Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes

Increased flux of glucose through the hexosamine biosynthetic pathway (HSP) is believed to mediate hyperglycemia-induced insulin resistance in diabetes. The end product of the HSP, UDP beta-N-acetylglucosamine (GlcNAc), is a donor sugar nucleotide for complex glycosylation in the secretory pathway and for O-linked GlcNAc (O-GlcNAc) addition to nucleocytoplasmic proteins. Cycling of the O-GlcNAc posttranslational modification was blocked by pharmacological inhibition of O-GlcNAcase, the enzyme that catalyzes O-GlcNAc removal from proteins, with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc). PUGNAc treatment increased levels of O-GlcNAc and caused insulin resistance in 3T3-L1 adipocytes. Insulin resistance induced through the HSP by glucosamine and chronic insulin treatment correlated with increased O-GlcNAc levels on nucleocytoplasmic proteins. Whereas insulin receptor autophosphorylation and insulin receptor substrate 2 tyrosine phosphorylation were not affected by PUGNAc inhibition of O-GlcNAcase, downstream phosphorylation of Akt at Thr-308 and glycogen synthase kinase 3 beta at Ser-9 was inhibited. PUGNAc-induced insulin resistance was associated with increased O-GlcNAc modification of several proteins including insulin receptor substrate 1 and beta-catenin, two important effectors of insulin signaling. These results suggest that elevation of O-GlcNAc levels attenuate insulin signaling and contribute to the mechanism by which increased flux through the HSP leads to insulin resistance in adipocytes.     

Glucose stimulates protein modification by O-linked GlcNAc in pancreatic beta cells: linkage of O-linked GlcNAc to beta cell death

The pancreatic beta cell can respond in the long term to hyperglycemia both with an increased capacity for insulin production and, in susceptible individuals, with apoptosis. When glucose-induced apoptosis offsets the increasing beta cell capacity, type 2 diabetes results. Here, we tested the idea that the pathway of glucose metabolism that leads to the modification of intracellular proteins with the O-linked monosaccharide N-acetylglucosamine (O-GlcNAc) is involved in the glucose-induced apoptosis. This idea is based on two recent observations. First, the beta cell expresses much more O-GlcNAc transferase than any other known cell, and second, that the beta cell-specific toxin, streptozotocin (STZ), itself a GlcNAc analog, specifically blocks the enzyme that cleaves O-GlcNAc from intracellular proteins. As a consequence, we now show that hyperglycemia leads to the rapid and reversible accumulation of O-GlcNAc specifically in beta cells in vivo. Animals pretreated with STZ also accumulate O-GlcNAc in their beta cells when hyperglycemic, but this change is sustained upon re-establishment of euglycemia. In concert with the idea that STZ toxicity results from the sustained accumulation of O-GlcNAc after a hyperglycemic episode, we established a low-dose STZ protocol in which the beta cells' toxicity of STZ was manifest only after glucose or glucosamine administration. Transgenic mice with impaired beta cell glucosamine synthesis treated with this protocol are resistant to the diabetogenic effect of STZ plus glucose yet succumb to STZ plus glucosamine. This study provides a causal link between apoptosis in beta cells and glucose metabolism through glucosamine to O-GlcNAc, implicating this pathway of glucose metabolism with beta cell glucose toxicity.     

Anatomical versus functional beta-cell mass in experimental diabetes

The ability of pancreatic beta-cell mass to vary according to insulin requirements is an important component of optimal long-term control of glucose homeostasis. It is generally assumed that alteration of this property largely contributes to the impairment of insulin secretion in type 2 diabetes. However, data in humans are scarce and it is impossible to correlate beta-cell mass and function with the various stages of the disease. Thus, the importance of animal models is obvious. In rodents, increased beta-cell mass associated with an increase in the function of individual beta-cells contributes to the adaptation of the insulin response to insulin resistance in late pregnancy and in obesity. A reduction in beta-cell mass always corresponds to an alteration in insulin secretory capacity of islet tissue (Zucker diabetic fatty and Goto-Kakisaki rats, db/db mice). During regenerative processes following experimental reduction of beta-cell mass [partial pancreatectomy, streptozocin (STZ) injection], beta-cell mass increase is not associated with a corresponding improvement of beta-cell function, thus indicating that regenerative beta-cells did not achieve functional maturity. The main lesson from experimental diabetes is therefore that beta-cell mass cannot always predict functional capacity of the beta-cell tissue and that the functional beta-cell mass rather than the anatomical beta-cell mass must be taken into account at all times.     

The pathophysiologic basis of efficacy and clinical experience with the new oral antidiabetic agents

Type 2 diabetes results from the abnormal resistance of peripheral tissues to insulin and from the progressive insulin secretory failure of the pancreatic beta-cells. Treatment of type 2 diabetes has greatly improved due to the availability of new classes of oral antidiabetic drugs (OADs) and new insulin analogs. Three types of oral medications exert their antidiabetic action without directly stimulating insulin release: alpha-glucosidase inhibitors (e.g., acarbose) interfere with the digestion of dietary glucose precursors and the absorption of glucose; biguanides (e.g., metformin) inhibit hepatic gluconeogenesis, thereby lowering fasting blood glucose concentrations and increasing peripheral insulin sensitivity; and thiazolidinediones (e.g., rosiglitazone) improve the sensitivity of tissues to insulin-stimulated glucose disposal. In contrast, two classes of OADs stimulate insulin release from pancreatic beta-cells. Sulfonylureas (e.g., glyburide) have been used successfully for many years to treat type 2 diabetes, but their prolonged action may result in hypoglycemia. The third-generation sulfonylurea glimepiride is associated with a reduced risk of hypoglycemia and less weight gain than other sulfonylureas. Finally, the meglitinides (e.g., repaglinide) and D-phenylalanine derivatives (e.g., nateglinide) are powerful prandial insulin secretagogues. If the pancreatic beta-cells deteriorate to such an extent that insulin secretion is significantly impaired, treatment with additional exogenous insulin may be required.     

Elevated beta-cell calmodulin produces a unique insulin secretory defect in transgenic mice.

Transgenic mice with elevated levels of beta-cell calmodulin develop severe diabetes even though pancreatic beta-cells contain reserve levels of insulin. Electron microscopic examination of transgenic pancreas confirmed the presence of abundant insulin secretory granules and failed to reveal obvious morphological abnormalities. These observations suggested that excess calmodulin may specifically impair the secretory process. To directly assess the effect of excess calmodulin on beta-cell function we have isolated pancreatic islets from transgenic animals. Transgenic islets from 6- to 8-day-old mice used 40% less glucose than normal islets and contained 58% of the normal insulin content, 90% of the normal glucagon content, and 5-fold higher levels of calmodulin than islets from control mice of the same age. Parallel perifusions of normal and transgenic islets confirmed that excess calmodulin inhibited glucose-stimulated insulin secretion; first phase secretion was reduced by 60%, and second phase secretion was essentially absent. Static assays were performed to assess the response to other secretagogues. All fuel secretagogues tested were ineffective in stimulating insulin secretion from transgenic islets. Secretion in response to depolarizing levels of potassium was also severely impaired. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine increased transgenic secretion, but not to the level obtained in normal islets. Of the compounds examined, only phorbol 12-myristate 13-acetate and carbachol, two substances thought to act in beta-cells by stimulation of protein kinase-C, produced equivalent secretion in normal and transgenic islets. Phorbol 12-myristate 13-acetate also appeared to restore second phase secretion in transgenic islets. These results indicate that the initial period of calmodulin-induced diabetes is due to a secretory defect. This defect appears to be distal to membrane depolarization and is selective for the second phase of insulin secretion.     

Distinct glucose lowering and beta cell protective effects of vanadium and food restriction in streptozotocin-diabetes

Vanadium is an oral insulin-mimetic agent that diminishes hyperglycemia, improves beta-cell insulin store and secretory function, and can reverse the diabetic state chronically after withdrawal from treatment. As food restriction has been reported to enhance insulin sensitivity and reduce insulin demand, we assessed the contribution of a reduced food intake to the glucose lowering and beta-cell protective effects of vanadium. Streptozotocin (STZ)-diabetic rats were untreated (D) or administered vanadyl sulfate in the drinking water (DT) at one week prior to and for 5 weeks following the administration of STZ. An additional group was pair-fed (DP) with an equal amount of food as that consumed by the DT group. Shortly after the induction of diabetes, hyperglycemic D rats demonstrated a significant rise in plasma insulin to levels that initially exceeded that of the controls. This was followed by a steady reduction over several weeks, suggesting a gradual depletion of functional beta-cells. Both vanadium treatment and pair-feeding abolished the insulin hypersecretory response following STZ administration. Glucose lowering was enhanced in DT animals when administered higher concentrations of vanadium, despite no further reduction in food intake, and all DT animals (10/10) were normoglycemic by 5 weeks. Mean pancreatic insulin content in DT rats was improved fourfold and was associated with a greater number of granulated beta-cells. Conversely, food restriction only modestly improved glycemia and the pancreatic insulin store and, unlike DT, DP rats remained highly glucose-intolerant. At 5 weeks of diabetes, fed circulating glucose and insulin levels were strongly correlated (P=0.0002) in the D and DP groups, supporting the notion that glucose lowering with food restriction is dependent on improved plasma insulin levels. A separate correlation was observed in DT animals within a lower range of plasma insulin, suggesting that vanadium, unlike food restriction, reduced plasma glucose by enhancing insulin sensitivity. Thus, vanadium preserves beta-cells in STZ-diabetes at least partially by abolishing the insulin hypersecretory response and the eventual exhaustion of residual insulin stores following a moderate dose of STZ. This property of vanadium would appear to be useful in the treatment of prediabetic and newly diagnosed insulin-dependent diabetes mellitus.     

Growth hormone signaling in pancreatic beta-cells--calcium handling regulated by growth hormone

Deficiency in insulin secretion is a fundamental part in the pathogenesis of all forms of diabetes, determined by impaired secretory function and inadequate beta-cell mass. Growth hormone (GH) is a multifunctional hormone, involved in cellular metabolism, mitogenesis and differentiation. In pancreatic islets, GH is involved in maintaining beta-cell mass, stimulating islet hormone production and insulin secretion, and, therefore, plays a role in maintaining normal insulin sensitivity and glucose homeostasis. The intracellular events that convey the GH signal into various cellular responses remain incompletely understood. In this review, we discuss GH signaling in the beta-cells, with emphasis on Ca(2+) handling and insulin secretion regulated by human GH (hGH). hGH-stimulated rise in [Ca(2+)](i) is dependent on extracellular Ca(2+) and is mediated by Ca(2+)-induced Ca(2+) release (CICR) in the beta-cell. This process is triggered by hGH-stimulated activation of the non-receptor tyrosine kinases JAK2 and c-Src, which causes tyrosine phosphorylation of RyRs, resulting in sensitization of CICR. The rise in [Ca(2+)](i) elicited by hGH is associated with an enhanced insulin secretion, effects that are mediated mainly through the prolactin receptor. These mechanisms indicate that a rise in [Ca(2+)](i) elicited by activation of PRLR is JAK2-dependent and is associated with enhanced insulin secretion. In contrast, GH receptor-mediated increase in [Ca(2+)](i) is JAK2-independent and is dissociated from insulin secretion.     

GLP-1 receptor signaling: effects on pancreatic beta-cell proliferation and survival

Type 2 diabetes is a metabolic disorder characterized by insulin resistance as well as a progressive deterioration of pancreatic beta-cell mass and function. Glucagon-like peptide 1 (GLP-1), an incretin hormone secreted by intestinal L cells, is a promising therapeutic agent in the treatment of diabetes. GLP-1 analogs and enhancers constitute a novel class of anti-diabetes medications which address both the insulin secretion defect as well as the decline in beta-cell mass. GLP-1 improves glucose-stimulated insulin secretion, restores glucose competence in glucose-resistant beta-cells, and stimulates insulin gene expression and biosynthesis. Furthermore, GLP-1 acts as a growth factor by promoting beta-cell proliferation, survival and neogenesis. This review focuses on the molecular mechanisms by which GLP-1 signaling induces beta-cell mass expansion.     

Oscillatory control of insulin secretion

Pancreatic beta-cells possess an inherent ability to generate oscillatory signals that trigger insulin release. Coordination of the secretory activity among beta-cells results in pulsatile insulin secretion from the pancreas, which is considered important for the action of the hormone in the target tissues. This review focuses on the mechanisms underlying oscillatory control of insulin secretion at the level of the individual beta-cell. Recent studies have demonstrated that oscillations of the cytoplasmic Ca(2+) concentration are synchronized with oscillations in beta-cell metabolism, intracellular cAMP concentration, phospholipase C activity and plasma membrane phosphoinositide lipid concentrations. There are complex interdependencies between the different messengers and signalling pathways that contribute to amplitude regulation and shaping of the insulin secretory response to nutrient stimuli and neurohormonal modulators. Several of these pathways may be important pharmacological targets for improving pulsatile insulin secretion in type 2 diabetes.     

Abnormal regulation by glucose of somatostatin secretion in the perfused pancreas of NIDDM rats

We have investigated the influence of non-insulin-dependent diabetes on the regulation of somatostatin secretion from the pancreatic D cell. These results were compared with the concomittantly measured secretory responses from A and B cells. Rats were rendered non-insulin-dependent diabetic by neonatal injection of streptozotocin (STZ). Secretion was studied in perfused pancreas at 6-10 weeks of age. At this age, STZ rats were mildly hyperglycemic, their nonfasting blood glucose being 9.0 +/- 0.8 vs. 5.6 +/- 0.2 mM in control rats. In perfused pancreas from the latter rats, high glucose, i.e., 16.7 mM, stimulated somatostatin secretion but completely failed to do so in STZ rats. Arginine (in the presence of low glucose, i.e., 3.3 mM) moderately stimulated somatostatin secretion in controls but fourfold more in STZ rats. Preperfusion with high glucose markedly potentiated subsequent arginine-induced somatostatin secretion in controls but failed to do so in STZ rats. Basal glucagon release was inhibited by ambient high glucose in control and STZ rats alike. Arginine-induced glucagon release was profoundly inhibited both by ambient and previous exposure to glucose in controls but only slightly and nonsignificantly in STZ rats. The insulin response to high glucose in controls was reduced by 90% in STZ. The insulin response to arginine (in the presence of low glucose) was 3.3-fold enhanced in STZ. Ambient and previous high glucose markedly enhanced arginine-induced insulin secretion in controls but only moderately so in STZ rats. We conclude that already mild hyperglycemia is associated with marked D-cell insensitivity to glucose that is qualitatively similar to A- and B-cell insensitivity.     

Role of the M3 muscarinic acetylcholine receptor in beta-cell function and glucose homeostasis

The release of insufficient amounts of insulin in the presence of elevated blood glucose levels is one of the key features of type 2 diabetes. Various lines of evidence indicate that acetylcholine (ACh), the major neurotransmitter of the parasympathetic nervous system, can enhance glucose-stimulated insulin secretion from pancreatic beta-cells. Studies with isolated islets prepared from whole body M(3) muscarinic ACh receptor knockout mice showed that cholinergic amplification of glucose-dependent insulin secretion is exclusively mediated by the M(3) muscarinic receptor subtype. To investigate the physiological relevance of this muscarinic pathway, we used Cre/loxP technology to generate mutant mice that lack M(3) receptors only in pancreatic beta-cells. These mutant mice displayed impaired glucose tolerance and significantly reduced insulin secretion. In contrast, transgenic mice overexpressing M(3) receptors in pancreatic beta-cells showed a pronounced increase in glucose tolerance and insulin secretion and were resistant to diet-induced glucose intolerance and hyperglycaemia. These findings indicate that beta-cell M(3) muscarinic receptors are essential for maintaining proper insulin secretion and glucose homeostasis. Moreover, our data suggest that enhancing signalling through beta-cell M(3) muscarinic receptors may represent a new avenue in the treatment of glucose intolerance and type 2 diabetes.     

Differentiation between the short and long term effects of glucose on the intracellular calcium content of the pancreatic beta-cell

The problem of how glucose affects the intracellular (La3+-nondisplaceable) calcium content of pancreatic beta-cells was approached by combining measurements of 45Ca in ob/ob-mouse islets loaded to isotopic equilibrium with determinations of calcium using electrothermal atomic absorption spectroscopy. Whereas short term changes of the glucose concentration induced marked alterations of insulin release, the islet content of intracellular 45Ca was remarkably stable. The chronic actions of glucose differed from the acute ones in being readily demonstrable and sometimes resulting even in a suppression of the calcium content. Thus, after 7 days of culture in 20 mM glucose, the amount of intracellular calcium was actually lower than when the islets were cultured at 5.5 mM glucose. The long term effect of glucose in suppressing the islet content of intracellular calcium was associated with degranulation and loss of immunoreactive insulin, indicated both from staining of the beta-cells and measurements of the extracted hormone by RIA. The previously unknown ability of glucose to suppress the islet content of intracellular calcium may consequently result from mobilization of the secretory granules.     

Anti-diabetic actions of glucagon-like peptide-1 on pancreatic beta-cells

Glucagon-like peptide-1 (GLP-1), an incretin hormone, is released from intestinal L-cells in response to nutrients. GLP-1 lowers blood glucose levels by stimulating insulin secretion from pancreatic beta-cells in a glucose-dependent manner. In addition, GLP-1 slows gastric emptying, suppresses appetite, reduces plasma glucagon, and stimulates glucose disposal, which are beneficial for glucose homeostasis. Therefore, incretin-based therapies such as GLP-1 receptor agonists and inhibitors of dipeptidyl peptidase IV, an enzyme which inactivates GLP-1, have been developed for treatment of diabetes. This review outlines our knowledge of the actions of GLP-1 on insulin secretion and biosynthesis, beta-cell proliferation and regeneration, and protection against beta-cell damage, as well as the involvement of recently discovered signaling pathways of GLP-1 action, mainly focusing on pancreatic beta-cells.     

Distinct intracellular Ca2+ response to extracellular adenosine triphosphate in pancreatic beta-cells in rats and mice

Extracellular adenosine triphosphate (ATP) has distinct effects on insulin secretion from pancreatic β-cells between rats and mice. Using a confocal microscope, we compared changes between rats and mice in cytosolic free calcium concentration ([Ca²⁺]_c) in pancreatic β-cells stimulated by extracellular ATP. Extracellular ATP (50 μM) induced calcium release from intracellular calcium stores by activating P2Y receptors in both rat and mouse β-cells. The intracellular calcium release stimulated by extracellular ATP is significantly smaller in amplitude and longer in duration in rat β-cells than in mouse. In response to extracellular ATP, rat β-cells activate store-operated calcium entry following intracellular calcium release. This response is lacking in mouse β-cells. Rat and mouse β-cells both responded to 9 mM glucose by increasing [Ca²⁺]_c. This increase, however, was pronounced only in the rat β-cells. In 9 mM glucose, extracellular ATP induced a pro-nounced calcium release above the increased level of [Ca²⁺]_c in rat β-cells. In mouse β-cells, however, extracellular ATP did not exhibit calcium release on top of the increased level of [Ca²⁺]_c in 9 mM glucose. These results demonstrate distinct responses between rat and mouse β-cells to extracellular ATP under the condition of low and high glucose. Considering that extracellular ATP inhibits insulin secretion from mouse β-cells but stimulates insulin secretion from rat β-cells, we suggest that store-operated Ca²⁺ entry may be related to exocytosis in pancreatic rat β-cells.     

Steroid effects on intracellular degradation of insulin and crinophagy in isolated pancreatic islets

Under physiological conditions substantial amounts of hormone may be degraded within endocrine cells by a crinophagic process comprising fusions of secretory granules with lysosomes. Glucocorticoids may stabilise and progesterone destabilise lysosomal membranes. The effects of corticosterone and progesterone on intracellular degradation of insulin and crinophagy were determined in pancreatic beta-cells, and possible pathways mediating these effects were evaluated. Pancreatic islets were isolated from mice, intracellular degradation of insulin was measured by a pulse-chase method, and crinophagy was studied by electron microscopy. The islets were exposed to 3.3, 5.5 or 28 mM glucose with or without corticosterone, progesterone or the receptor ligands A-224817.0 and WAY-161358. Mifepristone was used to block steroid receptors and indomethacin to inhibit prostaglandin synthesis. Corticosterone caused a concentration-dependent decrease of insulin degradation at the lower glucose concentrations. Progesterone effected a concentration-dependent stimulation of insulin degradation. These results were paralleled with changes of the crinophagic activity in the beta-cells. Corticosterone decreased and progesterone increased islet production of prostaglandin E(2). Mifepristone abolished the steroid actions on insulin degradation and prostaglandin production. The effects of corticosterone were mimicked by the selective glucocorticoid receptor modulator A-224817.0, but in contrast to progesterone, the selective progesterone receptor agonist WAY-161358 had no effect on insulin degradation or prostaglandin production. Inhibition of cyclooxygenase blocked insulin degradation. The findings indicate that both corticosterone and progesterone could affect intracellular insulin degradation and crinophagy solely via the glucocorticoid receptor, and that prostaglandins may have a regulatory role in intracellular turnover of secretory material in pancreatic islet beta-cells.     

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

Vertebrates express at least 15 different synaptotagmins with the same domain structure but diverse localizations and tissue distributions. Synaptotagmin-1,-2, and -9 act as calcium sensors for the fast phrase of neurotransmitter release, and synaptotagmin-12 acts as a calcium-independent modulator of release. The exact functions of the remaining 11 synaptotagmins, however, have not been established. By analogy to the role of synaptotagmin-1, -2, and -9 in neurotransmission, these other synaptotagmins may serve as Ca(2+) transducers regulating other Ca(2+)-dependent membrane processes, such as insulin secretion in pancreatic beta-cells. Of these other synaptotagmins, synaptotagmin-7 is one of the most abundant and is present in pancreatic beta-cells. To determine whether synaptotagmin-7 regulates Ca(2+)-dependent insulin secretion, we analyzed synaptotagmin-7 null mutant mice for glucose tolerance and insulin release. Here, we show that synaptotagmin-7 is required for the maintenance of systemic glucose tolerance and glucose-stimulated insulin secretion. Mutant mice have normal insulin sensitivity, insulin production, islet architecture and ultrastructural organization, and metabolic and calcium responses but exhibit impaired glucose-induced insulin secretion, indicating a calcium-sensing defect during insulin-containing secretory granule exocytosis. Taken together, our findings show that synaptotagmin-7 functions as a positive regulator of insulin secretion and may serve as a calcium sensor controlling insulin secretion in pancreatic beta cells.     

Elevation in intracellular long-chain acyl-coenzyme A esters lead to reduced beta-cell excitability via activation of adenosine 5'-triphosphate-sensitive potassium channels

Closure of pancreatic beta-cell ATP-sensitive potassium (K(ATP)) channels links glucose metabolism to electrical activity and insulin secretion. It is now known that saturated, but not polyunsaturated, long-chain acyl-coenyzme A esters (acyl-CoAs) can potently activate K(ATP) channels when superfused directly across excised membrane patches, suggesting a plausible mechanism to account for reduced beta-cell excitability and insulin secretion observed in obesity and type 2 diabetes. However, reduced beta-cell excitability due to elevation of endogenous saturated acyl-CoAs has not been confirmed in intact pancreatic beta-cells. To test this notion directly, endogenous acyl-CoA levels were elevated within primary mouse beta-cells using virally delivered overexpression of long-chain acyl-CoA synthetase-1 (AdACSL-1), and the effects on beta-cell K(ATP) channel activity and cell excitability was assessed using the perforated whole-cell and cell-attached patch-clamp technique. Data indicated a significant increase in K(ATP) channel activity in AdACSL-1-infected beta-cells cultured in medium supplemented with palmitate/oleate but not with the polyunsaturated fat linoleate. No changes in the ATP/ADP ratio were observed in any of the groups. Furthermore, AdACSL-1-infected beta-cells (with palmitate/oleate) showed a significant decrease in electrical responsiveness to glucose and tolbutamide and a hyperpolarized resting membrane potential at 5 mm glucose. These results suggest a direct link between intracellular fatty ester accumulation and K(ATP) channel activation, which may contribute to beta-cell dysfunction in type 2 diabetes.     

Conditional expression of hepatocyte nuclear factor-1beta, the maturity-onset diabetes of the young-5 gene product, influences the viability and functional competence of pancreatic beta-cells

Mutations in the gene encoding hepatocyte nuclear factor (HNF)1beta result in maturity-onset diabetes of the young-(MODY)5, by impairing insulin secretory responses and, possibly, by reducing beta-cell mass. The functional role of HNF1beta in normal beta-cells is poorly understood; therefore, in the present study, wild-type (WT) HNF1beta, or one of two naturally occurring MODY5 mutations (an activating mutation, P328L329del, or a dominant-negative form, A263insGG) were conditionally expressed in the pancreatic beta-cell line, insulin-1 (INS-1), and the functional consequences examined. Surprisingly, overexpression of the dominant-negative mutant did not modify any of the functional properties of the cells studied (including insulin secretion, cell growth and viability). By contrast, expression of WT HNF1beta was associated with a time- and dose-dependent inhibition of INS-1 cell proliferation and a marked increase in apoptosis. Induction of WT HNF1beta also inhibited the insulin secretory response to nutrient stimuli, membrane depolarisation or activation of protein kinases A and C and this correlated with a significant decrease in pancrease-duodenum homeobox-1 protein levels. The attenuation of insulin secretion was, however, dissociated from the inhibition of proliferation and loss of viability, since expression of the P328L329del mutant led to a reduced rate of cell proliferation, but failed to induce apoptosis or to alter insulin secretion. Taken together, the present results suggest that mature rodent beta-cells are sensitive to increased expression of WT HNF1beta and they imply that the levels of this protein are tightly regulated to maintain secretory competence and cell viability.