Decrease In Glucose-stimulated Insulin Secretion Research Articles

Vitamin A influences the incretin hormone profiles by activating the retinoic acid receptor β

BackgroundThis study aimed to investigate the impact of Vitamin A (VA) on intestinal glucose metabolic phenotypes. MethodsMale C57BL/6 mice were randomized assigned to a VA-normal diet (VAN) or a VA-deficient diet (VAD) for 12 weeks. After12 weeks, the VAD mice were given 30 IU/g/d retinol for 10 days and VAN diet (VADN) for 10 weeks. By using glucose tolerance tests, immunofluorescence staining, quantitative polymerase chain reaction, siRNA transduction, and enzyme-linked immunosorbent assay, the glucose metabolic phenotypes as well as secretory function and intracellular hormone changes of STC-1 were assessed. ResultsVAD mice showed a decrease of glucose-stimulated insulin secretion and a loss of intestinal glucagon-like peptide-1 (GLP-1) expression. Through reintroducing dietary VA to VAD mice, the intestinal VA levels, GLP-1 expression and normal glucose can be restored. The incubation with retinol increased VA signaling factors expression within STC-1 cells, especially retinoic acid receptor β (RARβ). The activation of RARβ restored intracellular incretin hormone synthesis and secretory function. ConclusionsVA deficiency leads to an imbalance of intestinal glucose metabolic phenotypes through a mechanism involving RARβ signaling pathway, suggesting a new method to achieve the treatment for VAD induced glucose metabolism impairment.

The m6A Methyltransferase METTL3 Ameliorates Methylglyoxal-Induced Impairment of Insulin Secretion in Pancreatic β Cells by Regulating MafA Expression.

Methylglyoxal, a major precursor of advanced glycation end products, is elevated in the plasma of patients with type 2 diabetes mellitus. Islet β-cell function was recently shown to be regulated by N6-methyladenosine (m6A), an RNA modification consisting of methylation at the N6 position of adenosine. However, the role of m6A methylation modification in methylglyoxal-induced impairment of insulin secretion in pancreatic β cells has not been clarified. In this study, we showed that treatment of two β-cell lines, NIT-1 and β-TC-6, with methylglyoxal reduced m6A RNA content and methyltransferase-like 3 (METTL3) expression levels. We also showed that silencing of METTL3 inhibited glucose-stimulated insulin secretion (GSIS) from NIT-1 cells, whereas upregulation of METTL3 significantly reversed the methylglyoxal-induced decrease in GSIS. The methylglyoxal-induced decreases in m6A RNA levels and METTL3 expression were not altered by knockdown of the receptor for the advanced glycation end product but were further decreased by silencing of glyoxalase 1. Mechanistic investigations revealed that silencing of METTL3 reduced m6A levels, mRNA stability, and the mRNA and protein expression levels of musculoaponeurotic fibrosarcoma oncogene family A (MafA). Overexpression of MafA greatly improved the decrease in GSIS induced by METTL3 silencing; silencing of MafA blocked the reversal of the MG-induced decrease in GSIS caused by METTL3 overexpression. The current study demonstrated that METTL3 ameliorates MG-induced impairment of insulin secretion in pancreatic β cells by regulating MafA.

Chebulic Acid Prevents Methylglyoxal-Induced Mitochondrial Dysfunction in INS-1 Pancreatic β-Cells.

To investigate the anti-diabetic properties of chebulic acid (CA) associated with the prevention of methyl glyoxal (MG)-induced mitochondrial dysfunction in INS-1 pancreatic β-cells, INS-1 cells were pre-treated with CA (0.5, 1.0, and 2.0 μM) for 48 h and then treated with 2 mM MG for 8 h. The effects of CA and MG on INS-1 cells were evaluated using the following: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; glyoxalase 1 (Glo-1) expression via Western blot and enzyme activity assays; Nrf-2, nuclear factor erythroid 2-related factor 2 protein expression via Western blot assay; reactive oxygen species (ROS) production assay; mRNA expression of mitochondrial dysfunction related components (UCP2, uncoupling protein 2; VDAC1, voltage-dependent anion-selective channel-1; cyt c, cytochrome c via quantitative reverse transcriptase-PCR; mitochondrial membrane potential (MMP); adenosine triphosphate (ATP) synthesis; glucose-stimulated insulin secretion (GSIS) assay. The viability of INS-1 cells was maintained upon pre-treating with CA before exposure to MG. CA upregulated Glo-1 protein expression and enzyme activity in INS-1 cells and prevented MG-induced ROS production. Mitochondrial dysfunction was alleviated by CA pretreatment; this occurred via the downregulation of UCP2, VDAC1, and cyt c mRNA expression and the increase of MMP and ATP synthesis. Further, CA pre-treatment promoted the recovery from MG-induced decrease in GSIS. These results indicated that CA could be employed as a therapeutic agent in diabetes due to its ability to prevent MG-induced development of insulin sensitivity and oxidative stress-induced dysfunction of β-cells.

VH+-ATPase-induced intracellular acidification is critical to glucose-stimulated insulin secretion in beta cells.

Swelling of secretory vesicles is critical for the regulated release of intra-vesicular contents from cells during secretion. At the secretory vesicle membrane of the exocrine pancreas and neurons, GTP-binding G proteins, vH+-ATPase, potassium channels and AQP water channels, are among the players implicated in vesicle volume regulation. Here we report in the endocrine insulin-secreting MIN6 cells, the similar requirement of vH+-ATPase-mediated intracellular acidification on glucose-stimulated insulin release. MIN6 cells exposed to the vH+-ATPase inhibitor Bafilomycin A show decreased acidification of the cytosolic compartment that include insulin-carrying granules. Additionally, a loss of insulin granules near the cell plasma membrane following Bafilomycin A treatment, suggests impaired transport of insulin granules and consequent decrease in glucose-stimulated insulin secretion and accumulation of intracellular insulin. These results suggest that vH+-ATPase-mediated intracellular acidification is required for insulin secretion in beta cells.

38-OR: Beta-Arrestin-1 Regulates Pancreatic Beta-Cell Function and Plays a Key Role in Maintaining Whole Body Glucose Homeostasis

Beta-arrestin-1 and -2 (barr1 and barr2, respectively) are major GPCR-associated signaling molecules that are widely expressed throughout the body. Studies with whole body barr1 and barr2 KO mice have shown that beta-arrestins play important roles in key metabolic functions. In this study, we inactivated the barr1 gene in a conditional fashion specifically in beta-cells of adult mice (beta-barr1-KO mice). In vivo studies showed that beta-barr1-KO mice exhibited a pronounced impairment in glucose tolerance and glucose-stimulated insulin secretion (GSIS), when mice were maintained on high-fat diet (HFD). Studies with isolated perifused pancreatic islets prepared from HFD beta-barr1-KO mice confirmed that barr1 deletion in beta-cells led to a significant decrease in GSIS. Interestingly, HFD beta-barr1-KO mice displayed pronounced reductions in beta-cell mass and the rate of beta-cell proliferation, suggesting that barr1 is required for the expansion of beta-cell mass that occurs during HFD feeding. To test whether enhanced barr1 signaling in beta-cells might ameliorate the metabolic deficits associated with the consumption of a HFD, we also generated transgenic mice over-expressing barr1 in beta-cells (RIPII-barr1 mice). These transgenic mice displayed metabolic phenotypes that were opposite to those observed with the beta-barr1-KO mice, including improved glucose tolerance and GSIS in vivo as well as increased beta-cell mass. This is the first study demonstrating that barr1 is critical for the proper function of pancreatic beta-cells and for maintaining whole body glucose homeostasis. Identification of the cellular mechanisms through which barr1 signaling promotes beta-cell function and proliferation may lead to the development of novel drugs useful for the treatment of type 2 diabetes. Disclosure L.F. Barella: None. M. Rossi: None. S. Pydi: None. S. Jain: None. L. Zhu: None. W. Chen: None. J. Wess: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases

180-OR: Tissue-Specific Functions of PPARα Overexpression in Liver, Adipose, and Pancreatic ß Cells

Peroxisome proliferator-activated receptor (PPAR) α is one of the master regulators of lipid metabolism and the major target of fibrates, a class of drugs improving dyslipidemia in metabolic syndromes. To understand the tissue-specific roles of PPARα and the coordination among multiple organs, we employed newly generated mouse models enabling doxycycline-inducible overexpression of PPARα specifically in liver, adipose or the insulin-producing pancreatic β-cells. Titrated by doxycycline dose, a moderate elevation of PPARα proteins in liver activated the transcription of its target genes. In mice with diet-induced obesity, hepatic overexpression of PPARα significantly reduced steatosis and improved systemic insulin sensitivity. Surprisingly, systemic triglyceride clearance was dramatically impaired, associated with a trend towards faster VLDL secretion. PPARα overexpression in adipose tissues had a marginal effect on systemic insulin sensitivity. However, inflammation was markedly ameliorated in white but not brown fat pads. Pancreatic β-cell-specific overexpression of PPARα resulted in a significant decrease in glucose-stimulated insulin secretion, suggesting a negative impact of PPARα induction on β-cell function. Taken together, our findings reveal the differential actions of enhanced PPARα on local tissues and whole-body metabolism and call for caution to the potential adverse effects from fibrate treatment. Further dissection of PPARα signaling with our mouse models may facilitate the discovery of novel therapeutic targets with high efficacy and specificity. Disclosure R. Ye: None. P.E. Scherer: None. Funding National Institutes of Health (R01DK55758, R01DK099110, P01-DK088761-01); JDRF (17-2012-36); Novo Nordisk Foundation (to P.E.S.)

Early deficits in insulin secretion, beta cell mass and islet blood perfusion precede onset of autoimmune type 1 diabetes in BioBreeding rats

Aims/hypothesisGenetic studies show coupling of genes affecting beta cell function to type 1 diabetes, but hitherto no studies on whether beta cell dysfunction could precede insulitis and clinical onset of type 1 diabetes are available.MethodsWe used 40-day-old BioBreeding (BB) DRLyp/Lyp rats (a model of spontaneous autoimmune type 1 diabetes) and diabetes-resistant DRLyp/+ and DR+/+ littermates (controls) to investigate beta cell function in vivo, and insulin and glucagon secretion in vitro. Beta cell mass was assessed by optical projection tomography (OPT) and morphometry. Additionally, measurements of intra-islet blood flow were performed using microsphere injections. We also assessed immune cell infiltration, cytokine expression in islets (by immunohistochemistry and qPCR), as well as islet Glut2 expression and ATP/ADP ratio to determine effects on glucose uptake and metabolism in beta cells.ResultsDRLyp/Lyp rats were normoglycaemic and without traces of immune cell infiltrates. However, IVGTTs revealed a significant decrease in the acute insulin response to glucose compared with control rats (1685.3 ± 121.3 vs 633.3 ± 148.7; p < 0.0001). In agreement, insulin secretion was severely perturbed in isolated islets, and both first- and second-phase insulin release were lowered compared with control rats, while glucagon secretion was similar in both groups. Interestingly, after 5–7 days of culture of islets from DRLyp/Lyp rats in normal media, glucose-stimulated insulin secretion (GSIS) was improved; although, a significant decrease in GSIS was still evident compared with islets from control rats at this time (7393.9 ± 1593.7 vs 4416.8 ± 1230.5 pg islet−1 h−1; p < 0.0001). Compared with controls, OPT of whole pancreas from DRLyp/Lyp rats revealed significant reductions in medium (4.1 × 109 ± 9.5 × 107 vs 3.8 × 109 ± 5.8 × 107 μm3; p = 0.044) and small sized islets (1.6 × 109 ± 5.1 × 107 vs 1.4 × 109 ± 4.5 × 107 μm3; p = 0.035). Finally, we found lower intra-islet blood perfusion in vivo (113.1 ± 16.8 vs 76.9 ± 11.8 μl min−1 [g pancreas]−1; p = 0.023) and alterations in the beta cell ATP/ADP ratio in DRLyp/Lyp rats vs control rats.Conclusions/interpretationThe present study identifies a deterioration of beta cell function and mass, and intra-islet blood flow that precedes insulitis and diabetes development in animals prone to autoimmune type 1 diabetes. These underlying changes in islet function may be previously unrecognised factors of importance in type 1 diabetes development.

Angiotensin-converting enzyme 2 regulates mitochondrial function in pancreatic β-cells

Mitochondrial metabolism plays an essential role in the regulation of insulin release and glucose homeostasis. Evidence demonstrated that the angiotensin-converting enzyme 2 (ACE2) participates in the regulation of glucose metabolism, however, its role in mitochondrial metabolism remains unclear. The purpose of our study was to determine if ACE2 can regulate mitochondrial function in pancreatic β-cells. We found that ACE2 over-expression restored glucose-stimulated insulin secretion (GSIS) and mitochondrial membrane potential (MMP) in the presence of H2O2 in INS-1 cells. PCR array demonstrated that ACE2 over-expression up-regulated 67 mitochondria-related genes in INS-1 cells. In pancreatic islets, ACE2 ablation attenuated intracellular calcium influx with a decrease in GSIS. Ace2−/y mice islets exhibited impaired mitochondrial respiration and lower production of ATP, along with decreased expression of genes involved in mitochondrial oxidation. In islets from db/db mice, ACE2 over-expression increased intracellular calcium influx and restored impaired mitochondrial oxidation, potentially causing an increase in GSIS. These results shed light on the potential roles of ACE2 in mitochondrial metabolism, moreover, may improve our understanding of diabetes.

Markers of Islet Endothelial Dysfunction Occur in Male B6.BKS(D)-Leprdb/J Mice and May Contribute to Reduced Insulin Release.

Islet endothelial cells produce paracrine factors that support β-cell function and growth. Endothelial dysfunction underlies diabetic microvascular complications; thus, we hypothesized that in diabetes, islet endothelial cells become dysfunctional, which may contribute to β-cell secretory dysfunction. Islets/islet endothelial cells were isolated from diabetic B6.BKS(D)-Leprdb/J male (db/db) mice, treated with or without the glucose-lowering agent phlorizin, or from C57BL/6J mice fed a high-fat diet for 18 weeks and appropriate controls. Messenger RNA (mRNA) and/or the protein levels of the cell adhesion molecule E-selectin (Sele), proinflammatory cytokine interleukin-6 (Il6), vasoconstrictor endothelin-1 (Edn1), and endothelial nitric oxide synthase (Nos3; Nos3) were evaluated, along with advanced glycation end product immunoreactivity. Furthermore, an islet endothelial cell line (MS-1) was exposed to diabetic factors (glucose, palmitate, insulin, and tumor necrosis factor-α) for six days. Conditioned media were collected from these cells, incubated with isolated islets, and glucose-stimulated insulin secretion and insulin content were assessed. Islet endothelial cells from db/db mice exhibited increased Sele, Il6, and Edn1 mRNA levels, decreased Nos3 protein, and accumulation of advanced glycation end products. Phlorizin treatment significantly increased Nos3 protein levels but did not alter expression of the other markers. High-fat feeding in C57BL/6J mice resulted in increased islet Sele, Il6, and Edn1 but no change in Nos3. Exposure of islets to conditioned media from MS-1 cells cultured in diabetic conditions resulted in a 50% decrease in glucose-stimulated insulin secretion and 30% decrease in insulin content. These findings demonstrate that, in diabetes, islet endothelial cells show evidence of a dysfunctional phenotype, which may contribute to loss of β-cell function.

The Glucotoxicity Protecting Effect of Ezetimibe in Pancreatic Beta Cells via Inhibition of CD36.

Inhibition of CD36, a fatty acid transporter, has been reported to prevent glucotoxicity and ameliorate high glucose induced beta cell dysfunction. Ezetimibe is a selective cholesterol absorption inhibitor that blocks Niemann Pick C1-like 1 protein, but may exert its effect through suppression of CD36. We attempted to clarify the beneficial effect of ezetimibe on insulin secreting cells and to determine whether this effect is related to change of CD36 expression. mRNA expression of insulin and CD36, intracellular peroxide level and glucose stimulated insulin secretion (GSIS) under normal (5.6 mM) or high glucose (30 mM) condition in INS-1 cells and primary rat islet cells were compared. Changes of the aforementioned factors with treatment with ezetimibe (20 μM) under normal or high glucose condition were also assessed. mRNA expression of insulin was decreased with high glucose, which was reversed by ezetimibe in both INS-1 cells and primary rat islets. CD36 mRNA expression was increased with high glucose, but decreased by ezetimibe in INS-1 cells and primary rat islets. Three-day treatment with high glucose resulted in an increase in intracellular peroxide level; however, it was decreased by treatment with ezetimibe. Decrease in GSIS by three-day treatment with high glucose was reversed by ezetimibe. Palmitate uptake following exposure to high glucose conditions for three days was significantly elevated, which was reversed by ezetimibe in INS-1 cells. Ezetimibe may prevent glucotoxicity in pancreatic β-cells through a decrease in fatty acid influx via inhibition of CD36.

The cannabinoid CB1 receptor and mTORC1 signalling pathways interact to modulate glucose homeostasis in mice.

ABSTRACTThe endocannabinoid system (ECS) is an intercellular signalling mechanism that is present in the islets of Langerhans and plays a role in the modulation of insulin secretion and expansion of the β-cell mass. The downstream signalling pathways mediating these effects are poorly understood. Mammalian target of rapamycin complex 1 (mTORC1) signalling is a key intracellular pathway involved in energy homeostasis and is known to importantly affect the physiology of pancreatic islets. We investigated the possible relationship between cannabinoid type 1 (CB1) receptor signalling and the mTORC1 pathway in the endocrine pancreas of mice by using pharmacological analysis as well as mice genetically lacking the CB1 receptor or the downstream target of mTORC1, the kinase p70S6K1. In vitro static secretion experiments on islets, western blotting, and in vivo glucose and insulin tolerance tests were performed. The CB1 receptor antagonist rimonabant decreased glucose-stimulated insulin secretion (GSIS) at 0.1 µM while increasing phosphorylation of p70S6K1 and ribosomal protein S6 (rpS6) within the islets. Specific pharmacological blockade of mTORC1 by 3 nM rapamycin, as well as genetic deletion of p70S6K1, impaired the CB1-antagonist-mediated decrease in GSIS. In vivo experiments showed that 3 mg/kg body weight rimonabant decreased insulin levels and induced glucose intolerance in lean mice without altering peripheral insulin sensitivity; this effect was prevented by peripheral administration of low doses of rapamycin (0.1 mg/kg body weight), which increased insulin sensitivity. These findings suggest a functional interaction between the ECS and the mTORC1 pathway within the endocrine pancreas and at the whole-organism level, which could have implications for the development of new therapeutic approaches for pancreatic β-cell diseases.

Sirtuin 6 regulates glucose-stimulated insulin secretion in mouse pancreatic beta cells.

Sirtuin 6 (SIRT6) has been implicated in ageing, DNA repair and metabolism; however, its function in pancreatic beta cells is unclear. The aim of this study is to elucidate the role of SIRT6 in pancreatic beta cells. To investigate the function of SIRT6 in pancreatic beta cells, we performed Sirt6 gene knockdown in MIN6 cells and generated pancreatic- and beta cell-specific Sirt6 knockout mice. Islet morphology and glucose-stimulated insulin secretion (GSIS) were analysed. Glycolysis and oxygen consumption rates in SIRT6-deficient beta cells were measured. Cytosolic calcium was monitored using the Fura-2-AM fluorescent probe (Invitrogen, Grand Island, NY, USA). Mitochondria were analysed by immunoblots and electron microscopy. Sirt6 knockdown in MIN6 beta cells led to a significant decrease in GSIS. Pancreatic beta cell Sirt6 knockout mice showed a ~50% decrease in GSIS. The knockout mouse islets had lower ATP levels compared with the wild-type controls. Mitochondrial oxygen consumption rates were significantly decreased in the SIRT6-deficient beta cells. Cytosolic calcium dynamics in response to glucose or potassium chloride were attenuated in the Sirt6 knockout islets. Numbers of damaged mitochondria were increased and mitochondrial complex levels were decreased in the SIRT6-deficient islets. These data suggest that SIRT6 is important for GSIS from pancreatic beta cells and activation of SIRT6 may be useful to improve insulin secretion in diabetes.

Abstract 483: Non-redundant and Opposing Roles of Group X and Group V Secretory Phospholipase A2s on Pancreatic Beta-cell Function

Background: Group X and group V secretory phospholipase A2s (GX and GV sPLA2s) potently release arachidonic acid (AA) from the plasma membrane of intact cells. AA is an activator of glucose-stimulated insulin secretion (GSIS) by β-islet cells. However, the AA metabolite prostaglandin E2 (PGE2) is a known inhibitor of GSIS. Both GX and GV sPLA2s are expressed in mouse pancreatic islet cells. We previously demonstrated that GX sPLA2 negatively regulates GSIS by a PGE2-dependent mechanism. In this study we investigated whether GV sPLA2 similarly regulates GSIS. Methods and Results: GSIS was measured in pancreatic islet cells isolated from WT and GV sPLA2-deficient (GV KO) mice. To complement these studies, GSIS was also assessed in vitro using MIN6 pancreatic beta cell lines with or without GV sPLA2 overexpression or silencing. In marked contrast to our findings in GX KO mice, GSIS was significantly decreased in islets isolated from GV KO mice compared to WT mice. Similarly, there was a significant decrease in GSIS in MIN6 cells with siRNA-mediated GV sPLA2 suppression. Consistent with these findings, MIN6 cells overexpressing GV sPLA2 (MIN6-GV) showed a significant increase in GSIS compared to control cells. As expected, the amount of AA released into the media by MIN6-GV cells was significantly increased compared to control MIN6 cells. However, unlike MIN6 cells overexpressing GX sPLA2, MIN6-GV cells did not exhibit enhanced PGE2 production or decreased cAMP content compared to control MIN6 cells, despite similar amounts of sPLA2 activity produced by the two cell lines. Conclusions: We conclude that GX and GV sPLA2s play opposing and non-redundant roles in pancreatic β-cell function. Whereas GV sPLA2 activates GSIS, GX sPLA2 suppresses this process. This functional difference appears to be due to the extent to which AA generated by the respective sPLA2’s is coupled to PGE2 production.

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone Protects the Impaired Insulin Secretion Induced by Glucotoxicity in Pancreatic β-Cells

2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), which is isolated and purified from the dried flower buds of Cleistocalyx operculatus (Roxb.) Merr. et Perry (Myrtaceae), was investigated for its insulinotropic benefits against glucotoxicity using in vitro methods. When exposed to high glucose at the cytotoxicity level for 48 h, RIN-5F β-cells experienced a significant viability loss and impaired insulin secretion function, whereas cotreating with DMC could protect β-cells against glucotoxicity-induced decrease in glucose-stimulated insulin secretion in a dose-dependent manner without affecting basal insulin secretion. It was demonstrated that DMC increased insulin secretion against glucotoxicity by simulating the effect of GLP-1 and enhancing the expression of GLP-1R, followed by activating the signal pathway of PDX-1, PRE-INS, and GLUT2-GCK. Another mechanism was that DMC avoided the pancreatic islet dysfunction resulting from cellular damage by suppressing the production of nitric oxide (NO) by iNOS, and the expression of MCP-1. The results indicated the potential application of DMC in the intervention against glucotoxicity-induced hyperglycemia.

Lipoproteins and β-Cell Functions: From Basic to Clinical Data.

Lipoproteins and β-Cell Functions: From Basic to Clinical Data.

Disruption of the histidine triad nucleotide-binding hint2 gene in mice affects glycemic control and mitochondrial function

The histidine triad nucleotide-binding (HINT2) protein is a mitochondrial adenosine phosphoramidase expressed in the liver and pancreas. Its physiological function is unknown. To elucidate the role of HINT2 in liver physiology, the mouse Hint2 gene was deleted. Hint2(-/-) and Hint2(+/+) mice were generated in a mixed C57Bl6/J × 129Sv background. At 20 weeks, the phenotypic changes in Hint2(-/-) relative to Hint2(+/+) mice were an accumulation of hepatic triglycerides, decreased tolerance to glucose, a defective counter-regulatory response to insulin-provoked hypoglycemia, and an increase in plasma interprandial insulin but a decrease in glucose-stimulated insulin secretion and defective thermoregulation upon fasting. Leptin messenger RNA (mRNA) in adipose tissue and plasma leptin were elevated. In mitochondria from Hint2(-/-) hepatocytes, state 3 respiration was decreased, a finding confirmed in HepG2 cells where HINT2 mRNA was silenced. The linked complex II-III electron transfer was decreased in Hint2(-/-) mitochondria, which was accompanied by a lower content of coenzyme Q. Hypoxia-inducible factor-2α expression and the generation of reactive oxygen species were increased. Electron microscopy of mitochondria in Hint2(-/-) mice aged 12 months revealed clustered, fused organelles. The hepatic activities of 3-hydroxyacyl-coenzyme A dehydrogenase short chain and glutamate dehydrogenase (GDH) were decreased by 68% and 60%, respectively, without a change in protein expression. GDH activity was similarly decreased in HINT2-silenced HepG2 cells. When measured in the presence of purified sirtuin 3, latent GDH activity was recovered (126% in Hint2(-/-) versus 83% in Hint2(+/+) ). This suggests a greater extent of acetylation in Hint2(-/-) than in Hint2(+/+) . Hint2/HINT2 positively regulates mitochondrial lipid metabolism and respiration and glucose homeostasis. The absence of Hint2 provokes mitochondrial deformities and a change in the pattern of acetylation of selected proteins.

MicroRNA‐29 expression in pancreatic islets correlates with the impairment of maternal glucose homeostasis induced by glucocorticoid excess in late pregnancy

We investigated the long‐term effects of dexamethasone (DEX) administration during late gestation in glucose homeostasis and in the expression of miRNAs in pancreatic islets. Pregnant Wistar rats received dexamethasone (DEX), 0.2 mg/kg daily in drinking water during the last five days of pregnancy (Preg‐DEX). Age‐matched virgin female rats either treated (CTL‐DEX) or not (CTL) with DEX, as well as untreated pregnant rats (Preg) were used as controls. All groups were subjected to glucose and insulin tolerance tests at weaning and at 3, 6, and 12 month after weaning. Pancreatic islets were isolated for glucose stimulated insulin secretion (GSIS) and miRNA expression. Compared to control groups, glucose intolerance was observed in Preg‐DEX rats from weaning to 12 months after weaning. In all periods insulin sensitivity was normal. An upregulation in miR‐29 family (miR‐29a/b/c) was observed in Preg‐DEX islets at the end of gestation and 12 months after weaning. In the later period, mir‐29 expression paralleled a decrease in GSIS. In conclusion, we show that the excess of glucocorticoid in late pregnancy permanently impairs pancreatic islets function, which is correlated with persistent upregulation of miR‐29 expression.Financial support: FAPESP 2011/01568–5, PRONEX‐FAPEMAT.

In Vitro Investigation of 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone for Glycemic Control

2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), a compound isolated and purified from the dried flower buds of Cleistocalyx operculatus (Roxb.) Merr. et Perry (Myrtaceae), was investigated for its glucose control benefits using in vitro methods. DMC showed strong noncompetitive (IC(50) of 43 μM) inhibition of pancreatic α-amylase; it was, however, ineffective against intestinal α-glucosidase. In addition, DMC exhibited remarkable glucose transport inhibition effects in both simulated fasting and fed states in Caco-2 cell monolayers (P < 0.05). Besides, exposure of MIN6 cells to 250 μM H(2)O(2) for 1 h caused a significant viability loss and insulin secretion reduction. Pretreatment of MIN6 cells with DMC for 2 h protected against the H(2)O(2)-induced decrease in glucose-stimulated insulin secretion in a dose-dependent manner and also enhanced the impaired basal insulin secretion. Such effects highlight the therapeutic potential of DMC in the management of hyperglycemia.

Abstract 143: Group X Secretory Phospholipase A2 Regulates Insulin Secretion by Mouse Pancreatic Beta Cells

Background: Group X secretory phospholipase A2 (GX sPLA2) potently releases arachidonic acid (AA) from the plasma membrane of intact cells. AA is a precursor of bioactive prostaglandins that are known to modulate insulin secretion by β-islet cells. C57BL/6 mice deficient in GX sPLA2 (GX KO) are protected from age-related defect in glucose tolerance. GX sPLA2 is expressed in mouse pancreatic islet cells. In this study we tested the hypothesis that GX sPLA2 regulates pancreatic insulin secretion. Methods and results: Glucose stimulated insulin secretion (GSIS) was measured in vivo in WT and GX KO mice and ex vivo using pancreatic islet cells isolated from WT and GX KO mice. To complement these studies, GSIS was also assessed in vitro using Min6 pancreatic beta cell lines with or without GX sPLA2 overexpression. GSIS was significantly increased in GX KO mice compared to WT mice, and in islet cells isolated from GX KO mice compared to WT mice. Consistent with this finding, Min6 cells overexpressing GX sPLA2 demonstrated significantly decreased GSIS compared to control cells. Expression of ABCA1 and ABCG1 mRNAs were significantly upregulated in islet cells from GX KO mice compared to WT mice, consistent with our previous report that GX sPLA2 negatively regulates LXR activity. However, there was no significant difference in cholesterol content between islets from WT and GX KO mice suggesting that altered LXR activity may not be the mechanism. Min6 cells overexpressing GX sPLA2 secrete significantly increased levels of PGE2- a known negative regulator of GSIS, in the medium compared to control cells. Treatment of these cells with NS398- an inhibitor of COX2 enzyme, abolished the decrease in GSIS shown by these cells. Conclusions: We conclude that GX sPLA2 plays a previously unrecognized role in negatively regulating pancreatic insulin secretion most likely by enhancing PGE2 production.

G6PD Up-Regulation Promotes Pancreatic β-Cell Dysfunction

Increased reactive oxygen species (ROS) induce pancreatic β-cell dysfunction during progressive type 2 diabetes. Glucose-6-phosphate dehydrogenase (G6PD) is a reduced nicotinamide adenine dinucleotide phosphate-producing enzyme that plays a key role in cellular reduction/oxidation regulation. We have investigated whether variations in G6PD contribute to β-cell dysfunction through regulation of ROS accumulation and β-cell gene expression. When the level of G6PD expression in pancreatic islets was examined in several diabetic animal models, such as db/db mice and OLEFT rats, G6PD expression was evidently up-regulated in pancreatic islets in diabetic animals. To investigate the effect of G6PD on β-cell dysfunction, we assessed the levels of cellular ROS, glucose-stimulated insulin secretion and β-cell apoptosis in G6PD-overexpressing pancreatic β-cells. In INS-1 cells, G6PD overexpression augmented ROS accumulation associated with increased expression of prooxidative enzymes, such as inducible nitric oxide synthase and reduced nicotinamide adenine dinucleotide phosphate oxidase. G6PD up-regulation also caused decrease in glucose-stimulated insulin secretion in INS-1 cells and primary pancreatic islets. Moreover, elevated G6PD expression led to β-cell apoptosis, concomitant with the increase in proapoptotic gene expression. On the contrary, suppression of G6PD with small interference RNA attenuated palmitate-induced β-cell apoptosis. Together, these data suggest that up-regulation of G6PD in pancreatic β-cells would induce β-cell dysregulation through ROS accumulation in the development of type 2 diabetes.