[Show abstract][Hide abstract] ABSTRACT: Blood glucose levels are tightly controlled, a process thought to be orchestrated primarily by peripheral mechanisms (insulin secretion by β cells, and insulin action on muscle, fat, and liver). The brain also plays an important, albeit less well-defined role. Subsets of neurons in the brain are excited by glucose; in many cases this involves ATP-mediated closure of K(ATP) channels. To understand the relevance of this, we are manipulating glucose sensing within glucose-excited neurons. In the present study, we demonstrate that glucose excitation of MCH-expressing neurons in the lateral hypothalamus is mediated by K(ATP) channels and is negatively regulated by UCP2 (a mitochondrial protein that reduces ATP production), and that glucose sensing by MCH neurons plays an important role in regulating glucose homeostasis. Combined, the glucose-excited neurons are likely to play key, previously unexpected roles in regulating blood glucose.
[Show abstract][Hide abstract] ABSTRACT: The current worldwide epidemic of obesity and metabolic diseases has energised the search for new approaches to treat these conditions. Type 2 diabetes appears to involve an interplay between susceptible genetic backgrounds and environmental factors including highly calorific westernised diets. The latter may generate 'glucolipotoxic' conditions which affect both the pancreatic beta-cell and insulin-sensitive tissues. Here we focus on efforts to better understand the basic signalling mechanisms through which the beta-cell senses changes in glucose concentration and how this process may become defective in type 2 diabetes. The recent demonstrations, through whole genome association studies, of important roles for genes involved in the control of cell cycle, as well as intracellular ion homeostasis, further highlight the central role of the beta-cell in both the pathogenesis of the disease and as a therapeutic target.
Frontiers of hormone research 02/2008; 36:118-34. DOI:10.1159/0000115360 · 3.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The current worldwide epidemic of obesity and metabolic diseases has energised the search for new approaches to treat these conditions. Type 2 diabetes appears to involve an interplay between susceptible genetic backgrounds and environmental factors including highly calorific westernised diets. The latter may generate ’glucolipotoxic’ conditions which affect both the pancreatic -cell and insulin-sensitive tissues. Here we focus on efforts to better understand the basic signalling mechanisms through which the -cell senses changes in glucose concentration and how this process may become defective in type 2 diabetes. The recent demonstrations, through whole genome association studies, of important roles for genes involved in the control of cell cycle, as well as intracellular ion homeostasis, further highlight the central role of the -cell in both the pathogenesis of the disease and as a therapeutic target.
[Show abstract][Hide abstract] ABSTRACT: Exercise-induced hyperinsulinism (EIHI) is a dominantly inherited hypoglycemic disorder characterized by inappropriate insulin secretion during anaerobic exercise or on pyruvate load. We aimed to identify the molecular basis of this novel disorder of beta -cell regulation. EIHI mapped to chromosome 1 (LOD score 3.6) in a genome scan performed for two families with 10 EIHI-affected patients. Mutational analysis of the promoter of the SLC16A1 gene, which encodes monocarboxylate transporter 1 (MCT1), located under the linkage peak, revealed changes in all 13 identified patients with EIHI. Patient fibroblasts displayed abnormally high SLC16A1 transcript levels, although monocarboxylate transport activities were not changed in these cells, reflecting additional posttranscriptional control of MCT1 levels in extrapancreatic tissues. By contrast, when examined in beta cells, either of two SLC16A1 mutations identified in separate pedigrees resulted in increased protein binding to the corresponding promoter elements and marked (3- or 10-fold) transcriptional stimulation of SLC16A1 promoter-reporter constructs. These studies show that promoter-activating mutations in EIHI induce SLC16A1 expression in beta cells, where this gene is not usually transcribed, permitting pyruvate uptake and pyruvate-stimulated insulin release despite ensuing hypoglycemia. These findings describe a novel disease mechanism based on the failure of cell-specific transcriptional silencing of a gene that is highly expressed in other tissues.
The American Journal of Human Genetics 10/2007; 81(3):467-74. DOI:10.1086/520960 · 10.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A subset of neurons in the brain, known as 'glucose-excited' neurons, depolarize and increase their firing rate in response to increases in extracellular glucose. Similar to insulin secretion by pancreatic beta-cells, glucose excitation of neurons is driven by ATP-mediated closure of ATP-sensitive potassium (K(ATP)) channels. Although beta-cell-like glucose sensing in neurons is well established, its physiological relevance and contribution to disease states such as type 2 diabetes remain unknown. To address these issues, we disrupted glucose sensing in glucose-excited pro-opiomelanocortin (POMC) neurons via transgenic expression of a mutant Kir6.2 subunit (encoded by the Kcnj11 gene) that prevents ATP-mediated closure of K(ATP) channels. Here we show that this genetic manipulation impaired the whole-body response to a systemic glucose load, demonstrating a role for glucose sensing by POMC neurons in the overall physiological control of blood glucose. We also found that glucose sensing by POMC neurons became defective in obese mice on a high-fat diet, suggesting that loss of glucose sensing by neurons has a role in the development of type 2 diabetes. The mechanism for obesity-induced loss of glucose sensing in POMC neurons involves uncoupling protein 2 (UCP2), a mitochondrial protein that impairs glucose-stimulated ATP production. UCP2 negatively regulates glucose sensing in POMC neurons. We found that genetic deletion of Ucp2 prevents obesity-induced loss of glucose sensing, and that acute pharmacological inhibition of UCP2 reverses loss of glucose sensing. We conclude that obesity-induced, UCP2-mediated loss of glucose sensing in glucose-excited neurons might have a pathogenic role in the development of type 2 diabetes.
[Show abstract][Hide abstract] ABSTRACT: Accumulation of intracellular lipid may contribute to defective insulin secretion in type 2 diabetes. Although Zucker diabetic fatty (ZDF; fa/fa) rat islets are fat-laden and overexpress the lipogenic master gene, sterol regulatory element binding protein 1c (SREBP-1c), the contribution of SREBP-1c to the secretory defects observed in this model remains unclear. Here we compare the gene expression profile of lean control (fa/+) and ZDF rat islets in the absence or presence of dominant-negative SREBP-1c (SREBP-1c DN). ZDF islets displayed elevated basal insulin secretion at 3 mmol/l glucose but a severely depressed response to 17 mmol/l glucose. While SREBP-1c DN reduced basal insulin secretion from ZDF islets, glucose-stimulated insulin secretion was not improved. Of 57 genes differentially regulated in ZDF islets and implicated in glucose metabolism, vesicle trafficking, ion fluxes, and/or exocytosis, 21 were upregulated and 5 were suppressed by SREBP-1c DN. Genes underrepresented in ZDF islets were either unaffected (Glut-2, Kir6.2, Rab3), stimulated (voltage-dependent Ca(2+) channel subunit alpha1D, CPT2, SUR2, rab9, syt13), or inhibited (syntaxin 7, secretogranin-2) by SREBP-1c inhibition. Correspondingly, SREBP-1c DN largely corrected decreases in the expression of the transcription factors Pdx-1 and MafA but did not affect the abnormalities in Pax6, Arx, hepatic nuclear factor-1alpha (HNF1alpha), HNF3beta/Forkhead box-a2 (Foxa2), inducible cyclic AMP early repressor (ICER), or transcription factor 7-like 2 (TCF7L2) expression observed in ZDF islets. We conclude that upregulation of SREBP-1c and mild increases in triglyceride content do not explain defective glucose-stimulated insulin secretion from ZDF rats. However, overexpression of SREBP-1c may contribute to enhanced basal insulin secretion in this model.
[Show abstract][Hide abstract] ABSTRACT: Uncoupling protein 2 (UCP2) negatively regulates insulin secretion. UCP2 deficiency (by means of gene knockout) improves obesity- and high glucose-induced beta cell dysfunction and consequently improves type 2 diabetes in mice. In the present study, we have discovered that the small molecule, genipin, rapidly inhibits UCP2-mediated proton leak. In isolated mitochondria, genipin inhibits UCP2-mediated proton leak. In pancreatic islet cells, genipin increases mitochondrial membrane potential, increases ATP levels, closes K(ATP) channels, and stimulates insulin secretion. These actions of genipin occur in a UCP2-dependent manner. Importantly, acute addition of genipin to isolated islets reverses high glucose- and obesity-induced beta cell dysfunction. Thus, genipin and/or chemically modified variants of genipin are useful research tools for studying biological processes thought to be controlled by UCP2. In addition, these agents represent lead compounds that comprise a starting point for the development of therapies aimed at treating beta cell dysfunction.
[Show abstract][Hide abstract] ABSTRACT: Defective insulin secretion from pancreatic islet beta-cells is a sine qua non of Type II (non-insulin-dependent) diabetes. Digital imaging analysis of the nanomechanics of individual exocytotic events, achieved using total internal reflection fluorescence microscopy, has allowed us to demonstrate that insulin is released via transient or 'cavicapture' events whereby the vesicle and plasma membranes fuse transiently and reversibly. Such studies reveal that an increase in the number of abortive fusion events contributes to defective insulin secretion in in vitro models of Type II diabetes. Complementary analyses of genome-wide changes in beta-cell gene expression, at both the mRNA and protein levels, are now facilitating the identification of key molecular players whose altered expression may contribute to the secretory defects in the diabetic beta-cell.
[Show abstract][Hide abstract] ABSTRACT: The mechanism(s) by which chronic hyperglycemia impairs glucose-stimulated insulin secretion is poorly defined. Here, we compare the "nanomechanics" of single exocytotic events in primary rat pancreatic beta-cells cultured for 48 h at optimal (10 mmol/l) or elevated (30 mmol/l) glucose concentrations. Cargo release was imaged by total internal reflection fluorescence microscopy of lumen-targeted probes (neuropeptide Y [NPY]-pH-insensitive yellow fluorescent protein [NPY-Venus] or NPY-monomeric red fluorescent protein), while the fate of the vesicle membrane was reported simultaneously with phosphatase-on-the-granule-of-insulinoma-enhanced green fluorescent protein. Under all conditions studied, exocytosis proceeded via a "cavity recapture" mechanism in which the vesicle and plasma membranes fused transiently. While essentially complete release of NPY-Venus was observed in 24 +/- 1% of glucose-stimulated exocytotic events in cells maintained at 10 mmol/l glucose, this value was reduced reversibly to 5 +/- 2% of events by culture at 30 mmol/l glucose, in line with decreases in Glut2 and glucokinase gene expression, and attenuated glucose-stimulated increases in NADPH and intracellular [Ca2+]. Since vesicle release in response to cell depolarization with KCl was not affected by culture at 30 mmol/l glucose, we conclude that hyperglycemia causes the abnormal termination of individual insulin release events principally by inhibiting glucose signaling.
[Show abstract][Hide abstract] ABSTRACT: Treatment of type 1 diabetes by islet transplantation is currently limited by loss of functional beta-cell mass after transplantation. We investigated here whether adenovirus-mediated changes in AMP-activated protein kinase (AMPK) activity, previously shown to affect insulin secretion in vitro, might affect islet graft function in vivo. In isolated mouse and rat islets, insulin secretion stimulated by 17 (vs 3) mmol/l glucose was inhibited by 36.5% (P<0.01) and 43% (P<0.02) respectively after over-expression of constitutively-active AMPK- (AMPK CA) versus null (eGFP-expressing) viruses, and glucose oxidation was decreased by 38% (P<0.05) and 26.6% (P<0.05) respectively. Increases in apoptotic index (terminal deoxynucleotide transferase-mediated deoxyuridine trisphosphate biotin nick end-labelling) (TUNEL)) were also observed in AMPK CA- (22.8 +/- 3.6% TUNEL-positive cells, P<0.001), but not AMPK DN- (2.72 +/- 3.9%, positive cells, P=0.05) infected islets, versus null adenovirus-treated islets (0.68 +/- 0.36% positive cells). Correspondingly, transplantation of islets expressing AMPK CA into streptozotocin-diabetic C57 BL/6 mice improved glycaemic control less effectively than transplantation with either null (P<0.02) or AMPK-DN-infected (P<0.01) islets. We conclude that activation of AMPK inhibits beta-cell function in vivo and may represent a target for therapeutic intervention during islet transplantation.
Journal of Endocrinology 11/2005; 187(2):225-35. DOI:10.1677/joe.1.06413 · 3.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Accumulation of triglyceride in islets may contribute to the loss of glucose-stimulated insulin secretion (GSIS) in some forms of type 2 diabetes (Diraison et al., Biochem J 373:769-778, 2004). Here, we use adenoviral vectors and oligonucleotide microarrays to determine the effects of the forced expression of SREBP1c on the gene expression profile of rat islets. Sterol regulatory element binding protein-1c (SREBP1c) overexpression led to highly significant (P <0.1 with respect to null adenovirus) changes in the expression of 1,238 genes or expressed sequence tags, of which 1,180 (95.3%) were upregulated. By contrast, overexpression of constitutively active AMP-activated protein kinase (AMPK), expected to promote lipolysis, altered the expression of 752 genes, of which 702 (93%) were upregulated. To identify specific targets for SREBP1c or AMPK, we eliminated messages that were 1) affected in the same direction by the expression of either protein, 2) changed by less than twofold, or 3) failed a positive false discovery test; 206 SREBP1c-regulated genes (195; 95% upregulated) and 48 AMPK-regulated genes (33; 69% upregulated) remained. As expected, SREBP1c-induced genes included those involved in cholesterol (6), fatty acid (3), and eicosanoid synthesis. Interestingly, somatostatin receptor (sstr1) expression was increased by SREBP1c, whereas AMPK induced the expression of peptide YY, the early endocrine pancreas marker.
[Show abstract][Hide abstract] ABSTRACT: Peroxisome proliferator-activated receptor-gamma (PPARgamma) serves as a target for the thiazolidinedione class of antidiabetic drugs and is an important regulator of adipose tissue differentiation. By contrast, the principal target genes for PPARgamma in the pancreatic islet and the impact of their induction on insulin secretion are largely undefined. Here, we show that mRNAs encoding both isoforms of rodent PPARgamma, gamma1 and gamma2, are expressed in primary rat islets and are upregulated by overexpresssion of the lipogenic transcription factor sterol response element-binding protein 1c. Unexpectedly, however, oligonucleotide microarray analysis demonstrates that graded activation of PPARgamma achieved with 1) the thiazolidinedione GW-347845, 2) transduction with adenoviral PPARgamma1, or 3) a combination of both treatments progressively enhances the expression of genes involved in fatty acid oxidation and transport. Moreover, maximal activation of PPARgamma1 reduces islet triglyceride levels and enhances the oxidation of exogenous palmitate while decreasing glucose oxidation, cellular ATP content, and glucose-, but not depolarization-stimulated, insulin secretion. We conclude that, in the context of the pancreatic islet, the principal response to PPARgamma expression and activation is the activation of genes involved in the disposal, rather than the synthesis, of fatty acids. Although fatty acid oxidation may have beneficial effects on beta-cell function in the longer term by countering beta-cell "lipotoxicity," the acute response to this metabolic shift is a marked inhibition of insulin secretion.
[Show abstract][Hide abstract] ABSTRACT: Accumulation of intracellular lipid by pancreatic islet beta-cells has been proposed to inhibit normal glucose-regulated insulin secretion ('glucolipotoxicity'). In the present study, we determine whether over-expression in rat islets of the lipogenic transcription factor SREBP1c (sterol-regulatory-element-binding protein-1c) affects insulin release, and whether changes in islet lipid content may be reversed by activation of AMPK (AMP-activated protein kinase). Infection with an adenovirus encoding the constitutively active nuclear fragment of SREBP1c resulted in expression of the protein in approx. 20% of islet cell nuclei, with a preference for beta-cells at the islet periphery. Real-time PCR (TaqMan) analysis showed that SREBP1c up-regulated the expression of FAS (fatty acid synthase; 6-fold), acetyl-CoA carboxylase-1 (2-fold), as well as peroxisomal-proliferator-activated receptor-gamma (7-fold), uncoupling protein-2 (1.4-fold) and Bcl2 (B-cell lymphocytic-leukaemia proto-oncogene 2; 1.3-fold). By contrast, levels of pre-proinsulin, pancreatic duodenal homeobox-1, glucokinase and GLUT2 (glucose transporter isoform-2) mRNAs were unaltered. SREBP1c-transduced islets displayed a 3-fold increase in triacylglycerol content, decreased glucose oxidation and ATP levels, and a profound inhibition of glucose-, but not depolarisation-, induced insulin secretion. Culture of islets with the AMPK activator 5-amino-4-imidazolecarboxamide riboside decreased the expression of the endogenous SREBP1c and FAS genes, and reversed the effect of over-expressing active SREBP1c on FAS mRNA levels and cellular triacylglycerol content. We conclude that SREBP1c over-expression, even when confined to a subset of beta-cells, leads to defective insulin secretion from islets and may contribute to some forms of Type II diabetes.