gonadotropin-releasing hormone (GnRH neurons regulate human puberty and reproduction. Modeling their development and function in vitro would be of interest for both basic research and clinical translation. Here, we report a three-step protocol to differentiate human pluripotent stem cells (hPSCs into GnRH-secreting neurons. Firstly, hPSCs were differentiated to FOXG1, EMX2, and PAX6 expressing anterior neural progenitor cells (NPCs by dual SMAD inhibition. Secondly, NPCs were treated for 10 days with FGF8, which is a key ligand implicated in GnRH neuron ontogeny, and finally, the cells were matured with Notch inhibitor to bipolar TUJ1-positive neurons that robustly expressed GNRH1 and secreted GnRH decapeptide into the culture medium. The protocol was reproducible both in human embryonic stem cells and induced pluripotent stem cells, and thus provides a translational tool for investigating the mechanisms of human puberty and its disorders.

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.

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

International Nuclear Information System (INIS)

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

1990-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

1990-10-01

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

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

International Nuclear Information System (INIS)

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

1990-01-01

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

Complexity of the influence of gangliosides on histamine release from human basophils and rat mast cells

DEFF Research Database (Denmark)

Jensen, C; Svendsen, U G; Thastrup, Ole

1987-01-01

The influence of exogenous addition of gangliosides on histamine release from human basophils and rat mast cells was examined in vitro. Gangliosides dose-dependently inhibited histamine release, and this inhibition was dependent on the ganglioside sialic acid content, since GT1b, having 3 sialic...... was reflected in the sensitivity of the cells to extracellular calcium, since inhibition of the release could be counteracted by increasing the extracellular concentration of calcium....

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.

Characterization of lipid metabolism in insulin-sensitive adipocytes differentiated from immortalized human mesenchymal stem cells

International Nuclear Information System (INIS)

Prawitt, Janne; Niemeier, Andreas; Kassem, Moustapha; Beisiegel, Ulrike; Heeren, Joerg

2008-01-01

There is a great demand for cell models to study human adipocyte function. Here we describe the adipogenic differentiation of a telomerase-immortalized human mesenchymal stem cell line (hMSC-Tert) that maintains numerous features of terminally differentiated adipocytes even after prolonged withdrawal of the peroxisome proliferator activated receptor γ (PPARγ) agonist rosiglitazone. Differentiated hMSC-Tert developed the characteristic monolocular phenotype of mature adipocytes. The expression of adipocyte specific markers was highly increased during differentiation. Most importantly, the presence of the PPARγ agonist rosiglitazone was not required for the stable expression of lipoprotein lipase, adipocyte fatty acid binding protein and perilipin on mRNA and protein levels. Adiponectin expression was post-transcriptionally down-regulated in the absence of rosiglitazone. Insulin sensitivity as measured by insulin-induced phosphorylation of Akt and S6 ribosomal protein was also independent of rosiglitazone. In addition to commonly used adipogenic markers, we investigated further PPARγ-stimulated proteins with a role in lipid metabolism. We observed an increase of lipoprotein receptor (VLDLR, LRP1) and apolipoprotein E expression during differentiation. Despite this increased expression, the receptor-mediated endocytosis of lipoproteins was decreased in differentiated adipocytes, suggesting that these proteins may have an additional function in adipose tissue beyond lipoprotein uptake

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

OpenAIRE

Aguer , Céline; Mercier , Jacques; Kitzmann , Magali

2009-01-01

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

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.

Thioredoxin reductase 1 upregulates MCP-1 release in human endothelial cells

Energy Technology Data Exchange (ETDEWEB)

Liu, Zhen-Bo [Institute of Biophysics, Chinese Academy of Sciences, and Graduate School of the Chinese Academy of Sciences, Beijing (China); Shen, Xun, E-mail: shenxun@sun5.ibp.ac.cn [Institute of Biophysics, Chinese Academy of Sciences, and Graduate School of the Chinese Academy of Sciences, Beijing (China)

2009-09-04

To know if thioredoxin reductase 1 (TrxR1) plays a role in antioxidant defense mechanisms against atherosclerosis, effect of TrxR1 on expression/release of monocyte chemoattractant protein (MCP-1) was investigated in activated human endothelial-like EAhy926 cells. The MCP-1 release and expression, cellular generation of reactive oxygen species (ROS), nuclear translocation and DNA-binding activity of NF-{kappa}B subunit p65 were assayed in cells either overexpressing recombinant TrxR1 or having their endogenous TrxR1 knocked down. It was found that overexpression of TrxR1 enhanced, while knockdown of TrxR1 reduced MCP-1 release and expression. Upregulation of MCP-1 by TrxR1 was associated with increasing generation of intracellular ROS generation, enhanced nuclear translocation and DNA-binding activity of NF-{kappa}B. Assay using NF-{kappa}B reporter revealed that TrxR1 upregulated transcriptional activity of NF-{kappa}B. This study suggests that TrxR1 enhances ROS generation, NF-{kappa}B activity and subsequent MCP-1 expression in endothelial cells, and may promote rather than prevent vascular endothelium from forming atherosclerotic plaque.

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.

Inhibition of human MCF-7 breast cancer cells and HT-29 colon cancer cells by rice-produced recombinant human insulin-like growth binding protein-3 (rhIGFBP-3.

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Stanley C K Cheung

Full Text Available BACKGROUND: Insulin-like growth factor binding protein-3 (IGFBP-3 is a multifunctional molecule which is closely related to cell growth, apoptosis, angiogenesis, metabolism and senescence. It combines with insulin-like growth factor-I (IGF-I to form a complex (IGF-I/IGFBP-3 that can treat growth hormone insensitivity syndrome (GHIS and reduce insulin requirement in patients with diabetes. IGFBP-3 alone has been shown to have anti-proliferation effect on numerous cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: We reported here an expression method to produce functional recombinant human IGFBP-3 (rhIGFBP-3 in transgenic rice grains. Protein sorting sequences, signal peptide and endoplasmic reticulum retention tetrapeptide (KDEL were included in constructs for enhancing rhIGFBP-3 expression. Western blot analysis showed that only the constructs with signal peptide were successfully expressed in transgenic rice grains. Both rhIGFBP-3 proteins, with or without KDEL sorting sequence inhibited the growth of MCF-7 human breast cancer cells (65.76 ± 1.72% vs 45.00 ± 0.86%, p < 0.05; 50.84 ± 1.97% vs 45.00 ± 0.86%, p < 0.01 respectively and HT-29 colon cancer cells (65.14 ± 3.84% vs 18.01 ± 13.81%, p < 0.05 and 54.7 ± 9.44% vs 18.01 ± 13.81%, p < 0.05 respectively when compared with wild type rice. CONCLUSION/SIGNIFICANCE: These findings demonstrated the feasibility of producing biological active rhIGFBP-3 in rice using a transgenic approach, which will definitely encourage more research on the therapeutic use of hIGFBP-3 in future.

Insulin resistance alters islet morphology in nondiabetic humans

DEFF Research Database (Denmark)

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

2014-01-01

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

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

FGF-2b and h-PL Transform Duct and Non-Endocrine Human Pancreatic Cells into Endocrine Insulin Secreting Cells by Modulating Differentiating Genes

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Giulia Donadel

2017-10-01

Full Text Available Background: Diabetes mellitus (DM is a multifactorial disease orphan of a cure. Regenerative medicine has been proposed as novel strategy for DM therapy. Human fibroblast growth factor (FGF-2b controls β-cell clusters via autocrine action, and human placental lactogen (hPL-A increases functional β-cells. We hypothesized whether FGF-2b/hPL-A treatment induces β-cell differentiation from ductal/non-endocrine precursor(s by modulating specific genes expression. Methods: Human pancreatic ductal-cells (PANC-1 and non-endocrine pancreatic cells were treated with FGF-2b plus hPL-A at 500 ng/mL. Cytofluorimetry and Immunofluorescence have been performed to detect expression of endocrine, ductal and acinar markers. Bromodeoxyuridine incorporation and annexin-V quantified cells proliferation and apoptosis. Insulin secretion was assessed by RIA kit, and electron microscopy analyzed islet-like clusters. Results: Increase in PANC-1 duct cells de-differentiation into islet-like aggregates was observed after FGF-2b/hPL-A treatment showing ultrastructure typical of islets-aggregates. These clusters, after stimulation with FGF-2b/hPL-A, had significant (p < 0.05 increase in insulin, C-peptide, pancreatic and duodenal homeobox 1 (PDX-1, Nkx2.2, Nkx6.1, somatostatin, glucagon, and glucose transporter 2 (Glut-2, compared with control cells. Markers of PANC-1 (Cytokeratin-19, MUC-1, CA19-9 were decreased (p < 0.05. These aggregates after treatment with FGF-2b/hPL-A significantly reduced levels of apoptosis. Conclusions: FGF-2b and hPL-A are promising candidates for regenerative therapy in DM by inducing de-differentiation of stem cells modulating pivotal endocrine genes.

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

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.

Directory of Open Access Journals (Sweden)

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.

Human multipotent adipose-derived stem cells differentiate into functional brown adipocytes

DEFF Research Database (Denmark)

Elabd, Christian; Chiellini, Chiara; Carmona, Mamen

2009-01-01

adipose-derived stem (hMADS) cells exhibit a normal karyotype and high self-renewal ability; they are known to differentiate into cells that exhibit the key properties of human white adipocytes, that is, uncoupling protein two expression, insulin-stimulated glucose uptake, lipolysis in response to beta......In contrast to the earlier contention, adult humans have been shown recently to possess active brown adipose tissue with a potential of being of metabolic significance. Up to now, brown fat precursor cells have not been available for human studies. We have shown previously that human multipotent......-agonists and atrial natriuretic peptide, and release of adiponectin and leptin. Herein, we show that, upon chronic exposure to a specific PPARgamma but not to a PPARbeta/delta or a PPARalpha agonist, hMADS cell-derived white adipocytes are able to switch to a brown phenotype by expressing both uncoupling protein one...

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

Science.gov (United States)

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.

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.

Science.gov (United States)

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...

**-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

Energy Technology Data Exchange (ETDEWEB)

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

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

Science.gov (United States)

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

2000-05-01

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

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

International Nuclear Information System (INIS)

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

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

Cryopreservation of human insulin expressing cells macro-encapsulated in a durable therapeutic immunoisolating device theracyte.

Science.gov (United States)

Yakhnenko, Ilya; Wong, Wallace K; Katkov, Igor I; Itkin-Ansari, Pamela

2012-01-01

Encapsulating insulin producing cells (INPCs) in an immunoisolation device have been shown to cure diabetes in rodents without the need for immunosuppression. However, micro-encapsulation in semi-solid gels raises longevity and safety concerns for future use of stem cell derived INPCs. We have focused on a durable and retrievable macro-encapsulation (> 10(6) cells) device (TheraCyte). Cryopreservation (CP) of cells preloaded into the device is highly desirable but may require prolonged exposure to cryoprotectants during loading and post-thaw manipulations. Here, we are reporting survival and function of a human islet cell line frozen as single cells or as islet-like cell clusters. The non-clusterized cells exhibited high cryosurvival after prolonged pre-freeze or post-thaw exposure to 10 percent DMSO. However, both clusterization and especially loading INPCs into the device reduced viable yield even without CP. The survived cryopreserved macro-encapsulated INPCs remained fully functional suggesting that CP of macro-encapsulated cells is a promising tool for cell based therapies.

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

International Nuclear Information System (INIS)

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

1982-01-01

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

Recombinant human bone morphogenetic protein-2 released from polyurethane-based scaffolds promotes early osteogenic differentiation of human mesenchymal stem cells

International Nuclear Information System (INIS)

Kim, Jinku; Hollinger, Jeffrey O

2012-01-01

The purposes of this study were to determine the pharmacokinetics of recombinant human bone morphogenetic protein-2 (rhBMP-2) from a polyurethane (PUR)-based porous scaffold and to determine the biological responses of human mesenchymal stem cells (hMSCs) to the rhBMP-2 released from those scaffolds. The rhBMP-2 was incorporated into the PUR three-dimensional (3D) porous scaffolds and release profiles were determined using enzyme-linked immunosorbent assay. The bioactivity of the rhBMP-2 containing releasates was determined using hMSCs and compared with exogenous rhBMP-2. Release of rhBMP-2 from PUR-based systems was bi-phasic and characterized by an initial burst followed by a sustained release for up to 21 days. Expression of alkaline phosphatase activity by hMSCs treated with the rhBMP-2 releasates was significantly greater than the cells alone (control) throughout the time periods. Furthermore, after 14 days of culture, the hMSCs cultured with rhBMP-2 releasate had a greater amount of mineralization compared to exogenous rhBMP-2. Overall, the rhBMP-2 release from the PUR-based scaffolds was sustained for 21 days and the releasates appeared to be bioactive and promoted earlier osteogenic differentiation and mineralization of hMSCs than the exogenous rhBMP-2. (paper)

Fasting Increases Human Skeletal Muscle Net Phenylalanine Release and This Is Associated with Decreased mTOR Signaling

Science.gov (United States)

Vendelbo, Mikkel Holm; Møller, Andreas Buch; Christensen, Britt; Nellemann, Birgitte; Clasen, Berthil Frederik Forrest; Nair, K. Sreekumaran; Jørgensen, Jens Otto Lunde; Jessen, Niels; Møller, Niels

2014-01-01

Aim Fasting is characterised by profound changes in energy metabolism including progressive loss of body proteins. The underlying mechanisms are however unknown and we therefore determined the effects of a 72-hour-fast on human skeletal muscle protein metabolism and activation of mammalian target of rapamycin (mTOR), a key regulator of cell growth. Methods Eight healthy male volunteers were studied twice: in the postabsorptive state and following 72 hours of fasting. Regional muscle amino acid kinetics was measured in the forearm using amino acid tracers. Signaling to protein synthesis and breakdown were assessed in skeletal muscle biopsies obtained during non-insulin and insulin stimulated conditions on both examination days. Results Fasting significantly increased forearm net phenylalanine release and tended to decrease phenylalanine rate of disappearance. mTOR phosphorylation was decreased by ∼50% following fasting, together with reduced downstream phosphorylation of 4EBP1, ULK1 and rpS6. In addition, the insulin stimulated increase in mTOR and rpS6 phosphorylation was significantly reduced after fasting indicating insulin resistance in this part of the signaling pathway. Autophagy initiation is in part regulated by mTOR through ULK1 and fasting increased expression of the autophagic marker LC3B-II by ∼30%. p62 is degraded during autophagy but was increased by ∼10% during fasting making interpretation of autophagic flux problematic. MAFbx and MURF1 ubiquitin ligases remained unaltered after fasting indicating no change in protesomal protein degradation. Conclusions Our results show that during fasting increased net phenylalanine release in skeletal muscle is associated to reduced mTOR activation and concomitant decreased downstream signaling to cell growth. PMID:25020061

Fasting increases human skeletal muscle net phenylalanine release and this is associated with decreased mTOR signaling.

Directory of Open Access Journals (Sweden)

Mikkel Holm Vendelbo

Full Text Available Fasting is characterised by profound changes in energy metabolism including progressive loss of body proteins. The underlying mechanisms are however unknown and we therefore determined the effects of a 72-hour-fast on human skeletal muscle protein metabolism and activation of mammalian target of rapamycin (mTOR, a key regulator of cell growth.Eight healthy male volunteers were studied twice: in the postabsorptive state and following 72 hours of fasting. Regional muscle amino acid kinetics was measured in the forearm using amino acid tracers. Signaling to protein synthesis and breakdown were assessed in skeletal muscle biopsies obtained during non-insulin and insulin stimulated conditions on both examination days.Fasting significantly increased forearm net phenylalanine release and tended to decrease phenylalanine rate of disappearance. mTOR phosphorylation was decreased by ∼50% following fasting, together with reduced downstream phosphorylation of 4EBP1, ULK1 and rpS6. In addition, the insulin stimulated increase in mTOR and rpS6 phosphorylation was significantly reduced after fasting indicating insulin resistance in this part of the signaling pathway. Autophagy initiation is in part regulated by mTOR through ULK1 and fasting increased expression of the autophagic marker LC3B-II by ∼30%. p62 is degraded during autophagy but was increased by ∼10% during fasting making interpretation of autophagic flux problematic. MAFbx and MURF1 ubiquitin ligases remained unaltered after fasting indicating no change in protesomal protein degradation.Our results show that during fasting increased net phenylalanine release in skeletal muscle is associated to reduced mTOR activation and concomitant decreased downstream signaling to cell growth.

Ghrelin stimulates angiogenesis in human microvascular endothelial cells: Implications beyond GH release

International Nuclear Information System (INIS)

Li Aihua; Cheng Guangli; Zhu Genghui; Tarnawski, Andrzej S.

2007-01-01

Ghrelin, a peptide hormone isolated from the stomach, releases growth hormone and stimulates appetite. Ghrelin is also expressed in pancreas, kidneys, cardiovascular system and in endothelial cells. The precise role of ghrelin in endothelial cell functions remains unknown. We examined the expression of ghrelin and its receptor (GHSR1) mRNAs and proteins in human microvascular endothelial cells (HMVEC) and determined whether ghrelin affects in these cells proliferation, migration and in vitro angiogenesis; and whether MAPK/ERK2 signaling is important for the latter action. We found that ghrelin and GHSR1 are constitutively expressed in HMVEC. Treatment of HMVEC with exogenous ghrelin significantly increased in these cells proliferation, migration, in vitro angiogenesis and ERK2 phosphorylation. MEK/ERK2 inhibitor, PD 98059 abolished ghrelin-induced in vitro angiogenesis. This is First demonstration that ghrelin and its receptor are expressed in human microvascular endothelial cells and that ghrelin stimulates HMVEC proliferation, migration, and angiogenesis through activation of ERK2 signaling

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

Energy Technology Data Exchange (ETDEWEB)

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.

Targeting development of incretin-producing cells increases insulin secretion

DEFF Research Database (Denmark)

Petersen, Natalia; Reimann, Frank; van Es, Johan H

2015-01-01

the number of intestinal L cells, which produce GLP-1, is an alternative strategy to augment insulin responses and improve glucose tolerance. Blocking the NOTCH signaling pathway with the γ-secretase inhibitor dibenzazepine increased the number of L cells in intestinal organoid-based mouse and human culture...... of the development of incretin-producing cells in the intestine has potential as a therapeutic strategy to improve glycemic control....

Sodium phenylbutyrate, a drug with known capacity to reduce endoplasmic reticulum stress, partially alleviates lipid-induced insulin resistance and beta-cell dysfunction in humans.

Science.gov (United States)

Xiao, Changting; Giacca, Adria; Lewis, Gary F

2011-03-01

Chronically elevated free fatty acids contribute to insulin resistance and pancreatic β-cell failure. Among numerous potential factors, the involvement of endoplasmic reticulum (ER) stress has been postulated to play a mechanistic role. Here we examined the efficacy of the chemical chaperone, sodium phenylbutyrate (PBA), a drug with known capacity to reduce ER stress in animal models and in vitro, on lipid-induced insulin resistance and β-cell dysfunction in humans. Eight overweight or obese nondiabetic men underwent four studies each, in random order, 4 to 6 weeks apart. Two studies were preceded by 2 weeks of oral PBA (7.5 g/day), followed by a 48-h i.v. infusion of intralipid/heparin or saline, and two studies were preceded by placebo treatment, followed by similar infusions. Insulin secretion rates (ISRs) and sensitivity (S(I)) were assessed after the 48-h infusions by hyperglycemic and hyperinsulinemic-euglycemic clamps, respectively. Lipid infusion reduced S(I), which was significantly ameliorated by pretreatment with PBA. Absolute ISR was not affected by any treatment; however, PBA partially ameliorated the lipid-induced reduction in the disposition index (DI = ISR × S(I)), indicating that PBA prevented lipid-induced β-cell dysfunction. These results suggest that PBA may provide benefits in humans by ameliorating the insulin resistance and β-cell dysfunction induced by prolonged elevation of free fatty acids.

Insulin resistance and chronic inflammation

Directory of Open Access Journals (Sweden)

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.

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

Science.gov (United States)

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

2013-12-05

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

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.

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

Science.gov (United States)

Das, Sromona; Bhattacharyya, Debasish

2017-12-01

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

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

Science.gov (United States)

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

2015-01-01

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

N-Acetylcysteine Amide Protects Against Oxidative Stress–Induced Microparticle Release From Human Retinal Pigment Epithelial Cells

Science.gov (United States)

Carver, Kyle A.; Yang, Dongli

2016-01-01

Purpose Oxidative stress is a major factor involved in retinal pigment epithelium (RPE) apoptosis that underlies AMD. Drusen, extracellular lipid- and protein-containing deposits, are strongly associated with the development of AMD. Cell-derived microparticles (MPs) are small membrane-bound vesicles shed from cells. The purpose of this study was to determine if oxidative stress drives MP release from RPE cells, to assess whether these MPs carry membrane complement regulatory proteins (mCRPs: CD46, CD55, and CD59), and to evaluate the effects of a thiol antioxidant on oxidative stress–induced MP release. Methods Retinal pigment epithelium cells isolated from human donor eyes were cultured and treated with hydrogen peroxide (H2O2) to induce oxidative stress. Isolated MPs were fixed for transmission electron microscopy or processed for component analysis by flow cytometry, Western blot analysis, and confocal microscopy. Results Transmission electron microscopy showed that MPs ranged in diameter from 100 to 1000 nm. H2O2 treatment led to time- and dose-dependent elevations in MPs with externalized phosphatidylserine and phosphatidylethanolamine, known markers of MPs. These increases were strongly correlated to RPE apoptosis. Oxidative stress significantly increased the release of mCRP-positive MPs, which were prevented by a thiol antioxidant, N-acetylcysteine amide (NACA). Conclusions This is the first evidence that oxidative stress induces cultured human RPE cells to release MPs that carry mCRPs on their surface. The levels of released MPs are strongly correlated with RPE apoptosis. N-acetylcysteine amide prevents oxidative stress–induced effects. Our findings indicate that oxidative stress reduces mCRPs on the RPE surface through releasing MPs. PMID:26842754

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

Directory of Open Access Journals (Sweden)

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.

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

DEFF Research Database (Denmark)

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

2000-01-01

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

Human prostatic cancer cells, PC3, elaborate mitogenic activity which selectively stimulates human bone cells

International Nuclear Information System (INIS)

Perkel, V.S.; Mohan, S.; Herring, S.J.; Baylink, D.J.; Linkhart, T.A.

1990-01-01

Prostatic cancer typically produces osteoblastic metastases which are not attended by marrow fibrosis. In the present study we sought to test the hypothesis that prostatic cancer cells produce factor(s) which act selectively on human osteoblasts. Such a paracrine mechanism would explain the observed increase in osteoblasts, unaccompanied by an increase in marrow fibroblasts. To test this hypothesis we investigated the mitogenic activity released by the human prostatic tumor cell line, PC3. PC3 cells have been reported previously to produce mitogenic activity for cells that was relatively specific for rat osteoblasts compared to rat fibroblasts. However, the effects of this activity on human cells has not been examined previously. PC3-conditioned medium (CM) (5-50 micrograms CM protein/ml) stimulated human osteoblast proliferation by 200-950% yet did not stimulate human fibroblast proliferation ([3H]thymidine incorporation). PC3 CM also increased cell numbers in human osteoblast but not fibroblast cell cultures. To determine whether the osteoblast-specific mitogenic activity could be attributed to known bone growth factors, specific assays for these growth factors were performed. PC3 CM contained 10 pg insulin-like growth factor (IGF) I, less than 2 pg IGF II, 54 pg basic fibroblast growth factor, and 16 pg transforming growth factor beta/microgram CM protein. None of these growth factors alone or in combination could account for the observed osteoblast-specific PC3 cell-derived mitogenic activity. Furthermore, when 5 micrograms/ml PC3 CM was tested in combination with maximally effective concentrations of either basic fibroblast growth factor, IGF I, IGF II, or transforming growth factor beta, it produced an additive effect suggesting that PC3 CM stimulates osteoblast proliferation by a mechanism independent of these bone mitogens

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

Science.gov (United States)

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

2014-08-01

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

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on adipogenic differentiation and insulin-induced glucose uptake in 3T3-L1 cells

International Nuclear Information System (INIS)

Hsu, Hsin-Fen; Tsou, Tsui-Chun; Chao, How-Ran; Kuo, Ya-Ting; Tsai, Feng-Yuan; Yeh, Szu-Ching

2010-01-01

Dioxin exposure has been positively associated with human type II diabetes. Because lipophilic dioxins accumulate mainly in adipose tissue, this study aimed to determine if dioxins induce metabolic dysfunction in fat cells. Using 3T3-L1 cells as an in vitro model, we analyzed the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a model dioxin, on adipogenic differentiation, glucose uptake, and lipolysis. TCDD inhibited adipogenic differentiation, as determined by using oil droplet formation and adipogenic marker gene expression, including PPARγ (peroxisome proliferator-activated receptor γ), C/EBPα (CCAAT/enhancer-binding protein α), and Glut4 (glucose transporter type 4). Effects of TCDD on glucose uptake were evaluated using fully differentiated 3T3-L1 adipocytes, revealing that TCDD significantly attenuated insulin-induced glucose uptake dose dependently. Inhibition of aryl hydrocarbon receptor (AhR) by α-naphthoflavone (α-NF), an AhR inhibitor, did not prevent the inhibitory effect of TCDD on glucose uptake, suggesting that TCDD attenuates insulin-induced glucose uptake in an AhR-independent manner. Effects of TCDD on lipolysis were determined using glycerol release assay. We found that TCDD had no marked effect on isoproterenol-induced glycerol release in fully differentiated 3T3-L1 adipocytes. These results provide in vitro evidence of TCDD's effects on fat cell metabolism, suggesting dioxin exposure in development of insulin resistance and type II diabetes.

Munc18-1 Regulates First-phase Insulin Release by Promoting Granule Docking to Multiple Syntaxin Isoforms

NARCIS (Netherlands)

Oh, E.; Kalwat, M.A.; Kim, M.J.; Verhage, M.; Thurmond, D.C.

2012-01-01

Attenuated levels of the Sec1/Munc18 (SM) protein Munc18-1 in human islet β-cells is coincident with type 2 diabetes, although how Munc18-1 facilitates insulin secretion remains enigmatic. Herein, using conventional Munc18-1

Expression profiling of insulin action in human myotubes

DEFF Research Database (Denmark)

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

2004-01-01

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

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.

Science.gov (United States)

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.

A novel dual-color reporter for identifying insulin-producing beta-cells and classifying heterogeneity of insulinoma cell lines.

Directory of Open Access Journals (Sweden)

Nan Sook Lee

Full Text Available Many research studies use immortalized cell lines as surrogates for primary beta- cells. We describe the production and use of a novel "indirect" dual-fluorescent reporter system that leads to mutually exclusive expression of EGFP in insulin-producing (INS(+ beta-cells or mCherry in non-beta-cells. Our system uses the human insulin promoter to initiate a Cre-mediated shift in reporter color within a single transgene construct and is useful for FACS selection of cells from single cultures for further analysis. Application of our reporter to presumably clonal HIT-T15 insulinoma cells, as well as other presumably clonal lines, indicates that these cultures are in fact heterogeneous with respect to INS(+ phenotype. Our strategy could be easily applied to other cell- or tissue-specific promoters. We anticipate its utility for FACS purification of INS(+ and glucose-responsive beta-like-cells from primary human islet cell isolates or in vitro differentiated pluripotent stem 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.)

Insulin action in human thighs after one-legged immobilization

DEFF Research Database (Denmark)

Richter, Erik; Kiens, Bente; Mizuno, M.

1989-01-01

Insulin action was assessed in thighs of five healthy young males who had one knee immobilized for 7 days by a splint. The splint was not worn in bed. Subjects also used crutches to prevent weight bearing of the immobilized leg. Immobilization decreased the activity of citrate synthase and 3-OH......-acyl-CoA-dehydrogenase in the vastus lateralis muscle by 9 and 14%, respectively, and thigh volume by 5%. After 7 days of immobilization, a two-step euglycemic hyperinsulinemic clamp procedure combined with arterial and bilateral femoral venous catheterization was performed. Insulin action on glucose uptake and tyrosine release...... of the thighs at mean plasma insulin concentrations of 67 (clamp step I) and 447 microU/ml (clamp step II) was decreased by immobilization, whereas immobilization did not affect insulin action on thigh exchange of free fatty acids, glycerol, O2, or potassium. Before and during the clamp step I, lactate release...

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

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

Science.gov (United States)

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.

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

International Nuclear Information System (INIS)

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

1984-01-01

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

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

Science.gov (United States)

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.

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

Interaction of Human Enterochromaffin Cells with Human Enteric Adenovirus 41 Leads to Serotonin Release and Subsequent Activation of Enteric Glia Cells.

Science.gov (United States)

Westerberg, Sonja; Hagbom, Marie; Rajan, Anandi; Loitto, Vesa; Persson, B David; Allard, Annika; Nordgren, Johan; Sharma, Sumit; Magnusson, Karl-Eric; Arnberg, Niklas; Svensson, Lennart

2018-04-01

Human adenovirus 41 (HAdV-41) causes acute gastroenteritis in young children. The main characteristics of HAdV-41 infection are diarrhea and vomiting. Nevertheless, the precise mechanism of HAdV-41-induced diarrhea is unknown, as a suitable small-animal model has not been described. In this study, we used the human midgut carcinoid cell line GOT1 to investigate the effect of HAdV-41 infection and the individual HAdV-41 capsid proteins on serotonin release by enterochromaffin cells and on enteric glia cell (EGC) activation. We first determined that HAdV-41 could infect the enterochromaffin cells. Immunofluorescence staining revealed that the cells expressed HAdV-41-specific coxsackievirus and adenovirus receptor (CAR); flow cytometry analysis supported these findings. HAdV-41 infection of the enterochromaffin cells induced serotonin secretion dose dependently. In contrast, control infection with HAdV-5 did not induce serotonin secretion in the cells. Confocal microscopy studies of enterochromaffin cells infected with HAdV-41 revealed decreased serotonin immunofluorescence compared to that in uninfected cells. Incubation of the enterochromaffin cells with purified HAdV-41 short fiber knob and hexon proteins increased the serotonin levels in the harvested cell supernatant significantly. HAdV-41 infection could also activate EGCs, as shown in the significantly altered expression of glia fibrillary acidic protein (GFAP) in EGCs incubated with HAdV-41. The EGCs were also activated by serotonin alone, as shown in the significantly increased GFAP staining intensity. Likewise, EGCs were activated by the cell supernatant of HAdV-41-infected enterochromaffin cells. IMPORTANCE The nonenveloped human adenovirus 41 causes diarrhea, vomiting, dehydration, and low-grade fever mainly in children under 2 years of age. Even though acute gastroenteritis is well described, how human adenovirus 41 causes diarrhea is unknown. In our study, we analyzed the effect of human adenovirus 41

Autocrine growth induced by the insulin-related factor in the insulin-independent teratoma cell line 1246-3A

International Nuclear Information System (INIS)

Yamada, Yukio; Serrero, G.

1988-01-01

An insulin-independent teratoma-derived cell line, called 1246-3A, has been isolated from the adipogenic cell line 1246, which stringently requires insulin for proliferation. The 1246-3A cell line, which can proliferate in the absence of exogenous insulin, produces in its conditioned medium a growth factor similar to pancreatic insulin by its biological and immunological properties. This factor, called insulin-related factor (IRF), was purified and iodinated to study its binding to cell surface receptors. 125 I-labeled IRF binding to intact 1246-3A cells is lower than to 1246 cells. Cell surface binding can be restored by culturing the 1246-3A cells in the presence of an anti-porcine insulin monoclonal antibody of by acid prewash of the cells prior to performing the binding. Scatchard analysis of binding indicates that IRF secreted by the 1246-3A cells partially occupies high-affinity binding sites on the producer cells. Moreover, insulin monoclonal antibody inhibits the proliferation of the IRF-producing 1246-3A cells, suggesting that these cells are dependent on the secreted IRF for growth in culture. The authors conclude that the insulin-related factor secreted by the insulin-independent 1246-3A cells stimulates their proliferation in an autocrine fashion

Insulin promotes cell migration by regulating PSA-NCAM

Energy Technology Data Exchange (ETDEWEB)

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.

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

DEFF Research Database (Denmark)

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

2011-01-01

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

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.

Pancreatic Endoderm-Derived From Diabetic Patient-Specific Induced Pluripotent Stem Cell Generates Glucose-Responsive Insulin-Secreting Cells.

Science.gov (United States)

Rajaei, Bahareh; Shamsara, Mehdi; Amirabad, Leila Mohammadi; Massumi, Mohammad; Sanati, Mohammad Hossein

2017-10-01

Human-induced pluripotent stem cells (hiPSCs) can potentially serve as an invaluable source for cell replacement therapy and allow the creation of patient- and disease-specific stem cells without the controversial use of embryos and avoids any immunological incompatibility. The generation of insulin-producing pancreatic β-cells from pluripotent stem cells in vitro provides an unprecedented cell source for personal drug discovery and cell transplantation therapy in diabetes. A new five-step protocol was introduced in this study, effectively induced hiPSCs to differentiate into glucose-responsive insulin-producing cells. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, primitive gut-tube endoderm, posterior foregut, pancreatic endoderm, and endocrine precursor. Each stage of differentiation were characterized by stage-specific markers. The produced cells exhibited many properties of functional β-cells, including expression of critical β-cells transcription factors, the potency to secrete C-peptide in response to high levels of glucose and the presence of mature endocrine secretory granules. This high efficient differentiation protocol, established in this study, yielded 79.18% insulin-secreting cells which were responsive to glucose five times higher than the basal level. These hiPSCs-derived glucose-responsive insulin-secreting cells might provide a promising approach for the treatment of type I diabetes mellitus. J. Cell. Physiol. 232: 2616-2625, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

Science.gov (United States)

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

1988-11-12

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

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

Energy Technology Data Exchange (ETDEWEB)

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

1984-09-01

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

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)

Human adipose cells in vitro are either refractory or responsive to insulin, reflecting host metabolic state.

Directory of Open Access Journals (Sweden)

Vladimir A Lizunov

Full Text Available While intercellular communication processes are frequently characterized by switch-like transitions, the endocrine system, including the adipose tissue response to insulin, has been characterized by graded responses. Yet here individual cells from adipose tissue biopsies are best described by a switch-like transition between the basal and insulin-stimulated states for the trafficking of the glucose transporter GLUT4. Two statistically-defined populations best describe the observed cellular heterogeneity, representing the fractions of refractive and responsive adipose cells. Furthermore, subjects exhibiting high systemic insulin sensitivity indices (SI have high fractions of responsive adipose cells in vitro, while subjects exhibiting decreasing SI have increasing fractions of refractory cells in vitro. Thus, a two-component model best describes the relationship between cellular refractory fraction and subject SI. Since isolated cells exhibit these different response characteristics in the presence of constant culture conditions and milieu, we suggest that a physiological switching mechanism at the adipose cellular level ultimately drives systemic SI.

Cytotoxic effects of glass ionomer cements on human dental pulp stem cells correlate with fluoride release.

Science.gov (United States)

Kanjevac, Tatjana; Milovanovic, Marija; Volarevic, Vladislav; Lukic, Miodrag L; Arsenijevic, Nebojsa; Markovic, Dejan; Zdravkovic, Nebojsa; Tesic, Zivoslav; Lukic, Aleksandra

2012-01-01

Glass ionomer cements (GICs) are commonly used as restorative materials. Responses to GICs differ among cell types and it is therefore of importance to thoroughly investigate the influence of these restorative materials on pulp stem cells that are potential source for dental tissue regeneration. Eight biomaterials were tested: Fuji I, Fuji II, Fuji VIII, Fuji IX, Fuji Plus, Fuji Triage, Vitrebond and Composit. We compared their cytotoxic activity on human dental pulp stem cells (DPSC) and correlated this activity with the content of Fluoride, Aluminium and Strontium ions in their eluates. Elution samples of biomaterials were prepared in sterile tissue culture medium and the medium was tested for toxicity by an assay of cell survival/proliferation (MTT test) and apoptosis (Annexin V FITC Detection Kit). Concentrations of Fluoride, Aluminium and Strontium ions were tested by appropriate methods in the same eluates. Cell survival ranged between 79.62% (Fuji Triage) to 1.5% (Fuji Plus) and most dead DPSCs were in the stage of late apoptosis. Fluoride release correlated with cytotoxicity of GICs, while Aluminium and Strontium ions, present in significant amount in eluates of tested GICs did not. Fuji Plus, Vitrebond and Fuji VIII, which released fluoride in higher quantities than other GICs, were highly toxic to human DPSCs. Opposite, low levels of released fluoride correlated to low cytotoxic effect of Composit, Fuji I and Fuji Triage.

UVB-Stimulated TNFα Release from Human Melanocyte and Melanoma Cells Is Mediated by p38 MAPK

Directory of Open Access Journals (Sweden)

Visalini Muthusamy

2013-08-01

Full Text Available Ultraviolet (UV radiation activates cell signaling pathways in melanocytes. As a result of altered signaling pathways and UV-induced cellular damage, melanocytes can undergo oncogenesis and develop into melanomas. In this study, we investigated the effect of UV-radiation on p38 MAPK (mitogen-activated protein kinase, JNK and NFκB pathways to determine which plays a major role in stimulating TNFα secretion in human HEM (melanocytes and MM96L (melanoma cells. MM96L cells exhibited 3.5-fold higher p38 activity than HEM cells at 5 min following UVA + B radiation and 1.6-fold higher JNK activity at 15–30 min following UVB+A radiation, while NFκB was minimally activated in both cells. Irradiated HEM cells had the greatest fold of TNFα secretion (UVB: 109-fold, UVA + B: 103-fold & UVB+A: 130-fold when co-exposed to IL1α. The p38 inhibitor, SB202190, inhibited TNFα release by 93% from UVB-irradiated HEM cells. In the UVB-irradiated MM96L cells, both SB202190 and sulfasalazine (NFκB inhibitor inhibited TNFα release by 52%. Although, anisomycin was a p38 MAPK activator, it inhibited TNFα release in UV-irradiated cells. This suggests that UV-mediated TNFα release may occur via different p38 pathway intermediates compared to those stimulated by anisomycin. As such, further studies into the functional role p38 MAPK plays in regulating TNFα release in UV-irradiated melanocyte-derived cells are warranted.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on adipogenic differentiation and insulin-induced glucose uptake in 3T3-L1 cells

Energy Technology Data Exchange (ETDEWEB)

Hsu, Hsin-Fen [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli County 35053, Taiwan (China); Tsou, Tsui-Chun, E-mail: tctsou@nhri.org.tw [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli County 35053, Taiwan (China); Chao, How-Ran [Department of Environmental Science and Engineering, National Pingtung University of Science and Technology, Neipu 912, Pingtung, Taiwan (China); Kuo, Ya-Ting; Tsai, Feng-Yuan; Yeh, Szu-Ching [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli County 35053, Taiwan (China)

2010-10-15

Dioxin exposure has been positively associated with human type II diabetes. Because lipophilic dioxins accumulate mainly in adipose tissue, this study aimed to determine if dioxins induce metabolic dysfunction in fat cells. Using 3T3-L1 cells as an in vitro model, we analyzed the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a model dioxin, on adipogenic differentiation, glucose uptake, and lipolysis. TCDD inhibited adipogenic differentiation, as determined by using oil droplet formation and adipogenic marker gene expression, including PPAR{gamma} (peroxisome proliferator-activated receptor {gamma}), C/EBP{alpha} (CCAAT/enhancer-binding protein {alpha}), and Glut4 (glucose transporter type 4). Effects of TCDD on glucose uptake were evaluated using fully differentiated 3T3-L1 adipocytes, revealing that TCDD significantly attenuated insulin-induced glucose uptake dose dependently. Inhibition of aryl hydrocarbon receptor (AhR) by {alpha}-naphthoflavone ({alpha}-NF), an AhR inhibitor, did not prevent the inhibitory effect of TCDD on glucose uptake, suggesting that TCDD attenuates insulin-induced glucose uptake in an AhR-independent manner. Effects of TCDD on lipolysis were determined using glycerol release assay. We found that TCDD had no marked effect on isoproterenol-induced glycerol release in fully differentiated 3T3-L1 adipocytes. These results provide in vitro evidence of TCDD's effects on fat cell metabolism, suggesting dioxin exposure in development of insulin resistance and type II diabetes.

Glycogen metabolism in the glucose-sensing and supply-driven β-cell.

Science.gov (United States)

Andersson, Lotta E; Nicholas, Lisa M; Filipsson, Karin; Sun, Jiangming; Medina, Anya; Al-Majdoub, Mahmoud; Fex, Malin; Mulder, Hindrik; Spégel, Peter

2016-12-01

Glycogen metabolism in β-cells may affect downstream metabolic pathways controlling insulin release. We examined glycogen metabolism in human islets and in the rodent-derived INS-1 832/13 β-cells and found them to express the same isoforms of key enzymes required for glycogen metabolism. Our findings indicate that glycogenesis is insulin-independent but influenced by extracellular glucose concentrations. Levels of glycogen synthase decrease with increasing glucose concentrations, paralleling accumulation of glycogen. We did not find cAMP-elicited glycogenolysis and insulin secretion to be causally related. In conclusion, our results reveal regulated glycogen metabolism in human islets and insulin-secreting cells. Whether glycogen metabolism affects insulin secretion under physiological conditions remains to be determined. © 2016 Federation of European Biochemical Societies.

Isotope release cytotoxicity assay applicable to human tumors: the use of 111-indium

Energy Technology Data Exchange (ETDEWEB)

Frost, P; Wiltrout, R; Maciorowski, Z; Rose, N R

1977-01-01

We have demonstrated that human tumors can be labelled efficiently with the 111indium-oxine chelate. Subsequently, this isotope can be released by cytotoxic lymphoid cells. Both natural and induced cytotoxicity can be demonstrated utilizing this isotope release method. Because of the slow spontaneous release of 111indium and its efficient labelling of human tumor cells, this isotope release assay can be utilized in long-term cytotoxic assays in the study of human tumor immunology.

Bio-engineering inslulin-secreting cells from embryonic stem cells: a review of progress.

Science.gov (United States)

Roche, E; Sepulcre, M P; Enseñat-Waser, R; Maestre, I; Reig, J A; Soria, B

2003-07-01

According to the Edmonton protocol, human islet transplantation can result in insulin independency for periods longer than 3 years. However, this therapy for type 1 diabetes is limited by the scarcity of cadaveric donors. Owing to the ability of embryonic stem cells to expand in vitro and differentiate into a variety of cell types, research has focused on ways to manipulate these cells to overcome this problem. It has been demonstrated that mouse embryonic stem cells can differentiate into insulin-containing cells, restoring normoglycaemia in diabetic mice. To this end, mouse embryonic stem cells were transfected with a DNA construct that provides resistance to neomycin under the control of the regulatory regions of the human insulin gene. However, this protocol has a very low efficiency, needing improvements for this technology to be transferred to human stem cells. Optimum protocols will be instrumental in the production of an unlimited source of cells that synthesise, store and release insulin in a physiological manner. The review focuses on the alternative source of tissue offered by embryonic stem cells for regenerative medicine in diabetes and some key points that should be considered in order for a definitive protocol for in vitro differentiation to be established.

Islet-like clusters derived from mesenchymal stem cells in Wharton's Jelly of the human umbilical cord for transplantation to control type 1 diabetes.

Directory of Open Access Journals (Sweden)

Kuo Ching Chao

Full Text Available BACKGROUND: There is a widespread interest in developing renewable sources of islet-replacement tissue for type I diabetes mellitus. Human mesenchymal cells isolated from the Wharton's jelly of the umbilical cord (HUMSCs, which can be easily obtained and processed compared with embryonic and bone marrow stem cells, possess stem cell properties. HUMSCs may be a valuable source for the generation of islets. METHODOLOGY AND PRINCIPAL FINDINGS: HUMSCs were induced to transform into islet-like cell clusters in vitro through stepwise culturing in neuron-conditioned medium. To assess the functional stability of the islet-like cell clusters in vivo, these cell clusters were transplanted into the liver of streptozotocin-induced diabetic rats via laparotomy. Glucose tolerance was measured on week 12 after transplantation accompanied with immunohistochemistry and electron microscopy analysis. These islet-like cell clusters were shown to contain human C-peptide and release human insulin in response to physiological glucose levels. Real-time RT-PCR detected the expressions of insulin and other pancreatic beta-cell-related genes (Pdx1, Hlxb9, Nkx2.2, Nkx6.1, and Glut-2 in these islet-like cell clusters. The hyperglycemia and glucose intolerance in streptozotocin-induced diabetic rats was significantly alleviated after xenotransplantation of islet-like cell clusters, without the use of immunosuppressants. In addition to the existence of islet-like cell clusters in the liver, some special fused liver cells were also found, which characterized by human insulin and nuclei-positive staining and possessing secretory granules. CONCLUSIONS AND SIGNIFICANCE: In this study, we successfully differentiate HUMSCs into mature islet-like cell clusters, and these islet-like cell clusters possess insulin-producing ability in vitro and in vivo. HUMSCs in Wharton's Jelly of the umbilical cord seem to be the preferential source of stem cells to convert into insulin

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...

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.

Science.gov (United States)

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

Regulation of lipoprotein lipase in primary cultures of isolated human adipocytes

International Nuclear Information System (INIS)

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

1985-01-01

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

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.

Efficient generation of functional pancreatic β-cells from human induced pluripotent stem cells.

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Yabe, Shigeharu G; Fukuda, Satsuki; Takeda, Fujie; Nashiro, Kiyoko; Shimoda, Masayuki; Okochi, Hitoshi

2017-02-01

Insulin-secreting cells have been generated from human embryonic or induced pluripotent stem cells (iPSCs) by mimicking developmental processes. However, these cells do not always secrete glucose-responsive insulin, one of the most important characteristics of pancreatic β-cells. We focused on the importance of endodermal differentiation from human iPSCs in order to obtain functional pancreatic β-cells. A six-stage protocol was established for the differentiation of human iPSCs to pancreatic β-cells using defined culture media without feeders or serum. The effects of CHIR99021, a selective glycogen synthase kinase-3β inhibitor, were examined in the presence of fibroblast growth factor 2, activin, and bone morphogenetic protein 4 (FAB) during definitive endodermal induction by immunostaining for SRY (sex determining region Y)-box 17 (SOX17) and Forkhead box protein A2 (FOXA2). Insulin secretion was compared between the last stage of monolayer culture and spheroid culture conditions. Cultured cells were transplanted under kidney capsules of streptozotocin-diabetic non-obese diabetic-severe combined immunodeficiency mice, and blood glucose levels were measured once a week. Immunohistochemical analyses were performed 4 and 12 weeks after transplantation. Addition of CHIR99021 (3 μmol/L) in the presence of FAB for 2 days improved endodermal cell viability, maintaining the high SOX17-positive rate. Spheroid formation after the endocrine progenitor stage showed more efficient insulin secretion than did monolayer culture. After cell transplantation, diabetic mice had lower blood glucose levels, and islet-like structures were detected in vivo. Functional pancreatic β-cells were generated from human iPSCs. Induction of definitive endoderm and spheroid formation may be key steps for producing these cells. © 2016 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

In vivo response of Mesocestoides vogae to human insulin.

Science.gov (United States)

Canclini, L; Esteves, A

2009-02-01

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

Human embryonic stem cell derived islet progenitors mature inside an encapsulation device without evidence of increased biomass or cell escape.

Science.gov (United States)

Kirk, Kaitlyn; Hao, Ergeng; Lahmy, Reyhaneh; Itkin-Ansari, Pamela

2014-05-01

There are several challenges to successful implementation of a cell therapy for insulin dependent diabetes derived from human embryonic stem cells (hESC). Among these are development of functional insulin producing cells, a clinical delivery method that eliminates the need for chronic immunosuppression, and assurance that hESC derived tumors do not form in the patient. We and others have shown that encapsulation of cells in a bilaminar device (TheraCyte) provides immunoprotection in rodents and primates. Here we monitored human insulin secretion and employed bioluminescent imaging (BLI) to evaluate the maturation, growth, and containment of encapsulated islet progenitors derived from CyT49 hESC, transplanted into mice. Human insulin was detectable by 7 weeks post-transplant and increased 17-fold over the course of 8 weeks, yet during this period the biomass of encapsulated cells remained constant. Remarkably, by 20 weeks post-transplant encapsulated cells secreted sufficient levels of human insulin to ameliorate alloxan induced diabetes. Further, bioluminescent imaging revealed for the first time that hESCs remained fully contained in encapsulation devices for up to 150 days, the longest period tested. Collectively, the data suggest that encapsulated hESC derived islet progenitors hold great promise as an effective and safe cell replacement therapy for insulin dependent diabetes. Copyright © 2014. Published by Elsevier B.V.

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

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

Science.gov (United States)

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.

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...

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...

Generation of L cells in mouse and human small intestine organoids

DEFF Research Database (Denmark)

Petersen, Natalia; Reimann, Frank; Bartfeld, Sina

2014-01-01

Upon a nutrient challenge, L cells produce glucagon-like peptide 1 (GLP-1), a powerful stimulant of insulin release. Strategies to augment endogenous GLP-1 production include promoting L-cell differentiation and increasing L-cell number. Here we present a novel in vitro platform to generate funct...

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...

NK cell-released exosomes: Natural nanobullets against tumors.

Science.gov (United States)

Fais, Stefano

2013-01-01

We have recently reported that human natural killer (NK) cells release exosomes that express both NK-cell markers and cytotoxic molecules. Similar results were obtained with circulating exosomes from human healthy donors. Both NK-cell derived and circulating exosomes exerted a full functional activity and killed both tumor and activated immune cells. These findings indicate that NK-cell derived exosomes might constitute a new promising therapeutic tool.

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...

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

Science.gov (United States)

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

2014-01-01

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

Rupatadine inhibits inflammatory mediator release from human laboratory of allergic diseases 2 cultured mast cells stimulated by platelet-activating factor.

Science.gov (United States)

Alevizos, Michail; Karagkouni, Anna; Vasiadi, Magdalini; Sismanopoulos, Nikolaos; Makris, Michael; Kalogeromitros, Dimitrios; Theoharides, Theoharis C

2013-12-01

Mast cells are involved in allergy and inflammation by the secretion of multiple mediators, including histamine, cytokines, and platelet-activating factor (PAF), in response to different triggers, including emotional stress. PAF has been associated with allergic inflammation, but there are no clinically available PAF inhibitors. To investigate whether PAF could stimulate human mast cell mediator release and whether rupatadine (RUP), a dual histamine-1 and PAF receptor antagonist, could inhibit the effect of PAF on human mast cells. Laboratory of allergic diseases 2 cultured mast cells were stimulated with PAF (0.001, 0.01, and 0.1 μmol/L) and substance P (1 μmol/L) with or without pretreatment with RUP (2.5 and 25 μmol/L), which was added 10 minutes before stimulation. Release of β-hexosaminidase was measured in supernatant fluid by spectrophotoscopy, and histamine, interleukin-8, and tumor necrosis factor were measured by enzyme-linked immunosorbent assay. PAF stimulated a statistically significant release of histamine, interleukin-8, and tumor necrosis factor (0.001-0.1 μmol/L) that was comparable to that stimulated by substance P. Pretreatment with RUP (25 μmol/L) for 10 minutes inhibited this effect. In contrast, pretreatment of laboratory of allergic diseases 2 cells with diphenhydramine (25 μmol/L) did not inhibit mediator release, suggesting that the effect of RUP was not due to its antihistaminic effect. PAF stimulates human mast cell release of proinflammatory mediators that is inhibited by RUP. This action endows RUP with additional properties in treating allergic inflammation. Copyright © 2013 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

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.

Metformin Ameliorates Dysfunctional Traits of Glibenclamide- and Glucose-Induced Insulin Secretion by Suppression of Imposed Overactivity of the Islet Nitric Oxide Synthase-NO System.

Directory of Open Access Journals (Sweden)

Ingmar Lundquist

Full Text Available Metformin lowers diabetic blood glucose primarily by reducing hepatic gluconeogenesis and increasing peripheral glucose uptake. However, possible effects by metformin on beta-cell function are incompletely understood. We speculated that metformin might positively influence insulin secretion through impacting the beta-cell nitric oxide synthase (NOS-NO system, a negative modulator of glucose-stimulated insulin release. In short-time incubations with isolated murine islets either glibenclamide or high glucose augmented insulin release associated with increased NO production from both neural and inducible NOS. Metformin addition suppressed the augmented NO generation coinciding with amplified insulin release. Islet culturing with glibenclamide or high glucose revealed pronounced fluorescence of inducible NOS in the beta-cells being abolished by metformin co-culturing. These findings were reflected in medium nitrite-nitrate levels. A glucose challenge following islet culturing with glibenclamide or high glucose revealed markedly impaired insulin response. Metformin co-culturing restored this response. Culturing murine islets and human islets from controls and type 2 diabetics with high glucose or high glucose + glibenclamide induced a pronounced decrease of cell viability being remarkably restored by metformin co-culturing. We show here, that imposed overactivity of the beta-cell NOS-NO system by glibenclamide or high glucose leads to insulin secretory dysfunction and reduced cell viability and also, importantly, that these effects are relieved by metformin inhibiting beta-cell NO overproduction from both neural and inducible NOS thus ameliorating a concealed negative influence by NO induced by sulfonylurea treatment and/or high glucose levels. This double-edged effect of glibenclamide on the beta-cellsuggests sulfonylurea monotherapy in type 2 diabetes being avoided.

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

Piezo1 regulates mechanotransductive release of ATP from human RBCs.

Science.gov (United States)

Cinar, Eyup; Zhou, Sitong; DeCourcey, James; Wang, Yixuan; Waugh, Richard E; Wan, Jiandi

2015-09-22

Piezo proteins (Piezo1 and Piezo2) are recently identified mechanically activated cation channels in eukaryotic cells and associated with physiological responses to touch, pressure, and stretch. In particular, human RBCs express Piezo1 on their membranes, and mutations of Piezo1 have been linked to hereditary xerocytosis. To date, however, physiological functions of Piezo1 on normal RBCs remain poorly understood. Here, we show that Piezo1 regulates mechanotransductive release of ATP from human RBCs by controlling the shear-induced calcium (Ca(2+)) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca(2+) influx decrease significantly. Remarkably, a critical extracellular Ca(2+) concentration is required to trigger significant ATP release, but membrane-associated ATP pools in RBCs also contribute to the release of ATP. Our results show how Piezo1 channels are likely to function in normal RBCs and suggest a previously unidentified mechanotransductive pathway in ATP release. Thus, we anticipate that the study will impact broadly on the research of red cells, cellular mechanosensing, and clinical studies related to red cell disorders and vascular disease.

Lesion-induced increase in survival and migration of human neural progenitor cells releasing GDNF

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Behrstock, Soshana; Ebert, Allison D.; Klein, Sandra; Schmitt, Melanie; Moore, Jeannette M.; Svendsen, Clive N.

2009-01-01

The use of human neural progenitor cells (hNPC) has been proposed to provide neuronal replacement or astrocytes delivering growth factors for brain disorders such as Parkinson’s and Huntington’s disease. Success in such studies likely requires migration from the site of transplantation and integration into host tissue in the face of ongoing damage. In the current study, hNPC modified to release glial cell line derived neurotrophic factor (hNPCGDNF) were transplanted into either intact or lesioned animals. GDNF release itself had no effect on the survival, migration or differentiation of the cells. The most robust migration and survival was found using a direct lesion of striatum (Huntington’s model) with indirect lesions of the dopamine system (Parkinson’s model) or intact animals showing successively less migration and survival. No lesion affected differentiation patterns. We conclude that the type of brain injury dictates migration and integration of hNPC which has important consequences when considering transplantation of these cells as a therapy for neurodegenerative diseases. PMID:19044202

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

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.

An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

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Massumi, Mohammad; Pourasgari, Farzaneh; Nalla, Amarnadh

2016-01-01

developed an abbreviated five-stage protocol (25-30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems......The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have...... positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux...

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

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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.

An Information Theoretical Analysis of Human Insulin-Glucose System Toward the Internet of Bio-Nano Things.

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Abbasi, Naveed A; Akan, Ozgur B

2017-12-01

Molecular communication is an important tool to understand biological communications with many promising applications in Internet of Bio-Nano Things (IoBNT). The insulin-glucose system is of key significance among the major intra-body nanonetworks, since it fulfills metabolic requirements of the body. The study of biological networks from information and communication theoretical (ICT) perspective is necessary for their introduction in the IoBNT framework. Therefore, the objective of this paper is to provide and analyze for the first time in the literature, a simple molecular communication model of the human insulin-glucose system from ICT perspective. The data rate, channel capacity, and the group propagation delay are analyzed for a two-cell network between a pancreatic beta cell and a muscle cell that are connected through a capillary. The results point out a correlation between an increase in insulin resistance and a decrease in the data rate and channel capacity, an increase in the insulin transmission rate, and an increase in the propagation delay. We also propose applications for the introduction of the system in the IoBNT framework. Multi-cell insulin glucose system models may be based on this simple model to help in the investigation, diagnosis, and treatment of insulin resistance by means of novel IoBNT applications.

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

Generation of Functional Beta-Like Cells from Human Exocrine Pancreas.

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Maria J Lima

Full Text Available Transcription factor mediated lineage reprogramming of human pancreatic exocrine tissue could conceivably provide an unlimited supply of islets for transplantation in the treatment of diabetes. Exocrine tissue can be efficiently reprogrammed to islet-like cells using a cocktail of transcription factors: Pdx1, Ngn3, MafA and Pax4 in combination with growth factors. We show here that overexpression of exogenous Pax4 in combination with suppression of the endogenous transcription factor ARX considerably enhances the production of functional insulin-secreting β-like cells with concomitant suppression of α-cells. The efficiency was further increased by culture on laminin-coated plates in media containing low glucose concentrations. Immunocytochemistry revealed that reprogrammed cultures were composed of ~45% islet-like clusters comprising >80% monohormonal insulin+ cells. The resultant β-like cells expressed insulin protein levels at ~15-30% of that in adult human islets, efficiently processed proinsulin and packaged insulin into secretory granules, exhibited glucose responsive insulin secretion, and had an immediate and prolonged effect in normalising blood glucose levels upon transplantation into diabetic mice. We estimate that approximately 3 billion of these cells would have an immediate therapeutic effect following engraftment in type 1 diabetes patients and that one pancreas would provide sufficient tissue for numerous transplants.

Selective insulin resistance in hepatocyte senescence

International Nuclear Information System (INIS)

Aravinthan, Aloysious; Challis, Benjamin; Shannon, Nicholas; Hoare, Matthew; Heaney, Judith; Alexander, Graeme J.M.

2015-01-01

Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence. - Highlights: • Senescent hepatocytes demonstrate selective insulin resistance. • GLUT changes act as markers of hepatocyte senescence and have prognostic value. • Study offers insight into long noticed intimacy of cirrhosis and insulin resistance

Selective insulin resistance in hepatocyte senescence

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Aravinthan, Aloysious [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Challis, Benjamin [Institute of Metabolic Sciences, University of Cambridge, Cambridge (United Kingdom); Shannon, Nicholas [Cancer Research UK Cambridge Institute, Cambridge (United Kingdom); Hoare, Matthew [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Cancer Research UK Cambridge Institute, Cambridge (United Kingdom); Heaney, Judith [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Foundation for Liver Research, Institute of Hepatology, London (United Kingdom); Alexander, Graeme J.M., E-mail: gja1000@doctors.org.uk [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom)

2015-02-01

Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence. - Highlights: • Senescent hepatocytes demonstrate selective insulin resistance. • GLUT changes act as markers of hepatocyte senescence and have prognostic value. • Study offers insight into long noticed intimacy of cirrhosis and insulin resistance.

The role of polyunsaturated fatty acids (n-3 PUFAs) on the pancreatic β-cells and insulin action.

Science.gov (United States)

Baynes, Habtamu Wondifraw; Mideksa, Seifu; Ambachew, Sintayehu

2018-03-14

Polyunsaturated Fatty acids have multiple effects in peripheral tissues and pancreatic beta cell function. The n-3 Polyunsaturated Fatty acids prevent and reverse high-fat-diet induced adipose tissue inflammation and insulin resistance. Insulin secretion is stimulated by glucose, amino acids, and glucagon- like peptide-1 in tissue containing high levels of n-3 Polyunsaturated Fatty acids than lower level of n-3 Polyunsaturated Fatty acids. Also, n-3 Polyunsaturated Fatty acids led to decreased production of prostaglandin, which in turn contributed to the elevation of insulin secretion. N-3 polyunsaturated fatty acids prevent cytokine-induced cell death in pancreatic islets. Supplementation of n-3 Polyunsaturated Fatty acids for human subjects prevent beta cell destruction and insulin resistance. It also enhances insulin secretion, reduction in lipid profiles and glucose concentration particularly in type II diabetes patients. Therefore there should be a focus on the treatment mechanism of insulin related obesity and diabetes by n-3 polyunsaturated fatty acids.

Histamine and chondroitin sulfate E proteoglycan released by cultured human colonic mucosa: indication for possible presence of E mast cells

International Nuclear Information System (INIS)

Eliakim, R.; Gilead, L.; Ligumsky, M; Okon, E.; Rachmilewitz, D.; Razin, E.

1986-01-01

An association between the release of histamine and chondroitin sulfate E proteoglycan (PG) was demonstrates in human colonic mucosa (HCM). Colonic biopsy samples incorporated [ 35 S]sulfate into PG, which was partially released into the culture medium during the incubation period. Ascending thin-layer chromatography of the released 35 S-labeled PG after its digestion by chondroitin ABC lyase (chondroitinase, EC 4.2.2.4) followed by autoradiography yielded three products that migrated in the position of monosulfated disaccharides of N-acetylgalactosamine 4-sulfate and N-acetylgalactosoamine 6-sulfate and of an oversulfated disaccharide possessing N-acetylgalatosamine 4,6-disulfate. Cultured colonic mucosa released 23.6 +/- 3.7ng of histamine per mg of wet tissue without any special trigger. Comparison by linear regression analysis of the release of histamine and chondroitin [ 35 S]sulfate E PG revealed a correlation coefficient (r) of 0.7. Histological examination of the colonic biopsies revealed the presence of many mast cells in various degrees of degranulation in the mucosa and submucosa. The above correlation, the observation that most of the mast cells showed various degrees of degranulation, and the lack of heparin synthesis as opposed to the synthesis and immunological release of chondroitin sulfate E strongly suggest that the E mast cell exists in the human colon

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.

Intermittent Hypoxia Disrupts Glucose Homeostasis in Liver Cells in an Insulin-Dependent and Independent Manner

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Chen Juan Gu

2018-05-01

Full Text Available Background/Aims: Obstructive sleep apnea is associated with diabetes and insulin resistance, but the underlying mechanisms remain unclear. The purpose of the current study was to determine the molecular effects of intermittent hypoxia (IH on hepatic insulin signaling and glucose homeostasis, and whether c-Jun NH2-terminal-kinase (JNK contributed to metabolic responses to IH in liver cells. Methods: The human HepG2 cells and rat FAO cells were exposed to 10, 30, 120, 240 or 360 cycles of IH (1% O2 for 60 s followed by 21% O2 for 60s, 7.5 cycles per hour or normoxia as a control. In a subgroup, we exposed cells to 360 cycles of IH with the JNK inhibitor SP600125. After IH exposure, cell glycogen content and glucose output were measured using colorimetric assay kits. Canonical insulin signaling and gluconeogenic genes were measured by western blot and quantitative polymerase chain reaction. Results: IH decreased insulin-stimulated protein kinase B (AKT/glycogen synthase kinase-3β (GSK-3β phosphorylation in a time-dependent manner, while inhibiting forkhead box protein O1 (FOXO1 expression and phosphoenolpyruvate carboxykinase (PEPCK transcription independent of insulin signaling. JNK inhibitor SP600125 partially restored AKT/ GSK-3β phosphorylation and glycogen synthesis, but did not affect other IH-induced glucose metabolic changes. Conclusion: IH in vitro impaired insulin signal transduction in liver cells as assessed by inhibited AKT/GSK-3β phosphorylation via JNK activation. IH inhibited FOXO1 and gluconeogenesis in an insulin-independent manner.

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

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Nisr, Raid B; Affourtit, Charles

2014-02-01

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

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

Science.gov (United States)

Nisr, Raid B.; Affourtit, Charles

2014-01-01

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

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

Science.gov (United States)

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

2015-06-01

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

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

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

Bacteria-induced histamine release from human bronchoalveolar cells and blood leukocytes

DEFF Research Database (Denmark)

Clementsen, P; Milman, N; Struve-Christensen, E

1991-01-01

23187 resulted in histamine release. S. aureus-induced histamine release from basophils was examined in leukocyte suspensions obtained from the same individuals, and in all experiments release was found. The dose-response curves were similar to those obtained with BAL cells. The bacteria...

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