Primary Rat Islets Research Articles

Expression of Cdk5r1 and not Cdk5r induces primary β‐cell proliferation

Decreased β‐cell mass is a hallmark of type 1 and type 2 diabetes. The use of islet transplantation as a diabetes therapy is hampered by the relative paucity of transplant ready islets. Greater understanding of the proliferative pathways controlling islet proliferation may be harnessed to increase functional beta‐cell mass through transplantation or by enhanced growth of endogenous beta‐cells. We have shown that the β‐cell transcription factor Nkx6.1 induces β‐cell proliferation by upregulating the orphan nuclear hormone receptors Nr4a1 and Nr4a3. Using expression analysis to elucidate the Nkx6.1 independent mechanism by which Nr4a1 and Nr4a3 induce beta‐cell proliferation, we demonstrated that cyclin‐dependent kinase 5 regulatory subunit 1 (Cdk5r1) is upregulated by Nr4a1 and Nr4a3 and not Nkx6.1. Adenovirus mediated overexpression of Cdk5r1 is sufficient to induce proliferation in primary rat islets. The observed proliferation is primarily in beta‐cells. Glucose stimulated insulin secretion is maintained with Cdk5r1 overexpression. The Cdk5 inhibitor roscovitine blocks islet proliferation, suggesting that Nr4a mediated β‐cell proliferation is a kinase dependent event. Overexpression of Cdk5r1 results in pRb phosphorylation, which is inhibited by roscovitine treatment. These data demonstrate that activation of the cdk5 complex is sufficient to induce β‐cell proliferation while maintaining glucose stimulated insulin secretion.

C‐Fos increases functional β‐cell mass

β‐cell mass is lost in both major forms of diabetes. Mature β‐cells have restricted proliferative capacity. Studies aimed at increasing β‐cell mass frequently have the unwanted side effects of decreased glucose stimulated insulin secretion (GSIS) or increased apoptotic rates. Understanding the molecular pathways that enhance β‐cell mass while maintaining or improving GSIS and the response to apoptotic stimuli could be used as a treatment for diabetes. Overexpression of the β‐cell transcription factor Nkx6.1 induces β‐cell proliferation, enhances GSIS and protects against apoptosis. Nkx6.1 induces expression of VGF, which is necessary for enhanced GSIS and apoptotic resistance. Nr4a1 and Nr4a3 are also induced in the Nkx6.1 molecular pathway, and are necessary for β‐cell proliferation. Microarray analysis of Nkx6.1 expressing primary rat islets demonstrates upregulation of c‐Fos at 24 and 48 hours after adenoviral transduction. We have shown that c‐Fos upregulates expression of VGF, Nr4a1 and Nr4a3. In addition c‐Fos is sufficient to induce β‐cell proliferation. Finally, preliminary data suggest that c‐Fos modulates GSIS and the β‐cell response to apoptotic insults. We present a model by which c‐Fos is necessary for Nkx6.1 mediated β‐cell proliferation, enhanced GSIS and protection from apoptosis by inducing expression of Nr4a1, Nr4a3 and VGF.

Compartmentalization of GABA synthesis by GAD67 differs between pancreatic beta cells and neurons.

The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD) in neurons and in pancreatic β-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization of the two non-allelic isoforms GAD65 and GAD67 to vesicular membranes is important for rapid delivery and accumulation of GABA for regulated secretion. While the membrane anchoring and trafficking of GAD65 are mediated by intrinsic hydrophobic modifications, GAD67 remains hydrophilic, and yet is targeted to vesicular membrane pathways and synaptic clusters in neurons by both a GAD65-dependent and a distinct GAD65-independent mechanism. Herein we have investigated the membrane association and targeting of GAD67 and GAD65 in monolayer cultures of primary rat, human, and mouse islets and in insulinoma cells. GAD65 is primarily detected in Golgi membranes and in peripheral vesicles distinct from insulin vesicles in β-cells. In the absence of GAD65, GAD67 is in contrast primarily cytosolic in β-cells; its co-expression with GAD65 is necessary for targeting to Golgi membranes and vesicular compartments. Thus, the GAD65-independent mechanism for targeting of GAD67 to synaptic vesicles in neurons is not functional in islet β-cells. Therefore, only GAD65:GAD65 homodimers and GAD67:GAD65 heterodimers, but not the GAD67:GAD67 homodimer gain access to vesicular compartments in β-cells to facilitate rapid accumulation of newly synthesized GABA for regulated secretion and fine tuning of GABA-signaling in islets of Langerhans.

Aurora Kinase A is critical for the Nkx6.1 mediated β-cell proliferation pathway

Type 1 and type 2 diabetes are ultimately characterized by depleted β-cell mass. Characterization of the molecular pathways that control β-cell proliferation could be harnessed to restore these cells. The homeobox β-cell transcription factor Nkx6.1 induces β-cell proliferation by activating the orphan nuclear receptors Nr4a1 and Nr4a3. Here, we demonstrate that Nkx6.1 localizes to the promoter of the mitotic kinase AURKA (Aurora Kinase A) and induces its expression. Adenovirus mediated overexpression of AURKA is sufficient to induce proliferation in primary rat islets while maintaining glucose stimulated insulin secretion. Furthermore, AURKA is necessary for Nkx6.1 mediated β-cell proliferation as demonstrated by shRNA mediated knock down and pharmacological inhibition of AURKA kinase activity. AURKA preferentially induces DNA replication in β-cells as measured by BrdU incorporation, and enhances the rate of histone H3 phosphorylation in primary β-cells, demonstrating that AURKA induces the replicative and mitotic cell cycle phases in rat β-cells. Finally, overexpression of AURKA results in phosphorylation of the cell cycle regulator p53, which targets p53 for degradation and permits cell cycle progression. These studies define a pathway by which AURKA upregulation by Nkx6.1 results in phosphorylation and degradation of p53, thus removing a key inhibitory factor and permitting engagement of the β-cell proliferation pathway.

Chronic effects of neuroendocrine regulatory peptide (NERP-1 and -2) on insulin secretion and gene expression in pancreatic β-cells

Neuroendocrine regulatory peptides (NERP-1 and -2) are novel amidated peptides derived from VGF, a polypeptide secreted from neurons and endocrine cells through a regulated pathway. Dr. Nakazato Masamitsu reported that NERP-1 and -2 may have a local modulator function on the human endocrine system, and clearly showed expression of NERP-1 and -2 in human pancreas islets. Based on these data, we investigated the alteration of insulin secretion, insulin granule-related protein, and pancreas-specific transcription factors in response to NERPs expression. We confirmed the expression of NERP-1 and -2 in the pancreas of a human diabetes patient, in addition to diabetic animal models. When INS1 cells and primary rat islets were incubated with 10nM NERPs for 3days, glucose-stimulated insulin secretion levels were blunted by NERP-1 and -2. The number of insulin granules released from the readily releasable pool, which is associated with the first phase of glucose-stimulated insulin release, was decreased by NERP-1 and -2. Insulin granule-related proteins and mRNAs were down-regulated by NERP-2 treatment. NERP-2 decreased the expression of BETA2/NeuroD and insulin and controlled the nucleo-cytoplasmic translocation of FOXO1 and Pdx-1. We observed that NERP-2 levels were dramatically increased in diabetic pancreas. In conclusion, NERP-2 may play an important role in insulin secretion through the regulation of insulin secretory granules and β-cell transcription factors. In addition, NERP-2 expression is increased in diabetic conditions. Therefore, we suggest that NERPs may be potent endogenous suppressors of glucose-dependent insulin secretion.

Dipeptidyl Peptidase IV Inhibitor Protects Against Tacrolimus-Induced Pancreatic and Renal Injury.

Background: Dipeptidyl peptidase IV (DPP IV) inhibitors had a protective effect in various disease models. This study was performed to evaluate whether sitagliptin, a DPP IV inhibitor, is effective against tacrolimus-induced pancreatic and renal injury. Method: Tacrolimus (TAC, 1.5mg/kg/day, s.c.) with or without sitagliptin (10, 20 mg/kg/day, gavage) were administered to rats for 4 weeks. We evaluated the effect of sitagliptin on DPP IV activity and GLP-1 level in tacrolimus-treated rats, and assessed the protective effect of sitagliptin in an experimental model of chronic tacrolimus-induced nephropathy and pancreatic injury. Oxidative stress and oxidative resistance were evaluated by measuring 8-hydroxy-2'-deoxyguanosine (8-OHdG) and heme oxygenase-1 (HO-1). The protective mechanism of sitagliptin was also tested based on the glucagon like peptide-1 (GLP-1)/ (forkhead box O) FoxO pathway. Results: Tacrolimus treatment for 4 weeks caused a 2-fold increase in plasma DPP4 activity and a 5-fold decrease in serum active GLP-1 levels; however, concomitant treatment with sitagliptin reversed these effects. Sitagliptin treatment attenuated tacrolimus-induced pancreatic islet and renal dysfunction, with improvement of islet size and renal tubulointerstitial fibrosis. Tacrolimus treatment increased oxidative stress marker, 8-OHdG and decreased oxidative resistance gene (reduced HO-1), and its underlying pathways (FoxO1 activation and FoxO3 deactivation), whereas sitagliptin treatment reversed these changes. Treatment of primary rat islet and HK-2 cells with GLP-1 also decreased the oxidative injury caused by tacrolimus. Consequently, sitagliptin upregulated antioxidant enzymes e.g. manganese superoxide dismutase, and downregulated proapoptotic proteins e.g. B-cell lymphoma 2-interacting mediator of cell death and apoptotic cell death assessed by the number of TDT-mediated dUTP-biotin nick end labeling-positive cells. Conclusion:DPP IV inhibition by sitagliptin attenuated TAC-induced pancreatic islet and renal dysfunction and enhanced the GLP-1 signaling pathway, which may be associated with a decrease in oxidative injury and apoptotic cell death. These beneficial effects of the DPP IV inhibitor may be helpful in delaying the onset of TAC-induced diabetes and nephropathy in renal transplant patients.

Exposure to high levels of glucose increases the expression levels of genes involved in cholesterol biosynthesis in rat islets.

Cells continually adjust their gene expression profiles in order to adapt to the availability of nutrients. Glucose is a major regulator of pxancreatic β-cell function and cell growth. However, the mechanism of β-cell adaptation to high levels of glucose remains uncertain. To identify the specific targets responsible for adaptation to high levels of glucose, the differentially expressed genes from primary rat islets treated with 3.3 and 16.7 mmol/l glucose for 24 h were detected by DNA microarray. The results revealed that the expression levels of genes that encode enzymes required for de novo cholesterol biosynthesis [3-hydroxy-3-methylglutaryl-CoA synthase 1 (Hmgcs1), 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), mevalonate (diphospho) decarboxylase (Mvd), isopentenyl-diphosphate δ-isomerase 1 (Idi1), squalene epoxidase (Sqle) and 7-dehydrocholesterol reductase (Dhcr7)] were significantly increased in islets treated with high levels of glucose compared with those in the islets treated with lower glucose levels. Quantitative polymerase chain reaction further confirmed that glucose stimulated the expression levels of these genes in a dose- and time-dependent manner. A similar result was obtained in islets isolated from rats subjected to 12, 24, 48 and 72 h of continuous glucose infusion. It has previously been recognized that cholesterol homeostasis is important for β-cell function. The present study provides, to the best of our knowledge, the first evidence for the involvement of the de novo cholesterol biosynthesis pathway in the adaptation of rat islets to high levels of glucose in vitro and in vivo.

Influence of cytokines on Dmt1 iron transporter and ferritin expression in insulin-secreting cells.

Free intracellular ferrous iron (Fe(2+)) is essential for the generation of the extremely toxic hydroxyl radicals, which contribute to β-cell destruction by cytokines. Therefore the expression of the different divalent metal transporter 1 (Dmt1) isoforms and ferritin (Ft) subunits, responsible for iron import and chelation, was analyzed under pro-inflammatory conditions (IL1β alone or together with TNFα+IFNγ). The Dmt1 isoforms (1A/1B and +IRE/-IRE) and the total Dmt1 expression in insulin-producing cells (RINm5F and INS-1E), in primary rat islets and, for comparison, in the neuroendocrine PC12 cell line were quantified by qRT-PCR. In addition, the expression of the light (L-Ft) and heavy Ft (H-Ft) subunits and the mitochondrial Ft isoform (Mtft) in insulin-producing cells under control conditions and after cytokine treatment was estimated. The 1B isoform was the predominant Dmt1 mRNA in all insulin-producing cells, accounting for almost 100% of the 1A/1B isoform expression. For the IRE variants, +IRE expression was higher than -IRE expression. Pro-inflammatory cytokines accelerated the expression of Dmt1 isoforms significantly with an overall 2.5- to 3-fold increase in the total Dmt1 expression. In contrast, the expression of the iron-buffering ferritin subunits L- and H-Ft was unaffected by IL1β and only slightly induced by the cytokine mixture. Mtft expression was also not increased. Dmt1 expression was significantly elevated through pro-inflammatory cytokines, whereas Ft expression was marginally increased. This imbalance between the increased iron transport capacity and the almost unaffected iron storage capacity can foster cytokine-mediated formation of hydroxyl radicals and thus pro-inflammatory cytokine toxicity through elevated free iron concentrations.

Humoral factors by human bone marrow-derived MSC enhance beta cell proliferation via ERK1/2 signalling

Background and aim: We previously demonstrated that human telomerase-immortalised MSC (hMSC-TERT) migrate towards injured rat INS-1E beta cells and promote their survival via preservation of functional active phospho (p)-AKT levels. Both cell lines display a valid model since they closely resemble results from primary rat MSC and islets. Since some studies indicate that MSC also mediate proliferation of beta cells, we here tested whether hMSC-TERT-derived factors promote proliferation of INS-1E and explored the signalling mechanims involved.

Increased expression of miR-187 in human islets from individuals with type 2 diabetes is associated with reduced glucose-stimulated insulin secretion

Aims/hypothesisType 2 diabetes is characterised by progressive beta cell dysfunction, with changes in gene expression playing a crucial role in its development. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and therefore alterations in miRNA levels may be involved in the deterioration of beta cell function.MethodsGlobal TaqMan arrays and individual TaqMan assays were used to measure islet miRNA expression in discovery (n = 20) and replication (n = 20) cohorts from individuals with and without type 2 diabetes. The role of specific dysregulated miRNAs in regulating insulin secretion, content and apoptosis was subsequently investigated in primary rat islets and INS-1 cells. Identification of miRNA targets was assessed using luciferase assays and by measuring mRNA levels.ResultsIn the discovery and replication cohorts miR-187 expression was found to be significantly increased in islets from individuals with type 2 diabetes compared with matched controls. An inverse correlation between miR-187 levels and glucose-stimulated insulin secretion (GSIS) was observed in islets from normoglycaemic donors. This correlation paralleled findings in primary rat islets and INS-1 cells where overexpression of miR-187 markedly decreased GSIS without affecting insulin content or apoptotic index. Finally, the gene encoding homeodomain-interacting protein kinase-3 (HIPK3), a known regulator of insulin secretion, was identified as a direct target of miR-187 and displayed reduced expression in islets from individuals with type 2 diabetes.Conclusions/interpretationOur findings suggest a role for miR-187 in the blunting of insulin secretion, potentially involving regulation of HIPK3, which occurs during the pathogenesis of type 2 diabetes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-013-3089-4) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Downregulation of Sfrp5 promotes beta cell proliferation during obesity in the rat

During obesity, the increment in beta cell mass in response to the rising demand for insulin is essential to maintain normal glucose homeostasis. However, the precise cellular and molecular mechanisms involved in beta cell mass plasticity remain poorly understood. The Wnt signalling pathway has been suggested as one possible modulator of beta cell proliferation, which represents the principal process involved in beta cell mass expansion. Here, we sought to determine the mechanisms involved in beta cell mass proliferation using diet-induced obese rats. Wistar rats aged 8 weeks old were fed a standard or cafeteria diet. Global transcriptomic analysis of pancreatic rat islets was performed using microarray analysis. Genetic loss-of-function approaches were performed in dispersed primary rat islets and the beta cell line INS1E. Gene expression was measured by real-time PCR, protein levels by immunoblot analysis, proliferation rates by ELISA and apoptosis by flow cytometry. Sfrp5, coding for secreted frizzled-related protein 5, is downregulated in the pancreatic islets of cafeteria-diet-fed rats as well as in the pancreatic islets of human obese patients. We demonstrate that silencing Sfrp5 increases beta cell proliferation, which correlates with activation of Wnt signalling and enhanced levels of proliferation markers. In addition, we show that expression of Sfrp5 in beta cells is modulated by IGF binding protein 3 (IGFBP3) secreted from visceral adipose tissue. Together, these findings reveal an important role for SFRP5 and Wnt signalling in the regulation of beta cell proliferation in obesity.

PPAR-γ Activation Increases Insulin Secretion through the Up-regulation of the Free Fatty Acid Receptor GPR40 in Pancreatic β-Cells

BackgroundIt has been reported that peroxisome proliferator-activated receptor (PPAR)-γ and their synthetic ligands have direct effects on pancreatic β-cells. We investigated whether PPAR-γ activation stimulates insulin secretion through the up-regulation of GPR40 in pancreatic β-cells.MethodsRat insulinoma INS-1 cells and primary rat islets were treated with rosiglitazone (RGZ) and/or adenoviral PPAR-γ overexpression. OLETF rats were treated with RGZ.ResultsPPAR-γ activation with RGZ and/or adenoviral PPAR-γ overexpression increased free fatty acid (FFA) receptor GPR40 expression, and increased insulin secretion and intracellular calcium mobilization, and was blocked by the PLC inhibitors, GPR40 RNA interference, and GLUT2 RNA interference. As a downstream signaling pathway of intracellular calcium mobilization, the phosphorylated levels of CaMKII and CREB, and the downstream IRS-2 and phospho-Akt were significantly increased. Despite of insulin receptor RNA interference, the levels of IRS-2 and phospho-Akt was still maintained with PPAR-γ activation. In addition, the β-cell specific gene expression, including Pdx-1 and FoxA2, increased in a GPR40- and GLUT2-dependent manner. The levels of GPR40, phosphorylated CaMKII and CREB, and β-cell specific genes induced by RGZ were blocked by GW9662, a PPAR-γ antagonist. Finally, PPAR-γ activation up-regulated β-cell gene expressions through FoxO1 nuclear exclusion, independent of the insulin signaling pathway. Based on immunohistochemical staining, the GLUT2, IRS-2, Pdx-1, and GPR40 were more strongly expressed in islets from RGZ-treated OLETF rats compared to control islets.ConclusionThese observations suggest that PPAR-γ activation with RGZ and/or adenoviral overexpression increased intracellular calcium mobilization, insulin secretion, and β-cell gene expression through GPR40 and GLUT2 gene up-regulation.

MiRNA-30a-5p-mediated silencing of Beta2/NeuroD expression is an important initial event of glucotoxicity-induced beta cell dysfunction in rodent models

The loss of beta cell function is a critical factor in the development of type 2 diabetes. Glucotoxicity plays a major role in the progressive deterioration of beta cell function and development of type 2 diabetes mellitus. Here we demonstrate that microRNA (miR)-30a-5p is a key player in early-stage glucotoxicity-induced beta cell dysfunction. We performed northern blots, RT-PCR and western blots in glucotoxicity-exposed primary rat islets and INS-1 cells. We also measured glucose-stimulated insulin secretion and insulin content. In vivo approaches were used to evaluate the role of miR-30a-5p in beta cell dysfunction. miR-30a-5p expression was increased in beta cells after exposure to glucotoxic conditions, and exogenous miR-30a-5p overexpression also induced beta cell dysfunction in vitro. miR-30a-5p directly suppressed expression of Beta2/NeuroD (also known as Neurod1) by binding to a specific binding site in its 3'-untranslated region. After restoration of Beta2/NeuroD expression by knockdown miR-30a-5p or transfection of the Beta2/NeuroD gene, beta cell dysfunction, including decreased insulin content, gene expression and glucose-stimulated insulin secretion, recovered. Glucose tolerance and beta cell dysfunction improved on direct injection of Ad-si30a-5p into the pancreas of diabetic mice. Our data demonstrate that miR-30a-5p-mediated direct suppression of Beta2/NeuroD gene expression is an important initiation step of glucotoxicity-induced beta cell dysfunction.

β-cell metabolic alterations under chronic nutrient overload in rat and human islets

The aim of this study was to assess multifactorial β-cell responses to metabolic perturbations in primary rat and human islets. Treatment of dispersed rat islet cells with elevated glucose and free fatty acids (FFAs, oleate:palmitate = 1:1 v/v) resulted in increases in the size and the number of lipid droplets in β-cells in a time- and concentration-dependent manner. Glucose and FFAs synergistically stimulated the nutrient sensor mammalian target of rapamycin complex 1 (mTORC1). A potent mTORC1 inhibitor, rapamycin (25 nM), significantly reduced triglyceride accumulation in rat islets. Importantly, lipid droplets accumulated only in β-cells but not in α-cells in an mTORC1-dependent manner. Nutrient activation of mTORC1 upregulated the expression of adipose differentiation related protein (ADRP), known to stabilize lipid droplets. Rat islet size and new DNA synthesis also increased under nutrient overload. Insulin secretion into the culture medium increased steadily over a 4-day period without any significant difference between glucose (10 mM) alone and the combination of glucose (10 mM) and FFAs (240 μM). Insulin content and insulin biosynthesis, however, were significantly reduced under the combination of nutrients compared with glucose alone. Elevated nutrients also stimulated lipid droplet formation in human islets in an mTORC1-dependent manner. Unlike rat islets, however, human islets did not increase in size under nutrient overload despite a normal response to nutrients in releasing insulin. The different responses of islet cell growth under nutrient overload appear to impact insulin biosynthesis and storage differently in rat and human islets.

Histone deacetylases 1 and 3 but not 2 mediate cytokine-induced beta cell apoptosis in INS-1 cells and dispersed primary islets from rats and are differentially regulated in the islets of type 1 diabetic children

Histone deacetylases (HDACs) are promising pharmacological targets in cancer and autoimmune diseases. All 11 classical HDACs (HDAC1-11) are found in the pancreatic beta cell, and HDAC inhibitors (HDACi) protect beta cells from inflammatory insults. We investigated which HDACs mediate inflammatory beta cell damage and how the islet content of these HDACs is regulated in recent-onset type 1 diabetes. The rat beta cell line INS-1 and dispersed primary islets from rats, either wild type or HDAC1-3 deficient, were exposed to cytokines and HDACi. Molecular mechanisms were investigated using real-time PCR, chromatin immunoprecipitation and ELISA assays. Pancreases from healthy children and children with type 1 diabetes were assessed using immunohistochemistry and immunofluorescence. Screening of 19 compounds with different HDAC selectivity revealed that inhibitors of HDAC1, -2 and -3 rescued INS-1 cells from inflammatory damage. Small hairpin RNAs against HDAC1 and -3, but not HDAC2, reduced pro-inflammatory cytokine-induced beta cell apoptosis in INS-1 and primary rat islets. The protective properties of specific HDAC knock-down correlated with attenuated cytokine-induced iNos expression but not with altered expression of the pro-inflammatory mediators Il1α, Il1β, Tnfα or Cxcl2. HDAC3 knock-down reduced nuclear factor κB binding to the iNos promoter and HDAC1 knock-down restored insulin secretion. In pancreatic sections from children with type 1 diabetes of recent onset, HDAC1 was upregulated in beta cells whereas HDAC2 and -3 were downregulated in comparison with five paediatric controls. These data demonstrate non-redundant functions of islet class I HDACs and suggest that targeting HDAC1 and HDAC3 would provide optimal protection of beta cell mass and function in clinical islet transplantation and recent-onset type 1 diabetic patients.

Glucose-Mediated Repression of Menin Promotes Pancreatic β-Cell Proliferation

Menin, encoded by the Men1 gene, is responsible for β-cell tumor formation in patients with multiple endocrine neoplasia type 1. Recently, menin has been proven to negatively regulate β-cell proliferation during pregnancy. However, it is unclear whether menin is involved in pancreatic β-cell proliferation in response to other physiological replication stimuli, such as glucose. In this study, we found that the menin level was significantly reduced in high glucose-treated INS1 cells and primary rat islets, both with increased proliferation. A similar observation was found in islets isolated from rats subjected to 72-h continuous glucose infusion. The glucose-induced proliferation was inhibited by menin overexpression. Further molecular studies showed that glucose-induced menin suppression was blocked by PI3K/Akt pathway inhibitors. A major PI3K/Akt substrate, Foxo1, was shown to enhance menin transcription levels by binding the promoter region of the Men1 gene. Therefore, we conclude that glucose inhibits menin expression via the PI3K/Akt/Foxo1 pathway and hence promotes pancreatic β-cell proliferation. Our study suggests that menin might serve as an important intracellular target of glucose to mediate the mitogenic effect that glucose exerts in pancreatic β-cells.

Survival of pancreatic beta cells is partly controlled by a TCF7L2-p53-p53INP1-dependent pathway

The transcription factor T-cell factor 7-like 2 (TCF7L2) confers type 2 diabetes risk mainly through impaired insulin secretion, perturbed incretin effect and reduced beta-cell survival. The aim of this study was to identify the molecular mechanism through which TCF7L2 influences beta-cell survival. TCF7L2 target genes in INS-1 cells were identified using Chromatin Immunoprecipitation. Validation of targets was obtained by: siRNA silencing, real-time quantitative polymerase chain reaction, electrophoretic mobility shift assay, luciferase reporter assays and western blot. Apoptosis rate was measured by DNA degradation and caspase-3 content. Islet viability was estimated by measuring metabolic rate. TCF7L2 binds to 3646 gene promoters in INS-1 cells in high or low glucose, including Tp53, Pten, Uggt1, Adamts9 and Fto. SiRNA-mediated reduction in TCF7L2 activity resulted in increased apoptosis and increased expression of Tp53, which resulted in elevated p53 protein activity and an increased expression of the p53 target gene Tp53inp1 (encoding p53-induced-nuclear-protein 1). Reversing the increase in p53INP1 protein expression, seen after Tcf7l2 silencing, protected INS-1 cells from Tcf7l2 depletion-induced apoptosis. This result was replicated in primary rat islets. The risk T-allele of rs7903146 is associated with increased TCF7L2 mRNA expression and transcriptional activity. On the other hand, in vitro silencing of TCF7L2 lead to increased apoptosis. One possibility is that the risk T-allele increases expression of an inhibitory TCF7L2 isoform with lower transcriptional activity. These results identify the p53-p53INP1 pathway as a molecular mechanism through which TCF7L2 may affect beta-cell survival and established a molecular link between Tcf7l2 and two type 2 diabetes-associated genes, Tp53inp1 and Adamts9.

TAK-875, an Orally Available G Protein-Coupled Receptor 40/Free Fatty Acid Receptor 1 Agonist, Enhances Glucose-Dependent Insulin Secretion and Improves Both Postprandial and Fasting Hyperglycemia in Type 2 Diabetic Rats

G protein-coupled receptor 40/free fatty acid receptor 1 (GPR40/FFA(1)) is highly expressed in pancreatic β cells and mediates free fatty acid-induced insulin secretion. This study examined the pharmacological effects and potential for avoidance of lipotoxicity of [(3S)-6-({2',6'-dimethyl-4'-[3-(methylsulfonyl)propoxy]biphenyl-3-yl}meth-oxy)-2,3-dihydro-1-benzofuran-3-yl]acetic acid hemi-hydrate) (TAK-875), a novel, orally available, selective GPR40 agonist. Insulinoma cell lines and primary rat islets were used to assess the effects of TAK-875 in vitro. The in vivo effects of TAK-875 on postprandial hyperglycemia, fasting hyperglycemia, and normoglycemia were examined in type 2 diabetic and normal rats. In rat insulinoma INS-1 833/15 cells, TAK-875 increased intracellular inositol monophosphate and calcium concentration, consistent with activation of the Gqα signaling pathway. The insulinotropic action of TAK-875 (10 μM) in INS-1 833/15 and primary rat islets was glucose-dependent. Prolonged exposure of cytokine-sensitive INS-1 832/13 to TAK-875 for 72 h at pharmacologically active concentrations did not alter glucose-stimulated insulin secretion, insulin content, or caspase 3/7 activity, whereas prolonged exposure to palmitic or oleic acid impaired β cell function and survival. In an oral glucose tolerance test in type 2 diabetic N-STZ-1.5 rats, TAK-875 (1-10 mg/kg p.o.) showed a clear improvement in glucose tolerance and augmented insulin secretion. In addition, TAK-875 (10 mg/kg, p.o.) significantly augmented plasma insulin levels and reduced fasting hyperglycemia in male Zucker diabetic fatty rats, whereas in fasted normal Sprague-Dawley rats, TAK-875 neither enhanced insulin secretion nor caused hypoglycemia even at 30 mg/kg. TAK-875 enhances glucose-dependent insulin secretion and improves both postprandial and fasting hyperglycemia with a low risk of hypoglycemia and no evidence of β cell toxicity.

Pleiotropic and Age-dependent Effects of Decreased Protein Modification by O-Linked N-Acetylglucosamine on Pancreatic β-Cell Function and Vascularization

The hexosamine biosynthesis pathway (HBP) regulates the post-translational modification of nuclear and cytoplasmic protein by O-linked N-acetylglucosamine (O-GlcNAc). Numerous studies have demonstrated increased flux through this pathway contributes to the development of β-cell dysfunction. The effect of decreased O-GlcNAc on the maintenance of normal β-cell function, however, is not well understood. We studied transgenic mice that over express β-N-acetylglucosaminidase (O-GlcNAcase), an enzyme that catalyzes the removal of O-GlcNAc from proteins, in the pancreatic β-cell under control of the rat insulin promoter. 3-4-Month-old O-GlcNAcase transgenic mice have higher glucose excursions with a concomitant decrease in circulating insulin levels, insulin mRNA levels, and total islet insulin content. In older (8-9-month-old) O-GlcNAcase transgenic mice glucose tolerance is no longer impaired. This is associated with increased serum insulin, islet insulin content, and insulin mRNA in the O-GlcNAcase transgenic mice. These improvements in β-cell function with aging are associated with increased angiogenesis and increased VEGF expression, with parallel increases in activation of Akt and expression of PGC1α. The biphasic effects as a function of age are consistent with published observations of mice with increased O-GlcNAc in islets and demonstrate that O-GlcNAc signaling exerts multiple effects on both insulin secretion and islet survival.

Syntaxin-1A Interacts with Distinct Domains within Nucleotide-binding Folds of Sulfonylurea Receptor 1 to Inhibit β-Cell ATP-sensitive Potassium Channels

The ATP-sensitive potassium (K(ATP)) channel regulates pancreatic β-cell function by linking metabolic status to electrical activity. Syntaxin-1A (Syn-1A), a SNARE protein mediating exocytotic fusion, binds and inhibits the K(ATP) channel via the nucleotide-binding folds (NBFs) of its sulfonylurea receptor-1 (SUR1) regulatory subunit. In this study, we elucidated the precise regions within the NBFs required for Syn-1A-mediated K(ATP) inhibition, using in vitro binding assays, whole cell patch clamp and FRET assay. Specifically, NBF1 and NBF2 were each divided into three subregions, Walker A (W(A)), signature sequence linker, and Walker B (W(B)), to make GST fusion proteins. In vitro binding assays revealed that Syn-1A associates with W(A) and W(B) regions of both NBFs. Patch clamp recordings on INS-1 and primary rat β-cells showed that Syn-1A-mediated channel inhibition was reversed by co-addition of NBF1-W(B) (not NBF1-W(A)), NBF2-W(A), and NBF2-W(B). The findings were corroborated by FRET studies showing that these truncates disrupted Syn-1A interactions with full-length SUR1. To further identify the binding sites, series single-site mutations were made in the Walker motifs of the NBFs. Only NBF1-W(A) (K719M) or NBF2-W(A) (K1385M) mutant no longer bound to Syn-1A; K1385M failed to disrupt Syn-1A-mediated inhibition of K(ATP) channels. These data suggest that NBF1-W(A) (Lys-719) and NBF2-W(A) (Lys-1385) are critical for Syn-1A-K(ATP) channel interaction. Taken together, Syn-1A intimately and functionally associates with the SUR1-NBF1/2 dimer via direct interactions with W(A) motifs and sites adjacent to W(B) motifs of NBF1 and NBF2 but transduces its inhibitory actions on K(ATP) channel activity via some but not all of these NBF domains.