Sphingosine 1-phosphate (S1P) regulates glucose-stimulated insulin secretion in pancreatic beta cells

Recent studies suggest that sphingolipid metabolism is altered during type 2 diabetes. Increased levels of the sphingolipid ceramide are associated with insulin resistance. However, a role for sphingolipids in pancreatic beta cell function, or insulin production, and release remains to be established. Our studies in MIN6 cells and mouse pancreatic islets demonstrate that glucose stimulates an intracellular rise in the sphingolipid, sphingosine 1-phosphate (S1P), whereas the levels of ceramide and sphingomyelin remain unchanged. The increase in S1P levels by glucose is due to activation of sphingosine kinase 2 (SphK2). Interestingly, rises in S1P correlate with increased glucose-stimulated insulin secretion (GSIS). Decreasing S1P levels by treatment of MIN6 cells or primary islets with the sphingosine kinase inhibitor reduces GSIS. Moreover, knockdown of SphK2 alone results in decreased GSIS, whereas knockdown of the S1P phosphatase, Sgpp1, leads to a rise in GSIS. Treatment of mice with the sphingosine kinase inhibitor impairs glucose disposal due to decreased plasma insulin levels. Altogether, our data suggest that glucose activates SphK2 in pancreatic beta cells leading to a rise in S1P levels, which is important for GSIS.     

Taurine regulates insulin release from pancreatic beta cell lines

Background

Pancreatic β-cells release insulin via an electrogenic response triggered by an increase in plasma glucose concentrations. The critical plasma glucose concentration has been determined to be ~3 mM, at which time both insulin and GABA are released from pancreatic β-cells. Taurine, a β-sulfonic acid, may be transported into cells to balance osmotic pressure. The taurine transporter (TauT) has been described in pancreatic tissue, but the function of taurine in insulin release has not been established. Uptake of taurine by pancreatic β-cells may alter membrane potential and have an effect on ion currents. If taurine uptake does alter β-cell current, it might have an effect on exocytosis of cytoplasmic vesicle. We wished to test the effect of taurine on regulating release of insulin from the pancreatic β-cell.

Methods

Pancreatic β-cell lines Hit-TI5 (Syrian hamster) and Rin-m (rat insulinoma) were used in these studies. Cells were grown to an 80% confluence on uncoated cover glass in RPMI media containing 10% fetal horse serum. The cells were then adapted to a serum-free, glucose free environment for 24 hours. At that time, the cells were treated with either 1 mM glucose, 1 mM taurine, 1 mM glucose + 1 mM taurine, 3 mM glucose, or 3 mM glucose + 1 mM taurine. The cells were examined by confocal microscopy for cytoplasmic levels of insulin.

Results

In both cell lines, 1 mM glucose had no effect on insulin levels and served as a control. Cells starved of glucose had a significant reduction (p<0.001) in the level of insulin, but this level was significantly higher than all other treatments. As expected, the 3 mM glucose treatment resulted in a statistically lower (p<0.001) insulin level than control cells. Interestingly, 1 mM taurine also resulted in a statistically lower level of insulin (p<0.001) compared to controls when either no glucose or 1 mM glucose was present. Cells treated with 1 mM taurine plus 3 mM glucose showed a level of insulin similar to that of 3 mM glucose alone.

Conclusions

Taurine administration can alter the electrogenic response in β-cell lines, leading to a change in calcium homeostasis and a subsequent decrease in intracellular insulin levels. The consequence of these actions could represent a method of increasing plasma insulin levels leading to a decrease in plasma glucose levels.

     

Metabolic concomitants in pure, pancreatic beta cells during glucose-stimulated insulin secretion

The role of the redox potential in insulin secretion by beta cells stimulated with high glucose was investigated using an in vitro pancreas perfusion system. To assess glycolytic flux the sum of fructose-1,6-P2 + triose-P was determined in pure beta cells microdissected from lyophilized sections of the isolated perfused pancreas quick frozen during the early insulin secretory response. L-Glycerol 3-phosphate and dihydroxyacetone phosphate were measured as indicators of the free cytosolic [NAD+]/[NADH] ratio and NADH and NADPH were also measured. Fructose-1,6-P2 + triose-P was increased in beta cells simultaneously with the onset of insulin secretion indicating an increase in glucose metabolism had occurred. The ratio of [dihydroxyacetone phosphate]/[L-glycerol 3-phosphate] increased simultaneously with the onset of insulin secretion. NADH content increased only after initiation of insulin secretion and NADPH levels remained unchanged during the early secretory response to high glucose. These data contradict the hypothesis that insulin secretion is triggered by a more reduced cytosolic redox state and instead indicate that insulin secretion is initiated by other metabolic coupling factor(s) generated in beta cells stimulated by high glucose.     

GAD65-mediated glutamate decarboxylation reduces glucose-stimulated insulin secretion in pancreatic beta cells

Mitochondrial metabolism plays a pivotal role in the pancreatic beta cell by generating signals that couple glucose sensing to insulin secretion. We have demonstrated previously that mitochondrially derived glutamate participates directly in the stimulation of insulin exocytosis. The aim of the present study was to impose altered cellular glutamate levels by overexpression of glutamate decarboxylase (GAD) to repress elevation of cytosolic glutamate. INS-1E cells infected with a recombinant adenovirus vector encoding GAD65 showed efficient overexpression of the GAD protein with a parallel increase in enzyme activity. In control cells glutamate levels were slightly increased by 7.5 mm glucose (1.4-fold) compared with the effect at 15 mm (2.3-fold) versus basal 2.5 mm glucose. Upon GAD overexpression, glutamate concentrations were no longer elevated by 15 mm glucose as compared with controls (-40%). Insulin secretion was stimulated in control cells by glucose at 7.5 mm (2.5-fold) and more efficiently at 15 mm (5.2-fold). INS-1E cells overexpressing GAD exhibited impaired insulin secretion on stimulation with 15 mm glucose (-37%). The secretory response to 30 mm KCl, used to raise cytosolic Ca(2+) levels, was unaffected. Similar results were obtained in perifused rat pancreatic islets following adenovirus transduction. This GAD65-mediated glutamate decarboxylation correlating with impaired glucose-induced insulin secretion is compatible with a role for glutamate as a glucose-derived factor participating in insulin exocytosis.     

Electrical activity and insulin release in pancreatic beta cells

The pancreatic beta cell, which is responsible for insulin secretion from the islets of Langerhans, exhibits a complex pattern of membrane-potential oscillations called bursting. These oscillations are induced by glucose and are strongly correlated with the release of insulin. Recently a number of the ion channels which are responsible for carrying membrane currents in the beta cell have been uncovered. This has led to several hypothesized mechanisms for bursting and a good deal of mathematical modeling. In this paper the progress in understanding bursting in the beta cell is reviewed, including the impact of the discovery of the ATP-inactivated, ADP-modulated channel on modeling and the effect of single-channel events on electrical activity.     

A role for transglutaminase in glucose-stimulated insulin release from the pancreatic beta-cell.

Preincubation of rat islets of Langerhans with the potent inhibitors of islet transglutaminase activity, monodansylcadaverine (30-100 microM) and N-(5-aminopentyl)-2-naphthalenesulphonamide (100-200 microM), led to significant inhibition of glucose-stimulated insulin release from islets. In contrast, the respective N'-dimethylated derivatives of these two compounds, which did not inhibit islet transglutaminase activity, were much less effective as inhibitors of glucose-stimulated insulin release. None of the compounds inhibited rat spleen protein kinase C activity at concentrations which gave rise to inhibition of glucose-stimulated insulin release. When tested for their effects on calmodulin-stimulated bovine heart phosphodiesterase activity, of the compounds that inhibited insulin release, only monodansylcadaverine did not act as an effective antagonist of calmodulin at concentrations (up to 50 microM) that gave rise to significant inhibition of glucose-stimulated insulin release. Furthermore, at 50 microM, monodansylcadaverine did not inhibit methylation of islet lipids. The inhibition of glucose-stimulated insulin release by monodansylcadaverine is therefore likely to be attributable to its interference with islet transglutaminase activity. The sensitivity of islet transglutaminase to activation by Ca2+ was investigated by using a modified assay incorporating dephosphorylated NN'-dimethylcasein as a substrate protein. The Km for Ca2+ obtained (approx. 3 microM) was an order of magnitude lower than previously reported for the islet enzyme [Bungay, Potter & Griffin (1984) Biochem. J. 219, 819-827]. Mg2+ (2 mM) was found to have little effect on the sensitivity of the enzyme to Ca2+. Investigation of the endogenous substrate proteins of islet transglutaminase by using the Ca2+-dependent incorporation of [14C]methylamine into proteins of islet homogenates demonstrated that most of the incorporated radiolabel was present in cross-linked polymeric aggregates which did not traverse 3% (w/v) acrylamide gels. The radiolabelled polymeric aggregates were present in 71 000 g-sedimented material of homogenates, and their formation was transglutaminase-mediated. These findings provide new evidence for the involvement of islet transglutaminase in the membrane-mediated events necessary for glucose-stimulated insulin release.     

Glucose-stimulated insulin release by individual pancreatic beta cells: potentiation by glyburide.

To investigate the effect of glyburide on insulin secretion by individual beta cells from normal rats, we employed a reverse hemolytic plaque assay. Pancreata were harvested from female Wistar-Furth rats, the pancreatic islets isolated, and the latter dispersed into single cells. These cells were mixed with protein A-coated ox erythrocytes, the mixture was placed in a Cunningham chamber in the presence of insulin antiserum, and the cells were exposed to the various test substances. Having developed hemolytic plaques around the insulin-secreting cells with complement, the percentage of plaque-forming cells was determined and the plaque areas (reflecting the amount of insulin secreted) were quantitated. For the purpose of validation, we demonstrated that (i) plaque-forming (but not nonplaque-forming) cells could be identified as insulin secreting by an independent immunofluorescent technique, (ii), plaques did not form if insulin antiserum was deleted from the preparation, (iii) plaques failed to develop if insulin antiserum was preabsorbed with insulin, and (iv) incubation with non-protein A-coated RBC or omission of complement resulted in no plaque formation. In addition, both the percentage of plaque-forming cells and the mean plaque are increased upon exposure to glucose (0.75-20 mM) in a concentration-dependent manner at 5- and 60-min incubation times. Moreover, somatostatin suppressed the percentage of plaque-forming cells and diminished the mean plaque area of cells which continued to secrete insulin in response to glucose. Exposure of cells to 100 nM glyburide in the presence of 5 mM or 20 mM glucose had no effect on the percentage of plaque-forming cells present at 5 min or 60 min. Similarly, glyburide did not alter mean plaque area at 5 or 60 min when cells were co-incubated with 5 mM glucose. However, mean plaque area was markedly enhanced at 5 and 60 min in response to glyburide and 20 mM glucose. These results demonstrate that glyburide (i) does appear to enhance insulin secretion by an effect directly on the pancreatic beta cell; (ii) does not act by recruiting previously noninsulin-secreting cells into a secretory pool; (iii) does not potentiate the effect of glucose, at fed concentrations, on insulin secretion by individual cells; but (iv) does augment insulin secretion by beta cells stimulated with supraphysiologic concentrations of glucose.     

Mitochondrial GTP regulates glucose-stimulated insulin secretion

Nucleotide-specific isoforms of the tricarboxylic acid (TCA) cycle enzyme succinyl-CoA synthetase (SCS) catalyze substrate-level synthesis of mitochondrial GTP (mtGTP) and ATP (mtATP). While mtATP yield from glucose metabolism is coupled with oxidative phosphorylation and can vary, each molecule of glucose metabolized within pancreatic beta cells produces approximately one mtGTP, making mtGTP a potentially important fuel signal. In INS-1 832/13 cells and cultured rat islets, siRNA suppression of the GTP-producing pathway (DeltaSCS-GTP) reduced glucose-stimulated insulin secretion (GSIS) by 50%, while suppression of the ATP-producing isoform (DeltaSCS-ATP) increased GSIS 2-fold. Insulin secretion correlated with increases in cytosolic calcium, but not with changes in NAD(P)H or the ATP/ADP ratio. These data suggest a role for mtGTP in controlling pancreatic GSIS through modulation of mitochondrial metabolism, possibly involving mitochondrial calcium. Furthermore, in light of its tight coupling to TCA oxidation rates, mtGTP production may serve as an important molecular signal of TCA-cycle activity.     

Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells

Sir2 and insulin/IGF-1 are the major pathways that impinge upon aging in lower organisms. In Caenorhabditis elegans a possible genetic link between Sir2 and the insulin/IGF-1 pathway has been reported. Here we investigate such a link in mammals. We show that Sirt1 positively regulates insulin secretion in pancreatic beta cells. Sirt1 represses the uncoupling protein (UCP) gene UCP2 by binding directly to the UCP2 promoter. In beta cell lines in which Sirt1 is reduced by SiRNA, UCP2 levels are elevated and insulin secretion is blunted. The up-regulation of UCP2 is associated with a failure of cells to increase ATP levels after glucose stimulation. Knockdown of UCP2 restores the ability to secrete insulin in cells with reduced Sirt1, showing that UCP2 causes the defect in glucose-stimulated insulin secretion. Food deprivation induces UCP2 in mouse pancreas, which may occur via a reduction in NAD (a derivative of niacin) levels in the pancreas and down-regulation of Sirt1. Sirt1 knockout mice display constitutively high UCP2 expression. Our findings show that Sirt1 regulates UCP2 in beta cells to affect insulin secretion.     

Class II phosphoinositide 3-kinase regulates exocytosis of insulin granules in pancreatic beta cells

Phosphoinositide 3-kinases (PI3Ks) are critical regulators of pancreatic β cell mass and survival, whereas their involvement in insulin secretion is more controversial. Furthermore, of the different PI3Ks, the class II isoforms were detected in β cells, although their role is still not well understood. Here we show that down-regulation of the class II PI3K isoform PI3K-C2α specifically impairs insulin granule exocytosis in rat insulinoma cells without affecting insulin content, the number of insulin granules at the plasma membrane, or the expression levels of key proteins involved in insulin secretion. Proteolysis of synaptosomal-associated protein of 25 kDa, a process involved in insulin granule exocytosis, is impaired in cells lacking PI3K-C2α. Finally, our data suggest that the mRNA for PI3K-C2α may be down-regulated in islets of Langerhans from type 2 diabetic compared with non-diabetic individuals. Our results reveal a critical role for PI3K-C2α in β cells and suggest that down-regulation of PI3K-C2α may be a feature of type 2 diabetes.     

Phospholipase D1 regulates secretagogue-stimulated insulin release in pancreatic beta-cells

Phospholipase D (PLD) has been strongly implicated in the regulation of Golgi trafficking as well as endocytosis and exocytosis. Our aim was to investigate the role of PLD in regulating the biphasic exocytosis of insulin from pancreatic beta-cells that is essential for mammalian glucose homeostasis. We observed that PLD activity in MIN6 pancreatic beta-cells is closely coupled to secretion. Cellular PLD activity was increased in response to a variety of secretagogues including the nutrient glucose and the cholinergic receptor agonist carbamoylcholine. Conversely, pharmacological or hormonal inhibition of stimulated secretion reduced PLD activity. Most importantly, blockade of PLD-catalyzed phosphatidic acid formation using butan-1-ol inhibited insulin secretion in both MIN6 cells and isolated pancreatic islets. It was further established that PLD activity was required for both the first and the second phase of glucose-stimulated insulin release, suggesting a role in the very distal steps of exocytosis, beyond granule recruitment into a readily releasable pool. Visualization of granules using green fluorescent protein-phogrin confirmed a requirement for PLD prior to granule fusion with the plasma membrane. PLD1 was shown to be the predominant isoform in MIN6 cells, and it was located at least partially on insulin granules. Overexpression of wild-type or a dominant negative catalytically inactive mutant of PLD1 augmented or inhibited secretagogue-stimulated secretion, respectively. The results suggest that phosphatidic acid formation on the granule membrane by PLD1 is essential for the regulated secretion of insulin from pancreatic beta-cells.     

Activation of TRPV4 channel in pancreatic INS-1E beta cells enhances glucose-stimulated insulin secretion via calcium-dependent mechanisms

Transient receptor potential channel vanilloid type 4 (TRPV4) is a Ca²⁺- and Mg²⁺-permeable cation channel that influences oxidative metabolism and insulin sensitivity. The role of TRPV4 in pancreatic beta cells is largely unknown. Here, we characterize the role of TRPV4 in controlling intracellular Ca²⁺ and insulin secretion in INS-1E beta cells. Osmotic, thermal or pharmacological activation of TRPV4 caused a rapid rise of intracellular Ca²⁺ and enhanced glucose-stimulated insulin secretion. In the presence of the TRPV channel blocker ruthenium red (RuR) or after suppression of TRPV4 protein production, TRPV4 activators failed to increase [Ca²⁺]_i and insulin secretion in INS-1E cells.     

Dynamin-mediated Nephrin Phosphorylation Regulates Glucose-stimulated Insulin Release in Pancreatic Beta Cells

We have previously demonstrated a role for Nephrin in glucose stimulated insulin release (GSIR). We now hypothesize that Nephrin phosphorylation is required for GSIR and that Dynamin influences Nephrin phosphorylation and function. MIN6-C3 Nephrin-deficient pancreatic beta cells and human islets were transfected with WT-Nephrin or with a mutant Nephrin in which the tyrosine residues responsible for SH2 domain binding were substituted with phenylalanine (3YF-Nephrin). GSIR and live images of Nephrin and vesicle trafficking were studied. Immunoprecipitation experiments and overexpression of WT-Dynamin or dominant negative Dynamin mutant (K44A-Dynamin) in WT-Nephrin, 3YF-Nephrin, or Nephrin siRNA-transfected cells were utilized to study Nephrin-Dynamin interaction. In contrast to WT-Nephrin or to single tyrosine mutants, 3YF-Nephrin did not positively affect GSIR and led to impaired cell-cell contacts and vesicle trafficking. K44A-Dynamin prevented the effect of Nephrin on GSIR in the absence of protein-protein interaction between Nephrin and Dynamin. Nephrin gene silencing abolished the positive effects of WT-Dynamin on GSIR. The effects of protamine sulfate and vanadate on Nephrin phosphorylation and GSIR were studied in MIN6 cells and human islets. WT-Nephrin phosphorylation after glucose occurred at Tyr-1176/1193 and resulted in improved GSIR. On the contrary, protamine sulfate-induced phosphorylation at Tyr-1176/1193/1217 was associated with Nephrin degradation and impaired GSIR. Vanadate, which prevented Nephrin dephosphorylation after glucose stimulation, improved GSIR in human islets and MIN6 cells. In conclusion, Dynamin-dependent Nephrin phosphorylation occurs in response to glucose and is necessary for Nephrin-mediated augmentation of GSIR. Pharmacological modulation of Nephrin phosphorylation may thus facilitate pancreatic beta cell function.     

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.     

Insulin-stimulated insulin secretion in single pancreatic beta cells

Functional insulin receptors are known to occur in pancreatic beta cells; however, except for a positive feedback on insulin synthesis, their physiological effects are unknown. Amperometric measurements at single, primary pancreatic beta cells reveal that application of exogenous insulin in the presence or absence of nonstimulatory concentrations of glucose evokes exocytosis mediated by the beta cell insulin receptor. Insulin also elicits increases in intracellular Ca2+ concentration in beta cells but has minimal effects on membrane potential. Conditions where the insulin receptor is blocked or cell surface concentration of free insulin is reduced during exocytosis diminishes secretion induced by other secretagogues, providing evidence for direct autocrine action of insulin upon secretion from the same cell. These results indicate that the beta cell insulin receptor can mediate positive feedback for insulin secretion. The presence of a positive feedback mechanism for insulin secretion mediated by the insulin receptor provides a potential link between impaired insulin secretion and insulin resistance.