Glucose-sensitive conductances in rat pancreatic beta-cells: contribution to electrical activity

The perforated patch technique was used to assess the relative contribution of K(ATP) channel activity, assessed from input conductance (G(input)), and volume-sensitive anion channel activity to the induction of electrical activity in single isolated rat pancreatic beta-cells by glucose, 2-ketoisocaproate and tolbutamide. In cells equilibrated in the absence of glucose, the membrane potential was -71 mV and G(input) 3.66 nS. Addition of 8 mM glucose resulted in depolarisation, electrical activity and a reduction in G(input), reflecting an inhibition of K(ATP) channels. Cells equilibrated in 4 mM glucose had a membrane potential of -59 mV and a G(input) of 0.88 nS. In this case, a rise in glucose concentration to 8-20 mM again resulted in depolarisation and electrical activity, but caused a small increase in G(input). 2-Ketoisocaproate also evoked electrical activity and an increase in G(input), whereas electrical activity elicited by addition of tolbutamide was accompanied by reduced G(input). Increasing the concentration of glucose from 4 to 8-20 mM generated a noisy inward current at -70 mV, reflecting activation of the volume-sensitive anion channel. The mean amplitude of this current was glucose-dependent within the range 4-20 mM. Addition of 2-ketoisocaproate or a 15% hypotonic solution elicited similar increases in inward current. In contrast, addition of tolbutamide failed to induce the inward current. It is concluded that K(ATP) channel activity is most sensitive to glucose within the range 0-4 mM. At higher glucose concentrations effective in generating electrical activity, activation of the volume-sensitive anion channel could contribute towards the nutrient-induced increase in G(input).     

Tranilast inhibits glucose-induced insulin secretion from pancreatic beta-cells.

Tranilast, N-(3,4-demethoxycinnamoyl)-anthranilic acid, is an anti-allergic agent identified as an inhibitor of mast cell degranulation. Recently, tranilast was shown to decrease albuminuria in a rat model of diabetic nephropathy and to ameliorate vascular hypertrophy in diabetic rats, suggesting that it may be clinically useful in the treatment of diabetic complications. However, the effects of tranilast on glucose tolerance have not been elucidated. Thus, the aim of this study is to investigate the effect of tranilast on insulin secretion in pancreatic beta-cells. Treatment with tranilast significantly suppressed insulin secretion in INS-1E cells and rat islets induced by 16.7 mmol/l glucose. Furthermore, tranilast inhibited tolbutamide-induced insulin secretion. Treatment with tranilast increased (86)Rb (+) efflux from COS-1 cells in which pancreatic beta-cell-type ATP-sensitive K (+) (K (ATP)) channels were reconstructed and suppressed the cytosolic ATP/ADP ratio in INS-1E cells. Interestingly, treatment with tranilast enhanced glucose uptake in INS-1E cells. In the present study, we demonstrated that tranilast inhibited glucose- and tolbutamide-induced insulin secretion through the activation of K (ATP) channels in pancreatic beta-cells.     

Inhibition of electrical activity in mouse pancreatic beta-cells by the ATP/ADP translocase inhibitor, bongkrekic acid

Bongkrekic acid causes fatal food poisoning which is associated with hyperglycaemia. Here we demonstrate that bongkrekic acid, a potent inhibitor of the mitochondrial ATP/ADP translocase, inhibits glucose-induced electrical activity in the pancreatic beta-cell through the stimulation of ATP-sensitive potassium channel (K-ATP-channel) activity. By comparison of its effects with those of oligomycin, we suggest that bongkrekic acid acts by the inhibition of glucose metabolism and may induce hyperglycaemia by impairing beta-cell function.     

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.     

Muscarinic receptor modulation of glucose-induced electrical activity in mouse pancreatic B-cells

Acetylcholine (1-10 microM) depolarized the membrane and stimulated glucose-induced bursts of electrical activity in mouse pancreatic B-cells. The acetylcholine effects were mimicked by muscarine while nicotine had no effect on membrane potential. Pirenzepine, an antagonist of the classical M1-type muscarinic receptors, but not gallamine (1-100 microM), an antagonist of the classical M2-type receptors, antagonized the acetylcholine action on glucose-induced electrical activity (IC50 = 0.25 microM). Bethanechol, an agonist of the classical M2-type muscarinic receptors, was approximately 100 times less effective than acetylcholine in stimulating the electrical activity. In addition, acetylcholine (1 microM) induced a marked increase (25%) in input resistance to the B-cell membrane. The results indicate that acetylcholine exerted its effects on the B-cell membrane by inhibiting K+ conductance via activation of a muscarinic receptor subtype distinct from the classical M2-type receptor.     

Nitric oxide-cyclic GMP system potentiates glucose-induced rise in cytosolic Ca2+ concentration in rat pancreatic beta-cells.

Involvement of nitric oxide (NO) in the regulation of insulin secretion from pancreatic beta-cells was investigated by measuring cytosolic Ca2+ concentration ([Ca2+]i) in isolated rat pancreatic beta-cells. At 7.0 mM glucose, L-arginine (0.1 mM) elevated [Ca2+]i in about 50% of the beta-cells examined. The response was partially inhibited by an NO synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMA; 0.1 mM), suggesting that part of the response was mediated by the production of NO from L-arginine. D-Arginine at higher concentrations (3 or 10 mM) also increased [Ca2+]i at 7.0 mM glucose; however, the response was not affected by L-NMA (0.1 mM). Similar [Ca2+]i elevation was produced by NO (10 nM) and sodium nitroprusside (SNP; 10 microM) at 7.0 mM glucose. The SNP-induced increase in [Ca2+]i was abolished by nicardipine (1 microM), suggesting that the [Ca2+]i response is mediated by Ca2+ influx through L-type voltage-operated Ca2+ channels. In the presence of oxyhemoglobin (1 microM), the [Ca2+]i elevation induced by NO (10 nM) was abolished. Neither degradation products of NO, NO2- nor NO3-, caused any changes in [Ca2+]i. 8-Bromo-cyclic GMP (8-Br-cGMP; 3 mM) and atrial natriuretic peptide (0.1 microM) elevated [Ca2+]i at 7.0 mM glucose. We conclude that NO, which is produced from L-arginine in pancreatic islets, facilitates glucose-induced [Ca2+]i increase via the elevation of cGMP in rat pancreatic beta-cells. NO-cGMP system may physiologically regulate insulin secretion from pancreatic beta-cells.     

Theoretical studies on the electrical activity of pancreatic beta-cells as a function of glucose.

The electrical activity of pancreatic beta-cells, which has been closely correlated both with intracellular Ca2+ concentration and insulin release, is characterized by a biphasic response to glucose and bursts of spiking action potentials. Recent voltage clamp and single channel patch clamp experiments have identified several transmembrane ionic channels that may play key roles in the electrophysiological behavior of beta-cells. There is a hypothesis that Ca2+-activated K+ channels are responsible for both the resting potential during low glucose concentration and the silent phase during bursting. The discovery of the ATP-inactivated K+ channel raises the possibility that the current for this latter K+ channel may dominate the resting potential, while the Ca2+-activated K+ current dominates the silent phase potential between bursts. The recent discovery that Ca2+-activated K+ channels are pH sensitive raises an interesting possibility for the biphasic electrical response. In this paper, numerical methods are presented for evaluating these hypotheses against experimental evidence.     

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.     

Alloxan cytotoxicity in vitro. Inhibition of rubidium ion pumping in pancreatic beta-cells.

Exposing micro-dissected pancreatic islets of non-inbred ob/ob mice to 2-5 mM-alloxan for 10 min decreased the ability of the islets to accumulate Rb+. Rb+ accumulation in pieces of exocrine pancreas was unaffected by alloxan. When islets were treated with alloxan in the presence of 2-20 mM-D-glucose, the Rb+-accumulating ability was protected in a dose-dependent manner. The protective action of D-glucose was reproduced with 3-O-methyl-D-glucose but not with L-glucose or D-mannoheptulose; mannoheptulose prevented D-glucose from exerting its protective action. The inhibition of Rb+ accumulation was due to a decreased inward pumping, since alloxan did not affect Rb+ efflux from pre-loaded islets. The inhibitory effect of alloxan had a latency of about 1 min, as revealed by experiments with dispersed islet cells in suspension. Alloxan-treated islets showed only a marginal decrease in ATP and no change in glucose 6-phosphate concentration. Although alloxan slightly decreased the hydrolysis of ATP in a subcellular fraction enriched in plasma membranes, this effect could not be attributed to a ouabain-sensitive adenosine triphosphatase. The plasma membranes exhibited a K+-activated hydrolysis of p-nitrophenyl phosphate; this enzyme activity too was insensitive to alloxan. Glucose may protect the univalent-cation pump by preventing permeation of alloxan via a path coupled to the hexose-transport system. Inhibition of the pump may be fundamental to the induction of alloxan-diabetes.     

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.     

A role for calcium release-activated current (CRAC) in cholinergic modulation of electrical activity in pancreatic beta-cells.

S. Bordin and colleagues have proposed that the depolarizing effects of acetylcholine and other muscarinic agonists on pancreatic beta-cells are mediated by a calcium release-activated current (CRAC). We support this hypothesis with additional data, and present a theoretical model which accounts for most known data on muscarinic effects. Additional phenomena, such as the biphasic responses of beta-cells to changes in glucose concentration and the depolarizing effects of the sarco-endoplasmic reticulum calcium ATPase pump poison thapsigargin, are also accounted for by our model. The ability of this single hypothesis, that CRAC is present in beta-cells, to explain so many phenomena motivates a more complete characterization of this current.     

Cytochalasin B: Its action on glucose-induced electrical and metabolic responses in rat pancreatic islets

Cytochalasin B was found to inhibit both glucose-induced spike activity and glucose utilization in isolated rat pancreatic islets under conditions previously shown to increase insulin release (Lacy, P.E., Klein N.J. and Fink. C.J. (1973) Endocrinology 92, 1458–1468). However, cytochalasin B did not prevent the depolarization of islet cells caused by high glucose. The results indicate that neither glycolysis nor spike activity are necessary events leading to insulin release.     

The effect of ATP-sensitive K+ channels on the electrical burst activity and insulin secretion in pancreatic beta-cells.

In recent years, the electrical burst activity of the insulin releasing pancreatic beta-cells has attracted many experimentalists and theoreticians, largely because of its functional importance, but also because of the nonlinear nature of the burst activity. The ATP-sensitive K+ channels are believed to play an important role in electrical activity and insulin release. In this paper, we show by computer simulation how ATP and antidiabetic drugs can lengthen the plateau fraction of bursting and how these chemicals can increase the intracellular Ca2+ level in the pancreatic beta-cell.     

Cell-attached recordings of the volume-sensitive anion channel in rat pancreatic beta-cells.

The cell-attached configuration of the patch-clamp technique was used to study the volume-sensitive anion conductance in isolated rat pancreatic beta-cells at the single-channel level. In unstimulated cells, current level was close to zero. Exposure of cells to a 33% hypotonic solution resulted in the generation of an inward current at 0 mV pipette potential. A similar inward current was elicited by a rise in glucose concentration or by addition of alpha-ketoisocaproate. In contrast, the sulphonylurea tolbutamide was ineffective. The inward current evoked by hypotonic solutions consisted of occasional discreet channel events interspersed with periods of current noise which could not be clearly resolved into unitary channel events. Stimulation with glucose resulted in a predominantly noisy pattern of current. With a reduced [Cl(-)] pipette solution, regular channel openings could be resolved in the presence of a stimulatory glucose concentration, with a calculated conductance of 215 pS. Channel activity could also be recorded in excised inside-out patches, though rapid 'rundown' occurred under such conditions. It is concluded that hypotonic solutions and glucose activate the volume-sensitive anion channel in the cell-attached configuration by increasing channel open probability. This generates an inward current in non-voltage-clamped cells. The channel showed complex kinetics which depended in part upon extracellular [Cl(-)].     

Inhibition of oxidative metabolism by nitric oxide restricts EMCV replication selectively in pancreatic beta-cells

Environmental factors, such as viral infection, are proposed to play a role in the initiation of autoimmune diabetes. In response to encephalomyocarditis virus (EMCV) infection, resident islet macrophages release the pro-inflammatory cytokine IL-1β, to levels that are sufficient to stimulate inducible nitric oxide synthase (iNOS) expression and production of micromolar levels of the free radical nitric oxide in neighboring β-cells. We have recently shown that nitric oxide inhibits EMCV replication and EMCV-mediated β-cell lysis and that this protection is associated with an inhibition of mitochondrial oxidative metabolism. Here we show that the protective actions of nitric oxide against EMCV infection are selective for β-cells and associated with the metabolic coupling of glycolysis and mitochondrial oxidation that is necessary for insulin secretion. Inhibitors of mitochondrial respiration attenuate EMCV replication in β-cells, and this inhibition is associated with a decrease in ATP levels. In mouse embryonic fibroblasts (MEFs), inhibition of mitochondrial metabolism does not modify EMCV replication or decrease ATP levels. Like most cell types, MEFs have the capacity to uncouple the glycolytic utilization of glucose from mitochondrial respiration, allowing for the maintenance of ATP levels under conditions of impaired mitochondrial respiration. It is only when MEFs are forced to use mitochondrial oxidative metabolism for ATP generation that mitochondrial inhibitors attenuate viral replication. In a β-cell selective manner, these findings indicate that nitric oxide targets the same metabolic pathways necessary for glucose stimulated insulin secretion for protection from viral lysis.     

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.     

Differential effects of the K+ channel blockers apamin and quinine on glucose-induced electrical activity in pancreatic beta-cells from a strain of ob/ob (obese) mice

The effects of apamin and quinine on glucose-induced electrical activity in pancreatic islets from ob/ob mice (Norwich colony) were compared. Apamin (40-400 nM) increased the duration of the bursts of electrical activity, whereas quinine (50-100 microM) affected only slightly the steady-state electrical response to glucose. This sensitivity to apamin and poor response to quinine contrast with the resistance to apamin and sensitivity to quinine previously reported for pancreatic islets from albino mice. The results give further support to the idea that pancreatic beta-cells from ob/ob mice have a modified Ca2+-activated K+ permeability.