Dehydroepiandrosterone increases beta-cell mass and improves the glucose-induced insulin secretion by pancreatic islets from aged rats

The effect of dehydroepiandrosterone (DHEA) on pancreatic islet function of aged rats, an animal model with impaired glucose-induced insulin secretion, was investigated. The following parameters were examined: morphological analysis of endocrine pancreata by immunohistochemistry; protein levels of insulin receptor, IRS-1, IRS-2, PI 3-kinase, Akt-1, and Akt-2; and static insulin secretion in isolated pancreatic islets. Pancreatic islets from DHEA-treated rats showed an increased beta-cell mass accompanied by increased Akt-1 protein level but reduced IR, IRS-1, and IRS-2 protein levels and enhanced glucose-stimulated insulin secretion. The present results suggest that DHEA may be a promising drug to prevent diabetes during aging.     

A role for G(z) in pancreatic islet beta-cell biology

Glucose-stimulated insulin secretion and beta-cell growth are important facets of pancreatic islet beta-cell biology. As a result, factors that modulate these processes are of great interest for the potential treatment of Type 2 diabetes. Here, we present evidence that the heterotrimeric G protein G(z) and its effectors, including some previously thought to be confined in expression to neuronal cells, are present in pancreatic beta-cells, the largest cellular constituent of the islets of Langerhans. Furthermore, signaling pathways upon which G alpha(z) impacts are intact in beta-cells, and G alpha(z) activation inhibits both cAMP production and glucose-stimulated insulin secretion in the Ins-1(832/13) beta-cell-derived line. Inhibition of glucose-stimulated insulin secretion by prostaglandin E (PGE1) is pertussis-toxin insensitive, indicating that other G alpha(i) family members are not involved in this process in this beta-cell line. Indeed, overexpression of a selective deactivator of G alpha(z), the RGS domain of RGSZ1, blocks the inhibitory effect of PGE1 on glucose-stimulated insulin secretion. Finally, the inhibition of glucose-stimulated insulin secretion by PGE1 is substantially blunted by small interfering RNA-mediated knockdown of G alpha(z) expression. Taken together, these data strongly imply that the endogenous E prostanoid receptor in the Ins-1(832/13) beta-cell line couples to G(z) predominantly and perhaps even exclusively. These data provide the first evidence for G(z) signaling in pancreatic beta-cells, and identify an endogenous receptor-mediated signaling process in beta-cells that is dependent on G alpha(z) function.     

Uric acid may inhibit glucose-induced insulin secretion via binding to an essential arginine residue in rat pancreatic beta-cells

Uric acid (1a) suppresses basal insulin release in isolated rat pancreatic islets and inhibition of glucose-stimulated insulin secretion (GSIS) occurs right at hyperuricaemic levels (0.4 mM). Conversely, 1 mM guanidinium urate (2a) was completely ineffective, strongly suggesting that binding to an essential arginine residue triggers the inhibitory effect. A specific recognition of 1a molecule at the crucial beta-cell receptor is probably involved in the blocking glucose signal transduction.     

Insulin receptor activation inhibits insulin secretion from human islets of Langerhans

There is no consensus on the role of insulin secreted from pancreatic β-cells in regulating its own secretion, either in rodent islets or in human islets. We have now investigated whether there is an autocrine signalling role for insulin in human islets by determining insulin receptor expression and assessing the effects of insulin receptor activation using a non-peptidyl insulin mimetic termed L-783,281. Human insulin receptor mRNA was detected by PCR amplification of human islet cDNA, and translation of the message in human islets was confirmed by Western blotting. Perifusion experiments revealed that both glucose-stimulated and basal insulin secretion were significantly inhibited following human islet insulin receptor activation with L-783,281, and that signalling through phosphatidylinositol 3-kinase (PI 3-kinase) was responsible, at least in part, for this inhibitory effect. These studies indicate that human islets express insulin receptors and that they are functionally coupled to a PI 3-kinase-dependent inhibition of insulin secretion.     

Metabolic consequence of long-term exposure of pancreatic beta cells to free fatty acid with special reference to glucose insensitivity.

Long-term exposure of the pancreatic beta cells to free fatty acid (FFA) reportedly inhibits glucose-stimulated insulin secretion. We here studied the impact of FFA on glucose and lipid metabolism in pancreatic beta cells with special reference to insulin secretion. Pancreatic beta-cell line MIN6 was exposed to various concentrations of palmitate for 3 days. Glucose-stimulated insulin secretion and insulin content were decreased corresponding to the concentration of the palmitate exposed. Glycolytic flux and ATP synthesis was unchanged, but pyruvate-stimulated change in NAD(P)H concentration was decreased. Pyruvate carboxylase was decreased at the protein level, which was restored by the removal of palmitate or the inhibition of beta-oxidation. Intracellular content of triglyceride and FFA were elevated, beta-oxidation was increased, and de novo lipogenesis from glucose was decreased. NADPH content and citrate output into the medium, which reflected pyruvate malate shuttle flux, were decreased, but malic enzyme activity was unaffected. The malic enzyme inhibitor alone inhibited insulin response to glucose. In conclusion, long-term exposure of FFA to beta cells inhibits glucose-stimulated insulin secretion via the decreased NADPH contents due to the inhibition of pyruvate carboxylase and malate pyruvate shuttle flux.     

EphA-Ephrin-A-mediated beta cell communication regulates insulin secretion from pancreatic islets

In vertebrates, beta cells are aggregated in the form of pancreatic islets. Within these islets, communication between beta cells inhibits basal insulin secretion and enhances glucose-stimulated insulin secretion, thus contributing to glucose homeostasis during fasting and feeding. In the search for the underlying molecular mechanism, we have discovered that beta cells communicate via ephrin-As and EphAs. We provide evidence that ephrin-A5 is required for glucose-stimulated insulin secretion. We further show that EphA-ephrin-A-mediated beta cell communication is bidirectional: EphA forward signaling inhibits insulin secretion, whereas ephrin-A reverse signaling stimulates insulin secretion. EphA forward signaling is downregulated in response to glucose, which indicates that, under basal conditions, beta cells use EphA forward signaling to suppress insulin secretion and that, under stimulatory conditions, they shift to ephrin-A reverse signaling to enhance insulin secretion. Thus, we explain how beta cell communication in pancreatic islets conversely affects basal and glucose-stimulated insulin secretion to improve glucose homeostasis.     

Preserved Pancreatic β-Cell Development and Function in Mice Lacking the Insulin Receptor-Related Receptor

Receptors of the insulin/insulinlike growth factor (IGF) family have been implicated in the regulation of pancreatic beta-cell growth and insulin secretion. The insulin receptor-related receptor (IRR) is an orphan receptor of the insulin receptor gene (Ir) subfamily. It is expressed at considerably higher levels in beta cells than either insulin or IGF-1 receptors, and it has been shown to engage in heterodimer formation with insulin or IGF-1 receptors. To address whether IRR plays a physiologic role in beta-cell development and regulation of insulin secretion, we have characterized mice lacking IRR and generated a combined knockout of Ir and Irr. We report that islet morphology, beta-cell mass, and secretory function are not affected in IRR-deficient mice. Moreover, lack of IRR does not impair compensatory beta-cell hyperplasia in insulin-resistant Ir(+/-) mice, nor does it affect beta-cell development and function in Ir(-/-) mice. We conclude that glucose-stimulated insulin secretion and embryonic beta-cell development occur normally in mice lacking Irr.     

Pancreatic islet beta-cells transiently metabolize pyruvate

Pancreatic beta-cell metabolism was followed during glucose and pyruvate stimulation of pancreatic islets using quantitative two-photon NAD(P)H imaging. The observed redox changes, spatially separated between the cytoplasm and mitochondria, were compared with whole islet insulin secretion. As expected, both NAD(P)H and insulin secretion showed sustained increases in response to glucose stimulation. In contrast, pyruvate caused a much lower NAD(P)H response and did not generate insulin secretion. Low pyruvate concentrations decreased cytoplasmic NAD(P)H without affecting mitochondrial NAD(P)H, whereas higher concentrations increased cytoplasmic and mitochondrial levels. However, the pyruvate-stimulated mitochondrial increase was transient and equilibrated to near-base-line levels. Inhibitors of the mitochondrial pyruvate-transporter and malate-aspartate shuttle were utilized to resolve the glucose- and pyruvate-stimulated NAD(P)H response mechanisms. These data showed that glucose-stimulated mitochondrial NAD(P)H and insulin secretion are independent of pyruvate transport but dependent on NAD(P)H shuttling. In contrast, the pyruvate-stimulated cytoplasmic NAD(P)H response was enhanced by both inhibitors. Surprisingly the malate-aspartate shuttle inhibitor enabled pyruvate-stimulated insulin secretion. These data support a model in which glycolysis plays a dominant role in glucose-stimulated insulin secretion. Based on these data, we propose a mechanism for glucose-stimulated insulin secretion that includes allosteric inhibition of tricarboxylic acid cycle enzymes and pH dependence of mitochondrial pyruvate transport.     

Reactive oxygen species stimulate insulin secretion in rat pancreatic islets: studies using mono-oleoyl-glycerol

Chronic exposure (24–72 hrs) of pancreatic islets to elevated glucose and fatty acid leads to glucolipoxicity characterized by basal insulin hypersecretion and impaired glucose-stimulated insulin secretion (GSIS). Our aim was to determine the mechanism for basal hypersecretion of insulin. We used mono-oleoyl-glycerol (MOG) as a tool to rapidly increase lipids in isolated rat pancreatic ß-cells and in the clonal pancreatic ß-cell line INS-1 832/13. MOG (25–400 µM) stimulated basal insulin secretion from ß-cells in a concentration dependent manner without increasing intracellular Ca²⁺ or O₂ consumption. Like GSIS, MOG increased NAD(P)H and reactive oxygen species (ROS). The mitochondrial reductant ß-hydroxybutyrate (ß-OHB) also increased the redox state and ROS production, while ROS scavengers abrogated secretion. Diazoxide (0.4 mM) did not prevent the stimulatory effect of MOG, confirming that the effect was independent of the K_ATP-dependent pathway of secretion. MOG was metabolized to glycerol and long-chain acyl-CoA (LC-CoA), whereas, acute oleate did not similarly increase LC-CoA. Inhibition of diacylglycerol kinase (DGK) did not mimic the effect of MOG on insulin secretion, indicating that MOG did not act primarily by inhibiting DGK. Inhibition of acyl-CoA synthetase (ACS) reduced the stimulatory effect of MOG on basal insulin secretion by 30% indicating a role for LC-CoA. These data suggest that basal insulin secretion is stimulated by increased ROS production, due to an increase in the mitochondrial redox state independent of the established components of GSIS.     

Impact of mitochondrial calcium on the coupling of metabolism to insulin secretion in the pancreatic beta-cell

Mitochondria play an essential role in metabolism-secretion coupling in the pancreatic beta-cell. Dysfunction of the organelle leads to impaired glucose-stimulated insulin secretion, as exemplified by the rare disease mitochondrial diabetes, which is caused by mutations in the mitochondrial DNA. In the excitable beta-cell, mitochondria generate ATP and possibly other coupling factors that promote plasma membrane depolarization and calcium influx triggering insulin exocytosis. Cytosolic calcium signals are relayed into the mitochondria, where the ion potentiates oxidative metabolism. Hormones such as glucagon-like peptide 1 (GLP-1) or neurotransmitter secretagogues stimulate the beta-cell by activating different signal transduction pathways eventually also raising mitochondrial calcium. Likewise, pharmacological inhibition of the Na(+)/Ca(2+) exchanger of the inner mitochondrial membrane augments intra-organellar calcium and insulin secretion. Islets obtained after autopsy from type 2 diabetic patients have altered mitochondrial morphology impaired glucose oxidation and reduced ATP generation, explaining defective insulin secretion. We hypothesize that the improvement of glucose-stimulated insulin secretion by sulfonylurea compounds in type 2 diabetic patients is in part due to their capacity to raise mitochondrial calcium, which is beneficial for the generation of metabolic coupling factors.     

Functional genomics of the beta-cell: short-chain 3-hydroxyacyl-coenzyme A dehydrogenase regulates insulin secretion independent of K+ currents

Recent advances in functional genomics afford the opportunity to interrogate the expression profiles of thousands of genes simultaneously and examine the function of these genes in a high-throughput manner. In this study, we describe a rational and efficient approach to identifying novel regulators of insulin secretion by the pancreatic beta-cell. Computational analysis of expression profiles of several mouse and cellular models of impaired insulin secretion identified 373 candidate genes involved in regulation of insulin secretion. Using RNA interference, we assessed the requirements of 10 of these candidates and identified four genes (40%) as being essential for normal insulin secretion. Among the genes identified was Hadhsc, which encodes short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD), an enzyme of mitochondrial beta-oxidation of fatty acids whose mutation results in congenital hyperinsulinism. RNA interference-mediated gene suppression of Hadhsc in insulinoma cells and primary rodent islets revealed enhanced basal but normal glucose-stimulated insulin secretion. This increase in basal insulin secretion was not attenuated by the opening of the KATP channel with diazoxide, suggesting that SCHAD regulates insulin secretion through a KATP channel-independent mechanism. Our results suggest a molecular explanation for the hyperinsulinemia hypoglycemic seen in patients with SCHAD deficiency.     

Protective effects of R-alpha-lipoic acid and acetyl-L-carnitine in MIN6 and isolated rat islet cells chronically exposed to oleic acid

Mitochondrial dysfunction due to oxidative stress and concomitant impaired beta-cell function may play a key role in type 2 diabetes. Preventing and/or ameliorating oxidative mitochondrial dysfunction with mitochondria-specific nutrients may have preventive or therapeutic potential. In the present study, the oxidative mechanism of mitochondrial dysfunction in pancreatic beta-cells exposed to sublethal levels of oleic acid (OA) and the protective effects of mitochondrial nutrients [R-alpha-lipoic acid (LA) and acetyl-L-carnitine (ALC)] were investigated. Chronic exposure (72 h) of insulinoma MIN6 cells to OA (0.2-0.8 mM) increased intracellular oxidant formation, decreased mitochondrial membrane potential (MMP), enhanced uncoupling protein-2 (UCP-2) mRNA and protein expression, and consequently, decreased glucose-induced ATP production and suppressed glucose-stimulated insulin secretion. Pretreatment with LA and/or ALC reduced oxidant formation, increased MMP, regulated UCP-2 mRNA and protein expression, increased glucose-induced ATP production, and restored glucose-stimulated insulin secretion. The key findings on ATP production and insulin secretion were verified with isolated rat islets. These results suggest that mitochondrial dysfunction is involved in OA-induced pancreatic beta-cell dysfunction and that pretreatment with mitochondrial protective nutrients could be an effective strategy to prevent beta-cell dysfunction.     

PIKfyve, a mammalian ortholog of yeast Fab1p lipid kinase, synthesizes 5-phosphoinositides. Effect of insulin.

One or more free hydroxyls of the phosphatidylinositol (PtdIns) head group undergo enzymatic phosphorylation, yielding phosphoinositides (PIs) with key functions in eukaryotic cellular regulation. Two such species, PtdIns 5-P and PtdIns 3,5-P(2), have now been identified in mammalian cells, but their biosynthesis remains unclear. We have isolated a novel mammalian PI kinase, p235, whose exact substrate specificity remained to be determined (Shisheva, A., Sbrissa, D., and Ikonomov, O. (1999) Mol. Cell. Biol. 19, 623-634). Here we report that recombinant p235 expressed in COS cells, like the authentic p235 in adipocytes, displays striking specificity for PtdIns over PI substrates and generates two products identified as PtdIns 5-P and PtdIns 3,5-P(2) by HPLC analyses. Synthetic PtdIns 3-P substrates were also converted to PtdIns 3,5-P(2) but to a substantially lesser extent than PtdIns isolated from natural sources. Important properties of the p235 PI 5-kinase include high sensitivity to nonionic detergents and relative resistance to wortmannin and adenosine. By analyzing deletion mutants in a heterologous cell system, we determined that in addition to the predicted catalytic domain other regions of the molecule are critical for the p235 enzymatic activity. HPLC resolution of monophosphoinositide products, generated by p235 immune complexes derived from lysates of 3T3-L1 adipocytes acutely stimulated with insulin, revealed essentially the same PtdIns 5-P levels as the corresponding p235 immune complexes of resting cells. However, the acute insulin action resulted in an increase of a wortmannin-sensitive PtdIns 3-P peak, suggestive of a plausible recruitment of wortmannin-sensitive PI 3-kinase(s) to p235. In conclusion, mouse p235 (renamed here PIKfyve) displays a strong in vitro activity for PtdIns 5-P and PtdIns 3,5-P(2) generation, implying PIKfyve has a key role in their biosynthesis.     

Circadian and age-dependent expression patterns of GLUT2 and glucokinase in the pancreatic beta-cell of diabetic and nondiabetic rats.

Alterations in glucose sensing are well-known in both humans and animal models of non-insulin-dependent diabetes mellitus. However, the circadian- and age-dependent expression of glucose-sensing genes has not previously been investigated in vivo. In the present paper, we show a progressive loss of beta-cell GLUT2-mRNA and, by immunocytochemistry, a gain of soluble, cytoplasmic GLUT2-protein in Goto-Kakizaki rat islets. We report that GLUT2-mRNA shows significant diurnal variation, which is stronger in metabolically healthy rats. We also demonstrate the significant diurnal variation of glucokinase-mRNA, with higher levels in the pancreas of 6-week-old Goto-Kakizaki rats than in Wistar rats. This leads to a maximum glucose phosphorylation capacity in-phase with food intake, enhanced glucose-stimulated insulin secretion, and prevents postprandial hyperglycemia. Perfusion experiments showed a reduction in glucose-stimulated insulin secretion in Goto-Kakizaki rat islets with an impaired first phase. Hyperglycemia and hypoinsulinemia in newborn and up to 3-week-old Goto-Kakizaki rats are thus probably due to reduced pancreatic beta-cell content, reduced beta-cell insulin content and impaired glucose sensing. The de-compensation of the metabolic situation in 42-week-old Goto-Kakizaki rats is likely to be caused by beta-cell destruction accompanied by negligible accumulation of GLUT2 in the cell membrane and further reduction of glucokinase expression.     

Alkali pH directly activates ATP-sensitive K+ channels and inhibits insulin secretion in beta-cells

Glucose stimulation of pancreatic beta-cells is reported to lead to sustained alkalization, while extracellular application of weak bases is reported to inhibit electrical activity and decrease insulin secretion. We hypothesize that beta-cell K(ATP) channel activity is modulated by alkaline pH. Using the excised patch-clamp technique, we demonstrate a direct stimulatory action of alkali pH on recombinant SUR1/Kir6.2 channels due to increased open probability. Bath application of alkali pH similarly activates native islet beta-cell K(ATP) channels, leading to an inhibition of action potentials, and hyperpolarization of membrane potential. In situ pancreatic perfusion confirms that these cellular effects of alkali pH are observable at a functional level, resulting in decreases in both phase 1 and phase 2 glucose-stimulated insulin secretion. Our data are the first to report a stimulatory effect of a range of alkali pH on K(ATP) channel activity and link this to downstream effects on islet beta-cell function.     

Attenuation of insulin secretion by insulin-like growth factor binding protein-1 in pancreatic beta-cells

IGFBP-1 is involved in glucohomeostasis, but the direct action of IGFBP-1 on the beta-cell remains unclear. Incubation of dispersed mouse beta-cells with IGFBP-1 for 30min inhibited insulin secretion stimulated by glucose, glucagon-like peptide 1 (GLP-1) or tolbutamide without changes in basal release of insulin and in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and NAD(P)H evoked by glucose. In contrast, IGFBP-1 augmented glucose-stimulated insulin secretion in intact islets, associated with a reduced somatostatin secretion. These results suggest a suppressive action of IGFBP-1 on insulin secretion in isolated beta-cells through a mechanism distal to energy generating steps and not involving regulation of [Ca(2+)](i). In contrast, IGFBP-1 amplifies glucose-stimulated insulin secretion in intact islets, possibly by suppressing somatostatin secretion. These direct modulatory influences of IGFBP-1 on insulin secretion may imply an important regulatory role of IGFBP-1 in vivo and in the pathogenesis of type 2 diabetes, in which loss of insulin release is an early pathogenetic event.     

Osmotic stress-induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p

Phosphatidylinositol 3,5-bisphosphate (PtdIns[3,5]P(2)) was first identified as a non-abundant phospholipid whose levels increase in response to osmotic stress. In yeast, Fab1p catalyzes formation of PtdIns(3,5)P(2) via phosphorylation of PtdIns(3)P. We have identified Vac14p, a novel vacuolar protein that regulates PtdIns(3,5)P(2) synthesis by modulating Fab1p activity in both the absence and presence of osmotic stress. We find that PtdIns(3)P levels are also elevated in response to osmotic stress, yet, only the elevation of PtdIns(3,5)P(2) levels are regulated by Vac14p. Under basal conditions the levels of PtdIns(3,5)P(2) are 18-28-fold lower than the levels of PtdIns(3)P, PtdIns(4)P, and PtdIns(4,5)P(2). After a 10 min exposure to hyperosmotic stress the levels of PtdIns(3,5)P(2) rise 20-fold, bringing it to a cellular concentration that is similar to the other phosphoinositides. This suggests that PtdIns(3,5)P(2) plays a major role in osmotic stress, perhaps via regulation of vacuolar volume. In fact, during hyperosmotic stress the vacuole morphology of wild-type cells changes dramatically, to smaller, more highly fragmented vacuoles, whereas mutants unable to synthesize PtdIns(3,5)P(2) continue to maintain a single large vacuole. These findings demonstrate that Vac14p regulates the levels of PtdIns(3,5)P(2) and provide insight into why PtdIns(3,5)P(2) levels rise in response to osmotic stress.     

Adaptations in pulsatile insulin secretion, hepatic insulin clearance, and beta-cell mass to age-related insulin resistance in rats

In health insulin is secreted in discrete insulin secretory bursts from pancreatic beta-cells, collectively referred to as beta-cell mass. We sought to establish the relationship between beta-cell mass, insulin secretory-burst mass, and hepatic insulin clearance over a range of age-related insulin sensitivity in adult rats. To address this, we used a novel rat model with chronically implanted portal vein catheters in which we recently established the parameters to permit deconvolution of portal vein insulin concentration profiles to measure insulin secretion and resolve its pulsatile components. In the present study, we examined total and pulsatile insulin secretion, insulin sensitivity, hepatic insulin clearance, and beta-cell mass in 35 rats aged 2-12 mo. With aging, insulin sensitivity declined, but euglycemia was sustained by an adaptive increase in fasting and glucose-stimulated insulin secretion through the mechanism of a selective augmentation of insulin pulse mass. The latter was attributable to a closely related increase in beta-cell mass (r=0.8, P<0.001). Hepatic insulin clearance increased with increasing portal vein insulin pulse amplitude, damping the delivery of insulin in the systemic circulation. In consequence, the curvilinear relationship previously reported between insulin secretion and insulin sensitivity was extended to both insulin pulse mass and beta-cell mass vs. insulin sensitivity. These data support a central role of adaptive changes in beta-cell mass to permit appropriate insulin secretion in the setting of decreasing insulin sensitivity in the aging animal. They emphasize the cooperative role of pancreatic beta-cells and the liver in regulating the secretion and delivery of insulin to the systemic circulation.