Crucial roles of neuronatin in insulin secretion and high glucose-induced apoptosis in pancreatic beta-cells

Neuronatin (Nnat) was initially identified as a selectively-expressed gene in neonatal brains, but its expression has been also identified in pancreatic beta-cells. Therefore, to investigate the possible functions that Nnat may serve in pancreatic beta-cells, two Nnat isotypes (alpha and beta) were expressed using adenoviruses in murine MIN6N8 pancreatic beta-cells, and the cellular fates and the effects of Nnat on insulin secretion, high glucose-induced apoptosis, and functional impairment were examined. Nnatalpha and Nnatbeta were primarily localized in the endoplasmic reticulum (ER), and their expressions increased insulin secretion by increasing intracellular calcium levels. However, under chronic high glucose conditions, the Nnatbeta to Nnatalpha ratio gradually increased in proportion to the length of exposure to high glucose levels. Moreover, adenovirally-expressed Nnatbeta was inclined to form aggresome-like structures, and we found that Nnatbeta aggregation inhibited the function of the proteasome. Therefore, when glucose is elevated, the expression of Nnatbeta sensitizes MIN6N8 cells to high glucose stress, which in turn, causes ER stress. As a result, expression of Nnatbeta increased hyperglycemia-induced apoptosis. In addition, the expression of Nnatbeta under high glucose conditions decreased the expression of genes important for beta-cell function, such as glucokinase (GCK), pancreas duodenum homeobox-1 (PDX-1), and insulin. Collectively, Nnat may play a critical factor in normal beta-cell function, as well as in the pathogenesis of type 2 diabetes.     

Immunocytochemical localization of alpha-protein kinase C in rat pancreatic beta-cells during glucose-induced insulin secretion

To investigate the role of protein kinase C (PKC) in the regulation of insulin secretion, we visualized changes in the intracellular localization of alpha-PKC in fixed beta-cells from both isolated rat pancreatic islets and the pancreas of awake unstressed rats during glucose-induced insulin secretion. Isolated, perifused rat islets were fixed in 4% paraformaldehyde, detergent permeabilized, and labeled with a mAb specific for alpha-PKC. The labeling was visualized by confocal immunofluorescent microscopy. In isolated rat pancreatic islets perifused with 2.75 mM glucose, alpha-PKC immunostaining was primarily cytoplasmic in distribution throughout the beta-cells. In islets stimulated with 20 mM glucose, there was a significant redistribution of alpha-PKC to the cell periphery. This glucose-induced redistribution was abolished when either mannoheptulose, an inhibitor of glucose metabolism, or nitrendipine, an inhibitor of calcium influx, were added to the perifusate. We also examined changes in the intracellular distribution of alpha-PKC in the beta-cells of awake, unstressed rats that were given an intravenous infusion of glucose. Immunocytochemical analysis of pancreatic sections from these rats demonstrated a glucose-induced translocation of alpha-PKC to the cell periphery of the beta-cells. These results demonstrate that the metabolism of glucose can induce the redistribution of alpha-PKC to the cell periphery of beta-cells, both in isolated islets and in the intact animal, and suggest that alpha-PKC plays a role in mediating glucose-induced insulin secretion.     

Syntaxin 4 facilitates biphasic glucose-stimulated insulin secretion from pancreatic beta-cells

Numerous overexpression studies have recently implicated Syntaxin 4 as an effector of insulin secretion, although its requirement in insulin granule exocytosis is unknown. To address this, islets from Syntaxin 4 heterozygous (-/+) knockout mice were isolated and compared with islets from wild-type mice. Under static incubation conditions, Syntaxin 4 (-/+) islets showed a 60% reduction in glucose-stimulated insulin secretion compared with wild-type islets. Perifusion analyses revealed that Syntaxin 4 (-/+) islets secreted 50% less insulin during the first phase of glucose-stimulated insulin secretion and that this defect could be fully restored by the specific replenishment of recombinant Syntaxin 4. This essential role for Syntaxin 4 in secretion from the islet was localized to the beta-cells because small interfering RNA-mediated depletion of Syntaxin 4 in MIN6 beta-cells abolished glucose-stimulated insulin secretion. Moreover, immunofluorescent confocal microscopy revealed that Syntaxin 4 was principally localized to the beta-cells and not the alpha-cells of the mouse islet. Remarkably, islets isolated from transgenic mice that express 2.4-fold higher levels of Syntaxin 4 relative to wild-type mice secreted approximately 35% more insulin during both phases of insulin secretion, suggesting that increased Syntaxin 4 may be beneficial for enhancing biphasic insulin secretion in a regulated manner. Taken together, these data support the notion that Syntaxin 4-based SNARE complexes are essential for biphasic insulin granule fusion in pancreatic beta-cells.     

TRPM4 controls insulin secretion in pancreatic beta-cells

TRPM4 is a calcium-activated non-selective cation channel that is widely expressed and proposed to be involved in cell depolarization. In excitable cells, TRPM4 may regulate calcium influx by causing the depolarization that drives the activation of voltage-dependent calcium channels. We here report that insulin-secreting cells of the rat pancreatic beta-cell line INS-1 natively express TRPM4 proteins and generate large depolarizing membrane currents in response to increased intracellular calcium. These currents exhibit the characteristics of TRPM4 and can be suppressed by expressing a dominant negative TRPM4 construct, resulting in significantly decreased insulin secretion in response to a glucose stimulus. Reduced insulin secretion was also observed with arginine vasopressin stimulation, a Gq-coupled receptor agonist in beta-cells. Moreover, the recruitment of TRPM4 currents was biphasic in both INS-1 cells as well as HEK-293 cells overexpressing TRPM4. The first phase is due to activation of TRPM4 channels localized within the plasma membrane followed by a slower secondary phase, which is caused by the recruitment of TRPM4-containing vesicles to the plasma membrane during exocytosis. The secondary phase can be observed during perfusion of cells with increasing [Ca(2+)](i), replicated with agonist stimulation, and coincides with an increase in cell capacitance, loss of FM1-43 dye, and vesicle fusion. Our data suggest that TRPM4 may play a key role in the control of membrane potential and electrical activity of electrically excitable secretory cells and the dynamic translocation of TRPM4 from a vesicular pool to the plasma membrane via Ca(2+)-dependent exocytosis may represent a key short- and midterm regulatory mechanism by which cells regulate electrical activity.     

Oleanolic acid enhances insulin secretion in pancreatic beta-cells

We investigated the effect of oleanolic acid, a plant-derived triterpenoid, on insulin secretion and content in pancreatic beta-cells and rat islets. Oleanolic acid significantly enhanced insulin secretion at basal and stimulatory glucose concentrations in INS-1 832/13 cells and enhanced acute glucose-stimulated insulin secretion in isolated rat islets. In the cell line the effects of oleanolic acid on insulin secretion were comparable to that of the sulfonylurea tolbutamide at basal glucose levels and with the incretin mimetic Exendin-4 under glucose-stimulated conditions, yet neither Ca(2+) nor cAMP rose in response to oleanolic acid. Chronic treatment with oleanolic acid increased total cellular insulin protein and mRNA levels. These effects may contribute to the anti-diabetic properties of this natural product.     

Insulin granule trafficking in beta-cells: mathematical model of glucose-induced insulin secretion

A mathematical model that represents the dynamics of intracellular insulin granules in beta-cells is proposed. Granule translocation and exocytosis are controlled by signals assumed to be essentially related to ATP-to-ADP ratio and cytosolic Ca(2+) concentration. The model provides an interpretation of the roles of the triggering and amplifying pathways of glucose-stimulated insulin secretion. Values of most of the model parameters were inferred from available experimental data. The numerical simulations represent a variety of experimental conditions, such as the stimulation by high K(+) and by different time courses of extracellular glucose, and the predicted responses agree with published experimental data. Model capacity to represent data measured in a hyperglycemic clamp was also tested. Model parameter changes that may reflect alterations of beta-cell function present in type 2 diabetes are investigated, and the action of pharmacological agents that bind to sulfonylurea receptors is simulated.     

A role for malonyl-CoA in glucose-stimulated insulin secretion from clonal pancreatic beta-cells

To gain insight into the relationship between acyl coenzyme A (CoA) esters and glucose-induced insulin release, acyl-CoA profiles were determined in clonal pancreatic beta-cells (HIT). A high sensitivity high performance liquid chromatography method was used to measure malonyl, succinyl, beta-hydroxy beta-methylglutaryl and acetyl-CoA esters and free CoASH. Malonyl-CoA content increased more than 3-fold following exposure of HIT cells to 10 mM glucose. The rise in malonyl-CoA, which preceded insulin secretion, was evident 2 min after exposure to glucose and was sustained for at least 30 min. The increase in malonyl-CoA was associated with inhibition of fatty acid oxidation, increased de novo lipid synthesis and a rise in diacylglycerol content. Succinyl-CoA levels, which may reflect anaplerotic influx into the citric acid cycle, were elevated in the presence of glucose. The concentration of acetyl-CoA and the ratio of free CoASH to acetyl-CoA was unchanged. The data are consistent with a metabolic model in which malonyl-CoA mediates the switch from fatty acid catabolism to lipid synthesis during glucose stimulation of beta-cells. We suggest that these changes in lipid metabolism, by leading to increased diacylglycerol synthesis or protein acylation could play a pivotal role in the regulation of the sustained phase of insulin secretion.     

Mitochondrial reactive oxygen species reduce insulin secretion by pancreatic beta-cells

Pancreatic beta-cells exposed to hyperglycemia produce reactive oxygen species (ROS). Because beta-cells are sensitive to oxidative stress, excessive ROS may cause dysfunction of beta-cells. Here we demonstrate that mitochondrial ROS suppress glucose-induced insulin secretion (GIIS) from beta-cells. Intracellular ROS increased 15min after exposure to high glucose and this effect was blunted by inhibitors of the mitochondrial function. GIIS was also suppressed by H(2)O(2), a chemical substitute for ROS. Interestingly, the first-phase of GIIS could be suppressed by 50 microM H(2)O(2). H(2)O(2) or high glucose suppressed the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, and inhibitors of the mitochondrial function abolished the latter effects. Our data suggested that high glucose induced mitochondrial ROS, which suppressed first-phase of GIIS, at least in part, through the suppression of GAPDH activity. We propose that mitochondrial overwork is a potential mechanism causing impaired first-phase of GIIS in the early stages of diabetes mellitus.     

Neurotensin is a regulator of insulin secretion in pancreatic beta-cells

Neurotensin (NT) is secreted from neurons and gastrointestinal endocrine cells. We previously reported that the three NT receptors (NTSRs) are expressed in pancreatic islets and beta cell lines on which we observed a protective effect of NT against cytotoxic agents. In this study, we explored the role of NT on insulin secretion in the endocrine pancreatic beta cells. We observed that NT stimulates insulin secretion at low glucose level and has a small inhibiting effect on stimulated insulin secretion from isolated islets or INS-1E cells. We studied the mechanisms by which NT elicited calcium concentration changes using fura-2 loaded islets or INS-1E cells. NT increases calcium influx through the opening of cationic channels. Similar calcium influxes were observed after treatment with NTSR selective ligands. NT-evoked calcium regulation involves PKC and the translocation of PKCα and PKC? to the plasma membrane. Part of NT effects appears to be also mediated by PKA but not via the Erk pathway. Taken together, these data provide evidence for an important endocrine role of NT in the regulation of the secretory function of beta cells.     

Protein prenylation in glucose-induced insulin secretion from the pancreatic islet beta cell: a perspective.

Insulin secretion from the pancreatic beta-cell is regulated principally by the ambient concentration of glucose. However, the molecular and cellular mechanisms underlying the stimulus-secretion coupling of glucose-stimulated insulin secretion (GSIS) remain only partially understood. Emerging evidence from multiple laboratories suggests key regulatory roles for GTP-binding proteins (G-proteins) in the cascade of events leading to GSIS. This class of signaling proteins undergo a series of requisite post-translational modifications (e.g., prenylation) at their C-terminal cysteines, which appear to be necessary for their targeting to respective membranous sites for optimal interaction with their respective effector proteins. This communication represents a perspective on potential regulatory roles for protein prenylation steps (i.e., protein farnesylation and protein geranylgeranylation) in GSIS from the islet beta cell. Possible consequences of protein prenylation and potential mechanisms underlying glucose-induced regulation of prenylation, specifically in the context of GSIS are also discussed.     

Difference in mechanism between glyceraldehyde- and glucose-induced insulin secretion from isolated rat pancreatic islets

The effects of D-glyceraldehyde and glucose on islet function were compared in order to investigate the difference between them in the mechanism by which they induce insulin secretion. The stimulation of insulin secretion from isolated rat islets by 10 mM glyceraldehyde was not completely inhibited by either 150 microM diazoxide (an opener of ATP-sensitive K(+) channels) or 5 microM nitrendipine (an L-type Ca(2+)-channel blocker), whereas the stimulation of insulin secretion by 20 mM glucose was completely inhibited by either drug. The insulin secretion induced by glyceraldehyde was less augmented by 100 microM carbachol (a cholinergic agonist) than that induced by glucose. The stimulation of myo-inositol phosphate production by 100 microM carbachol was more marked in islets incubated with the hexose than with the triose. The content of glyceraldehyde 3-phosphate, a glycolytic intermediate, in islets incubated with glyceraldehyde was far higher than that in islets incubated with glucose, whereas the ATP content in islets incubated with the triose was significantly lower than that in islets incubated with the hexose. These results suggest that glyceraldehyde not only mimics the effect of glucose on insulin secretion but also has the ability to cause the secretion of insulin without the influx of Ca(2+ )through voltage-dependent Ca(2+) channels. The reason for the lower potency of the triose than the hexose in stimulating insulin secretion is also discussed.     

Metabolic activation-driven mitochondrial hyperpolarization predicts insulin secretion in human pancreatic beta-cells

Mitochondrial metabolism plays a central role in insulin secretion in pancreatic beta-cells. Generation of protonmotive force and ATP synthesis from glucose-originated pyruvate are critical steps in the canonical pathway of glucose-stimulated insulin secretion. Mitochondrial metabolism is intertwined with pathways that are thought to amplify insulin secretion with mechanisms distinct from the canonical pathway, and the relative importance of these two pathways is controversial. Here I show that glucose-induced mitochondrial membrane potential (MMP) hyperpolarization is necessary for, and predicts, the rate of insulin secretion in primary cultured human beta-cells. When glucose concentration is elevated, increased metabolism results in a substantial MMP hyperpolarization, as well as in increased rates of ATP synthesis and turnover marked by faster cell respiration. Using modular kinetic analysis I explored what properties of cellular energy metabolism enable a large glucose-induced change in MMP in human beta-cells. I found that an ATP-dependent pathway activates glucose or substrate oxidation, acting as a positive feedback in energy metabolism. This activation mechanism is essential for concomitant fast respiration and high MMP, and for a high magnitude glucose-induced MMP hyperpolarization and therefore for insulin secretion.     

Glutamate inhibits protein phosphatases and promotes insulin exocytosis in pancreatic beta-cells

In human type 2 diabetes mellitus, loss of glucose-sensitive insulin secretion from the pancreatic beta-cell is an early pathogenetic event, but the mechanisms involved in glucose sensing are poorly understood. A messenger role has been postulated for L-glutamate in linking glucose stimulation to sustained insulin exocytosis in the beta-cell, but the precise nature by which L-glutamate controls insulin secretion remains elusive. Effects of L-glutamate on the activities of ser/thr protein phosphatases (PPase) and Ca(2+)-regulated insulin exocytosis in INS-1E cells were investigated. Glucose increases L-glutamate contents and promotes insulin secretion from INS-1E cells. L-glutamate also dose-dependently inhibits PPase enzyme activities analogous to the specific PPase inhibitor, okadaic acid. L-glutamate and okadaic acid directly and non-additively promote insulin exocytosis from permeabilized INS-1E cells in a Ca(2+)-independent manner. Thus, an increase in phosphorylation state, through inhibition of protein dephosphorylation by glucose-derived L-glutamate, may be a novel regulatory mechanism linking glucose sensing to sustained insulin exocytosis.     

Islet amyloid polypeptide inhibits glucose-stimulated insulin secretion from isolated rat pancreatic islets

Islet amyloid polypeptide has 37 amino acids and is a major component of amyloid deposition in pancreatic islets of patients with type 2 diabetes mellitus. To determine whether the peptide is involved in the impaired insulin secretion in this type of diabetes mellitus, we synthesized islet amyloid polypeptide and its fragments and examined its effect on insulin secretion. Islet amyloid polypeptide inhibited the glucose-stimulated insulin secretion from isolated rat pancreatic islets, as calcitonin gene-related peptide did, but the fragments failed to inhibit the secretion. Thus, we propose that amyloid deposition may be an important factor in the impairment of insulin secretion in type 2 diabetes mellitus.     

Hexamminecobalt(III) chloride inhibits glucose-induced insulin secretion at the exocytotic process

Hexamminecobalt(III) (HAC) chloride was found to have a potent inhibitory effect on glucose-induced insulin secretion from pancreatic islets. HAC at 2 mm inhibited the secretion in response to 22.2 mm glucose by 90% in mouse islets. Perifusion experiments revealed that the first phase of insulin secretion was severely suppressed and that the second phase of secretion was completely abrogated. Removal of HAC from the perifusate immediately restored insulin secretion with a transient overshooting above the normal level. However, HAC failed to affect glucose-induced changes in d-[6-(14)C]glucose oxidation, levels of reduced forms of NAD and NADP, mitochondrial membrane potential, ATP content, cytosolic calcium concentration, or calcium influx into mitochondria. Furthermore, HAC inhibited 50 mm potassium-stimulated insulin secretion by 77% and 10 microm mastoparan-stimulated insulin secretion in the absence of extracellular Ca(2+) by 80%. The results of a co-immunoprecipitation study of lysates from insulin-secreting betaHC9 cells using anti-syntaxin and anti-vesicle-associated membrane protein antibodies for immunoprecipitation or Western blotting suggested that HAC inhibited disruption of the SNARE complex, which is normally observed upon glucose challenge. These results suggest that the inhibitory effect of HAC on glucose-induced insulin secretion is exerted at a site(s) distal to the elevation of cytosolic [Ca(2+)], possibly in the exocytotic machinery per se; and thus, HAC may serve as a useful tool for dissecting the molecular mechanism of insulin exocytotic processes.     

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.