Different effects of tolbutamide and diazoxide in alpha, beta-, and delta-cells within intact islets of Langerhans

Interaction between the different types of cells within the islet of Langerhans is vital for adequate control of insulin release. Once insulin secretion becomes defective, as in type 2 diabetes, the most useful drugs to increase insulin release are sulfonylureas. It is well-known that sulfonylureas block K(ATP) channels, which results in depolarization of the membrane that provokes calcium influx and increases intracellular calcium concentration ([Ca2+]i), which thereby triggers insulin secretion. The sulfonamide diazoxide produces the opposite effect: it activates K(ATP) channels, resulting in a decreased insulin secretion. Despite such evidence, little is known about the effect of sulfonylureas and sulfonamides in non-beta-cells of the islet of Langerhans. In this article, we describe the effects of tolbutamide and diazoxide on [Ca2+]i in alpha-, beta-, and delta-cells within intact islets of Langerhans. Tolbutamide elicits an increase in [Ca2+li in beta- and delta-cells, regardless of glucose concentrations. Remarkably, tolbutamide is without effect in alpha-cells. When diazoxide is applied, glucose-induced [Ca2+]i oscillations in beta- and delta-cells are abolished, whereas [Ca2+]i oscillations in alpha-cells remain unaltered. Furthermore, the existence of sulfonylurea receptors is demonstrated in beta-cells but not in alpha-cells by using binding of glybenclamide-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) combined with immunostaining for insulin and glucagon.     

PIP2 and ATP cooperatively prevent cytosolic Ca2+-induced modification of ATP-sensitive K+ channels in rat pancreatic beta-cells

The factors that influence functional coupling between the sulfonylurea receptor (SUR1) and Kir6.2 subunits of ATP-sensitive K+ (K+(ATP)) channels were studied in rat pancreatic beta-cells using patch clamp and microfluorometric techniques. Tolbutamide at 10 micromol/l inhibited K+(ATP) channels in association with occurrence of action currents, but further exposure of beta-cells to the drug for 30 min or longer resulted in reappearance of K+(ATP) channel events. Half-maximal inhibition concentration (IC50) for tolbutamide was 1.5 microl/mol in 2.8 mmol/l glucose, and it was increased to 13.3 micromol/l when the cellular metabolism was inhibited by 0.5 mmol/l 2,4-dinitrophenol (DNP) for 5 min. Tolbutamide at 10 micromol/l induced an increase in cytosolic Ca2+ concentration ([Ca2+]i), and its amplitude was markedly reduced following exposure to 0.5 mmol/l DNP or long-term (30 min) exposure to 10 micromol/l tolbutamide. This tolbutamide insensitivity, as assessed by the [Ca2+]i response, was not observed when the external Ca2+ was omitted during the long-term exposure to tolbutamide. In cell-attached membrane patches, the tolbutamide insensitivity was also produced by treatment of cells with 150 micromol/l diazoxide and 25 mmol/l KCl in the presence, but not absence, of 2 mmol/l Ca2+ in the external solution. When the cytoplasmic face of inside-out membrane patches was treated with higher Ca2+ concentrations (2 micromol/l), both ADP-evoked activation and tolbutamide-induced inhibition of K+ ATP channels were attenuated with retaining ATP-induced inhibition, indicating the modification of K+(ATP) channels. The Ca2+-induced channel modification was prevented partially by phosphatidylinositol 4,5-bisphosphate (PIP2) and completely by ATP and PIP2 together, but not by ATP alone. Treatment of the channel with cytochalasin D, a disrupter of F-actin, evoked channel modification similar to that induced by Ca2+. The modification was prevented completely by phalloidin, a stabilizer of F-actin. In conclusion, long-term exposure to tolbutamide or metabolic inhibition causes modification of K+ ATP channels via mechanisms involving Ca2+-dependent reaction. The modification, which may reflect functional disconnection between SUR1 and Kir6.2, is prevented by ATP and PIP2, which may act cooperatively to stabilize membrane cytoskeletons (F-actin structures).     

Hyperinsulinism of infancy: the regulated release of insulin by KATP channel-independent pathways

Hyperinsulinism of infancy (HI) is a congenital defect in the regulated release of insulin from pancreatic beta-cells. Here we describe stimulus-secretion coupling mechanisms in beta-cells and intact islets of Langerhans isolated from three patients with a novel SUR1 gene defect. 2154+3 A to G SUR1 (GenBank accession number L78207) is the first report of familial HI among nonconsanguineous Caucasians identified in the U.K. Using patch-clamp methodologies, we have shown that this mutation is associated with both a decrease in the number of operational ATP-sensitive K+ channels (KATP channels) in beta-cells and impaired ADP-dependent regulation. There were no apparent defects in the regulation of Ca2+- and voltage-gated K+ channels or delayed rectifier K+ channels. Intact HI beta-cells were spontaneously electrically active and generating Ca2+ action currents that were largely insensitive to diazoxide and somatostatin. As a consequence, when intact HI islets were challenged with glucose and tolbutamide, there was no rise in intracellular free calcium ion concentration ([Ca2+]i) over basal values. Capacitance measurements used to monitor exocytosis in control and HI beta-cells revealed that there were no defects in Ca2+-dependent exocytotic events. Finally, insulin release studies documented that whereas tolbutamide failed to cause insulin secretion as a consequence of impaired [Ca2+]i signaling, glucose readily promoted insulin release. Glucose was also found to augment the actions of protein kinase C- and protein kinase A-dependent agonists in the absence of extracellular Ca2+. These findings document the relationship between SUR1 gene defects and insulin secretion in vivo and in vitro and describe for the first time KATP channel-independent pathways of regulated insulin secretion in diseased human beta-cells.     

Palmitate stimulation of glucagon secretion in mouse pancreatic alpha-cells results from activation of L-type calcium channels and elevation of cytoplasmic calcium

We have investigated the short-term effects of the saturated free fatty acid (FFA) palmitate on pancreatic alpha-cells. Palmitate (0.5 or 1 mmol/l bound to fatty acid-free albumin) stimulated glucagon secretion from intact mouse islets 1.5- to 2-fold when added in the presence of 1-15 mmol/l glucose. Palmitate remained stimulatory in islets depolarized with 30 mmol/l extracellular K(+) or exposed to forskolin, but it did not remain stimulatory after treatment with isradipine or triacsin C. The stimulatory action of palmitate on secretion correlated with a 3.5-fold elevation of intracellular free Ca(2+) when applied in the presence of 15 mmol/l glucose, a 40% stimulation of exocytosis (measured as increases in cell capacitance), and a 25% increase in whole-cell Ca(2+) current. The latter effect was abolished by isradipine, suggesting that palmitate selectively modulates l-type Ca(2+) channels. The effect of palmitate on exocytosis was not mediated by palmitoyl-CoA, and intracellular application of this FFA metabolite decreased rather than enhanced Ca(2+)-induced exocytosis. The stimulatory effects of palmitate on glucagon secretion were paralleled by a approximately 50% inhibition of somatostatin release. We conclude that palmitate increases alpha-cell exocytosis principally by enhanced Ca(2+) entry via l-type Ca(2+) channels and, possibly, relief from paracrine inhibition by somatostatin released by neighboring delta-cells.     

Sulfonylurea compounds uncouple the glucose dependence of the insulinotropic effect of glucagon-like peptide 1

Glucagon-like peptide (GLP)-1 mimetics have been reported to cause hypoglycemia when combined with sulfonylureas. This study investigated the impact of tolbutamide on the glucose dependence of the GLP-1-mediated effects on insulin, glucagon, and somatostatin secretion in the in situ perfused rat pancreas. At 3 mmol/l glucose, GLP-1 alone did not augment insulin secretion, whereas tolbutamide alone caused a rapid increase in insulin secretion. However, when GLP-1 and tolbutamide were administered simultaneously, insulin secretion increased significantly to 43.7 +/- 6.2 pmol/min (means +/- SE), exceeding the sum of the responses to GLP-1 (2.0 +/- 0.6 pmol/min; P = 0.019) and tolbutamide (11.3 +/- 3.8; P = 0.005) alone by a factor of 3.3. At 11 mmol/l glucose, co-infusion of GLP-1 and tolbutamide augmented insulin secretion to 141.7 +/- 10.3 vs. 115.36 +/- 14.1 (GLP-1) and 42.5 +/- 7.3 pmol/min (tolbutamide). Interestingly, increases in somatostatin secretion, both by glucose and GLP-1, were consistently paralleled by suppression of glucagon release. In conclusion, we demonstrate uncoupling of GLP-1 from its glucose dependence by tolbutamide. This uncoupling probably explains the tendency of GLP-1 to provoke hypoglycemia in combination with sulfonylureas. The results suggest that closure of ATP-sensitive K(+) channels by glucose might be involved in the glucose dependence of GLP-1's insulinotropic effect and that somatostatin acts as a paracrine regulator of glucagon release.     

Glucose or insulin, but not zinc ions, inhibit glucagon secretion from mouse pancreatic alpha-cells

The mechanisms by which hypoglycemia stimulates glucagon release are still poorly understood. In particular, the relative importance of direct metabolic coupling versus paracrine regulation by beta-cell secretory products is unresolved. Here, we compare the responses to glucose of 1) alpha-cells within the intact mouse islet, 2) dissociated alpha-cells, and 3) clonal alphaTC1-9 cells. Free cytosolic concentrations of ATP ([ATP](c)) or Ca(2+) ([Ca(2+)](c)) were imaged using alpha-cell-targeted firefly luciferase or a green fluorescent protein-based Ca(2+) probe ("pericam"), respectively. Consistent with a direct effect of glucose on alpha-cell oxidative metabolism, an increase in glucose concentration (from 0 or 3 mmol/l to 20 mmol/l) increased [ATP](c) by 7-9% in alpha-cells within the intact islet and by approximately 4% in alphaTC1-9 cells. Moreover, glucose also dose-dependently decreased the frequency of [Ca(2+)](c) oscillations in both dissociated alpha-cells and alphaTC1-9 cells. Although the effects of glucose were mimicked by exogenous insulin, they were preserved when insulin signaling was blocked with wortmannin. Addition of ZnCl(2) slightly increased the frequency of [Ca(2+)](c) oscillations but failed to affect glucagon release from either islets or alphaTC1-9 cells under most conditions. We conclude that glucose and insulin, but not Zn(2+) ions, independently suppress glucagon secretion in the mouse.     

Triggering and amplifying pathways of regulation of insulin secretion by glucose

Glucose stimulates insulin secretion by generating triggering and amplifying signals in beta-cells. The triggering pathway is well characterized. It involves the following sequence of events: entry of glucose by facilitated diffusion, metabolism of glucose by oxidative glycolysis, rise in the ATP-to-ADP ratio, closure of ATP-sensitive K+ (KATP) channels, membrane depolarization, opening of voltage-operated Ca2+ channels, Ca2+ influx, rise in cytoplasmic free Ca2+ concentration ([Ca2+]i), and activation of the exocytotic machinery. The amplifying pathway can be studied when beta-cell [Ca2+]i is elevated and clamped by a depolarization with either a high concentration of sulfonylurea or a high concentration of K+ in the presence of diazoxide (K(ATP) channels are then respectively blocked or held open). Under these conditions, glucose still increases insulin secretion in a concentration-dependent manner. This increase in secretion is highly sensitive to glucose (produced by as little as 1-6 mmol/l glucose), requires glucose metabolism, is independent of activation of protein kinases A and C, and does not seem to implicate long-chain acyl-CoAs. Changes in adenine nucleotides may be involved. The amplification consists of an increase in efficacy of Ca2+ on exocytosis of insulin granules. There exists a clear hierarchy between both pathways. The triggering pathway predominates over the amplifying pathway, which remains functionally silent as long as [Ca2+]i has not been raised by the first pathway; i.e., as long as glucose has not reached its threshold concentration. The alteration of this hierarchy by long-acting sulfonylureas or genetic inactivation of K(ATP) channels may lead to inappropriate insulin secretion at low glucose. The amplifying pathway serves to optimize the secretory response not only to glucose but also to nonglucose stimuli. It is impaired in beta-cells of animal models of type 2 diabetes, and indirect evidence suggests that it is altered in beta-cells of type 2 diabetic patients. Besides the available drugs that act on K(ATP) channels and increase the triggering signal, novel drugs that correct a deficient amplifying pathway would be useful to restore adequate insulin secretion in type 2 diabetic patients.     

Paradoxical stimulation of glucagon secretion by high glucose concentrations

Hypersecretion of glucagon contributes to the dysregulation of glucose homeostasis in diabetes. To clarify the underlying mechanism, glucose-regulated glucagon secretion was studied in mouse pancreatic islets and clonal hamster In-R1-G9 glucagon-releasing cells. Apart from the well-known inhibition of secretion with maximal effect around 7 mmol/l glucose, we discovered that mouse islets showed paradoxical stimulation of glucagon release at 25-30 mmol/l and In-R1-G9 cells at 12-20 mmol/l sugar. Whereas glucagon secretion in the absence of glucose was inhibited by hyperpolarization with diazoxide, this agent tended to further enhance secretion stimulated by high concentrations of the sugar. Because U-shaped dose-response relationships for glucose-regulated glucagon secretion were observed in normal islets and in clonal glucagon-releasing cells, both the inhibitory and stimulatory components probably reflect direct effects on the alpha-cells. Studies of isolated mouse alpha-cells indicated that glucose inhibited glucagon secretion by lowering the cytoplasmic Ca(2+) concentration. However, stimulation of glucagon release by high glucose concentrations did not require elevation of Ca(2+), indicating involvement of novel mechanisms in glucose regulation of glucagon secretion. A U-shaped dose-response relationship for glucose-regulated glucagon secretion may explain why diabetic patients with pronounced hyperglycemia display paradoxical hyperglucagonemia.     

Kir6.2 polymorphisms sensitize beta-cell ATP-sensitive potassium channels to activation by acyl CoAs: a possible cellular mechanism for increased susceptibility to type 2 diabetes?

The commonly occurring E23K and I337V Kir6.2 polymorphisms in the ATP-sensitive potassium (KATP) channel are more frequent in Caucasian type 2 diabetic populations. However, the underlying cellular mechanisms contributing to the pathogenesis of type 2 diabetes remain uncharacterized. Chronic elevation of plasma free fatty acids observed in obese and type 2 diabetic subjects leads to cytosolic accumulation of long-chain acyl CoAs (LC-CoAs) in pancreatic beta-cells. We postulated that the documented stimulatory effects of LC-CoAs on KATP channels might be enhanced in polymorphic KATP channels. Patch-clamp experiments were performed on inside-out patches containing recombinant KATP channels (Kir6.2/SUR1) to record macroscopic currents. KATP channels containing Kir6.2 (E23K/I337V) showed significantly increased activity in response to physiological palmitoyl-CoA concentrations (100-1,000 nmol/l) compared with wild-type KATP channels. At physiological intracellular ATP concentrations (mmol/l), E23K/I337V polymorphic KATP channels demonstrated significantly enhanced activity in response to palmitoyl-CoA. The observed increase in KATP channel activity may result in multiple defects in glucose homeostasis, including impaired insulin and glucagon-like peptide-1 secretion and increased glucagon release. In summary, these results suggest that the E23K/I337V polymorphism may have a diabetogenic effect via increased KATP channel activity in response to endogenous levels of LC-CoAs in tissues involved in the maintenance of glucose homeostasis.     

Measurements of cytoplasmic Ca2+ in islet cell clusters show that glucose rapidly recruits beta-cells and gradually increases the individual cell response

The proportion of isolated single beta-cells developing a metabolic, biosynthetic, or secretory response increases with glucose concentration (recruitment). It is unclear whether recruitment persists in situ when beta-cells are coupled. We therefore measured the cytoplasmic free Ca2+ correction ([Ca2+]i) (the triggering signal of glucose-induced insulin secretion) in mouse islet single cells or clusters cultured for 1-2 days. In single cells, the threshold glucose concentration ranged between 6 and 10 mmol/l, at which concentration a maximum of approximately 65% responsive cells was reached. Only 13% of the cells did not respond to glucose plus tolbutamide. The proportion of clusters showing a [Ca2+]i rise increased from approximately 20 to 95% between 6 and 10 mmol/l glucose, indicating that the threshold sensitivity to glucose differs between clusters. Within responsive clusters, 75% of the cells were active at 6 mmol/l glucose and 95-100% at 8-10 mmol/l glucose, indicating that individual cell recruitment is not prominent within clusters; in clusters responding to glucose, all or almost all cells participated in the response. Independently of cell recruitment, glucose gradually augmented the magnitude of the average [Ca2+]i rise in individual cells, whether isolated or associated in clusters. When insulin secretion was measured simultaneously with [Ca2+]i, a good temporal and quantitative correlation was found between both events. However, beta-cell recruitment was maximal at 10 mmol/l glucose, whereas insulin secretion increased up to 15-20 mmol/l glucose. In conclusion, beta-cell recruitment by glucose can occur at the stage of the [Ca2+]i response. However, this type of recruitment is restricted to a narrow range of glucose concentrations, particularly when beta-cell association decreases the heterogeneity of the responses. Glucose-induced insulin secretion by islets, therefore, cannot entirely be ascribed to recruitment of beta-cells to generate a [Ca2+]i response. Modulation of the amplitude of the [Ca2+]i response and of the action of Ca2+ on exocytosis (amplifying actions of glucose) may be more important.     

P2Y purinergic potentiation of glucose-induced insulin secretion and pancreatic beta-cell metabolism

Purine nucleotides and their analogs increase insulin secretion through activation of pancreatic beta-cell P2Y receptors. The present study aimed at determining the role of glucose metabolism in the response to P2Y agonists and whether ATP-activated K+ channels (KATP channels) are involved in this response. The experiments were performed in the rat isolated pancreas, perfused with a Krebs-bicarbonate buffer supplemented with 2 g/l bovine serum albumin under dynamic glucose conditions from 5 mmol/l baseline to 11 mmol/l. ADPbetaS (0.5 micromol/l) was selected as a stable and selective P2Y agonist. This compound, ineffective on the 5 mmol/l glucose background, induced a significant threefold increase in insulin release triggered by the glucose challenge. The effect of ADPbetaS was markedly reduced (P <0.001) in the presence of an inhibitor of glucose metabolism. In addition to glucose, the ADP analog also amplified the beta-cell insulin response to 15 mmol/l methyl pyruvate (P <0.05), but it was ineffective on the insulin response to 2.5 mmol/l methyl succinate. A nonmetabolic stimulus was applied using tolbutamide (185 micromol/l). Insulin secretion induced by the KATP channel blocker was strongly reinforced by ADPbetaS (P <0.001), which prompted us to check a possible interplay of KATP channels in the effect of ADPbetaS. In the presence of diazoxide 250 micromol/l and 21 mmol/l KCl, ADPbetaS still amplified the second phase of glucose-induced insulin secretion (P <0.001). We conclude that P2Y receptor activation is able to promote insulin secretion through a mechanism, involving beta-cell metabolism and a rise in intracellular calcium; this effect does not result from a direct inhibitory effect on KATP channels.     

Roles of ATP-sensitive K+ channels as metabolic sensors: studies of Kir6.x null mice

ATP-sensitive K+ channels (KATP channels) are present in various tissues, including pancreatic beta-cells, heart, skeletal muscles, vascular smooth muscles, and brain. KATP channels are hetero-octameric proteins composed of inwardly rectifying K+ channel (Kir6.x) and sulfonylurea receptor (SUR) subunits. Different combinations of Kir6.x and SUR subunits comprise KATP channels with distinct electrophysiological and pharmacological properties. Recent studies of genetically engineered mice have provided insight into the physiological and pathophysiological roles of Kir6.x-containing KATP channels. Analysis of Kir6.2 null mice has shown that Kir6.2/SUR1 channels in pancreatic beta-cells and the hypothalamus are essential in glucose-induced insulin secretion and hypoglycemia-induced glucagon secretion, respectively, and that Kir6.2/SUR2 channels are involved in glucose uptake in skeletal muscles. Kir6.2-containing KATP channels in brain also are involved in protection from hypoxia-induced generalized seizure. In cardiovascular tissues, Kir6.1-containing KATP channels are involved in regulation of vascular tonus. In addition, the Kir6.1 null mouse is a model of Prinzmetal angina in humans. Our studies of Kir6.2 null and Kir6.1 null mice reveal that KATP channels are critical metabolic sensors in acute metabolic changes, including hyperglycemia, hypoglycemia, ischemia, and hypoxia.     

Tight coupling between electrical activity and exocytosis in mouse glucagon-secreting alpha-cells

alpha-Cells were identified in preparations of dispersed mouse islets by immunofluorescence microscopy. A high fraction of alpha-cells correlated with a small cell size measured as the average cell diameter (10 microm) and whole-cell capacitance (<4 pF). The alpha-cells generated action potentials at a low frequency (1 Hz) in the absence of glucose. These action potentials were reversibly inhibited by elevation of the glucose concentration to 20 mmol/l. The action potentials originated from a membrane potential more negative than -50 mV, had a maximal upstroke velocity of 5 V/s, and peaked at +1 mV. Voltage-clamp experiments revealed the ionic conductances underlying the generation of action potentials. alpha-Cells are equipped with a delayed tetraethyl-ammonium-blockable outward current (activating at voltages above -20 mV), a large tetrodotoxin-sensitive Na+ current (above -30 mV; peak current 200 pA at +10 mV), and a small Ca2+ current (above -50 mV; peak current 30 pA at +10 mV). The latter flowed through omega-conotoxin GVIA (25%)- and nifedipine-sensitive (50%) Ca(2+)-channels. Mouse alpha-cells contained, on average, 7,300 granules, which undergo Ca(2+)-induced exocytosis when the alpha-cell is depolarized. Three functional subsets of granules were identified, and the size of the immediately releasable pool was estimated as 80 granules, or 1% of the total granule number. The maximal rate of exocytosis (1.5 pF/s) was observed 21 ms after the onset of the voltage-clamp depolarization, which is precisely the duration of Ca(2+)-influx during an action potential. Our results suggest that the secretory machinery of the alpha-cell is optimized for maximal efficiency in the use of Ca2+ for exocytosis.     

Glucose dependence of imidazoline-induced insulin secretion: different characteristics of two ATP-Sensitive K+ channel-blocking compounds

The glucose dependence of the insulinotropic action of KATP channel-blocking imidazoline compounds was investigated. Administration of 100 micromol/l phentolamine, but not 100 micromol/l efaroxan, markedly increased insulin secretion of freshly isolated mouse islets when the perifusion medium contained 5 mmol/l glucose. When the glucose concentration was raised to 10 mmol/l in the continued presence of either imidazoline, a clear potentiation of secretion occurred as compared with 10 mmol/l glucose alone. In the presence of efaroxan, a brisk first-phase-like increase was followed by a sustained phase, whereas a more gradual increase resulted in the presence of phentolamine. Administration of 100 micromol/l phentolamine was somewhat more effective than 100 micromol/l efaroxan to inhibit KATP channel activity in intact cultured beta-cells (reduction by 96 vs. 83%). Both compounds were similarly effective to depolarize the beta-cells. When measured by the perforated patch-technique, the depolarization by efaroxan was often oscillatory, whereas that by phentolamine was sustained. In perifused cultured islets, both compounds increased the cytosolic calcium concentration ([Ca2+]c) in the presence of 5 and 10 mmol/l glucose. Efaroxan induced large amplitude oscillations of [Ca2+]c, whereas phentolamine induced a sustained increase. It appears that a KATP channel block by imidazolines is not incompatible with a glucose-selective enhancement of insulin secretion. The glucose selectivity of efaroxan may involve an inhibitory effect distal to [Ca2+]c increase and/or the generation of [Ca2+]c oscillations.     

Intraislet hyperinsulinemia prevents the glucagon response to hypoglycemia despite an intact autonomic response

Because absence of the glucagon response to falling plasma glucose concentrations plays a key role in the pathogenesis of iatrogenic hypoglycemia in patients with insulin-deficient diabetes and the mechanism of this defect is unknown, and given evidence in experimental animals that a decrease in intraislet insulin is a signal to increased glucagon secretion, we examined the role of endogenous insulin in the physiological glucagon response to hypoglycemia. We tested the hypothesis that intraislet hyperinsulinemia prevents the glucagon response to hypoglycemia despite an intact autonomic-adrenomedullary, sympathetic neural, and parasympathetic neural-response and a low alpha-cell glucose concentration. Twelve healthy young adults were studied on three separate occasions. Insulin was infused in hourly steps in relatively low doses (1.5, 3.0, 4.5, and 6.0 pmol.kg(-1).min(-1)) from 60 through 300 min on all three occasions. Plasma glucose levels were clamped at euglycemia ( approximately 5.0 mmol/l, approximately 90 mg/dl) on one occasion and at hourly steps of approximately 4.7, 4.2, 3.6, and 3.0 mmol/l ( approximately 85, 75, 65, and 55 mg/dl) from 60 through 300 min on the other two occasions. On one of the latter occasions, the beta-cell secretagogue tolbutamide was infused in a dose of 1.0 g/h from 60 through 300 min. Hypoglycemia with tolbutamide infusion, compared with similar hypoglycemia alone, was associated with higher (P < 0.0001) C-peptide levels (final values of 1.0 +/- 0.2 vs. 0.1 +/- 0.0 nmol/l), higher (P < 0.0001) rates of insulin secretion (final values of 198 +/- 60 vs. 15 +/- 4 pmol/min), and higher (P < 0.0001) insulin levels (final values of 325 +/- 30 vs. 245 +/- 20 pmol/l) as expected. The glucagon response to hypoglycemia was prevented during tolbutamide infusion (P < 0.0001). Glucagon levels were 17 +/- 1 pmol/l at baseline on both occasions, 14 +/- 1 vs. 15 +/- 1 pmol/l, respectively, during the initial hyperinsulinemic euglycemia, and 15 +/- 1 vs. 22 +/- 2 pmol/l, respectively, during hypoglycemia with and without tolbutamide infusion. Autonomic-adrenomedullary (plasma epinephrine), sympathetic neural (plasma norepinephrine), and parasympathetic neural (plasma pancreatic polypeptide)-responses to hypoglycemia were not reduced during tolbutamide infusion. We conclude that intraislet hyperinsulinemia prevents the glucagon response to hypoglycemia despite an intact autonomic response and a low alpha-cell glucose concentration.     

Glucose-dependent regulation of gamma-aminobutyric acid (GABA A) receptor expression in mouse pancreatic islet alpha-cells

The mechanism(s) by which glucose regulates glucagon secretion both acutely and in the longer term remain unclear. Added to isolated mouse islets in the presence of 0.5 mmol/l glucose, gamma-aminobutyric acid (GABA) inhibited glucagon release to a similar extent (46%) as 10 mmol/l glucose (55%), and the selective GABA(A) receptor (GABA(A)R) antagonist SR95531 substantially reversed the inhibition of glucagon release by high glucose. GABA(A)R alpha4, beta3, and gamma2 subunit mRNAs were detected in mouse islets and clonal alphaTC1-9 cells, and immunocytochemistry confirmed the presence of GABA(A)Rs at the plasma membrane of primary alpha-cells. Glucose dose-dependently increased GABA(A)R expression in both islets and alphaTC1-9 cells such that mRNA levels at 16 mmol/l glucose were approximately 3.0-fold (alpha4), 2.0-fold (beta3), or 1.5-fold (gamma2) higher than at basal glucose concentrations (2.5 or 1.0 mmol/l, respectively). These effects were mimicked by depolarizing concentrations of K(+) and reversed by the L-type Ca(2+) channel blocker nimodipine. We conclude that 1) release of GABA from neighboring beta-cells contributes substantially to the acute inhibition of glucagon secretion from mouse islets by glucose and 2) that changes in GABA(A)R expression, mediated by changes in intracellular free Ca(2+) concentration, may modulate this response in the long term.     

Stimulation of insulin secretion by denatonium,one of the most bitter-tasting substances known

Denatonium, one of the most bitter-tasting substances known, stimulated insulin secretion in clonal HIT-T15 beta-cells and rat pancreatic islets. Stimulation of release began promptly after exposure of the beta-cells to denatonium, reached peak rates after 4-5 min, and then declined to near basal values after 20-30 min. In islets, no effect was observed at 2.8 mmol/;l glucose, whereas a marked stimulation was observed at 8.3 mmol/;l glucose. No stimulation occurred in the absence of extracellular Ca(2+) or in the presence of the Ca(2+)-channel blocker nitrendipine. Stimulated release was inhibited by alpha(2)-adrenergic agonists. Denatonium had no direct effect on voltage-gated calcium channels or on cyclic AMP levels. There was no evidence for the activation of gustducin or transducin in the beta-cell. The results indicate that denatonium stimulates insulin secretion by decreasing KATP channel activity, depolarizing the beta-cell, and increasing Ca(2+) influx. Denatonium did not displace glybenclamide from its binding sites on the sulfonylurea receptor (SUR). Strikingly, it increased glybenclamide binding by decreasing the K(d). It is concluded that denatonium, which interacts with K(+) channels in taste cells, most likely binds to and blocks Kir6.2. A consequence of this is a conformational change in SUR to increase the SUR/glybenclamide binding affinity.     

Glucose-sensing in glucagon-like peptide-1-secreting cells

Glucagon-like peptide-1 (GLP-1) is released from intestinal L-cells in response to carbohydrate and fat in the diet. Despite the interest in GLP-1 as an antidiabetic agent, very little is known about the mechanism of stimulus-secretion coupling in L-cells. We investigated the electrophysiological events underlying glucose-induced GLP-1 release in the GLP-1-secreting cell line, GLUTag. Cells were studied using perforated-patch and standard whole-cell patch clamp recordings. GLUTag cells were largely quiescent and hyperpolarized in the absence of glucose. Increasing the glucose concentration between 0 and 20 mmol/l decreased the membrane conductance, caused membrane depolarization, and triggered the generation of action potentials. Action potentials were also triggered by tolbutamide (500 micro mol/l) and were suppressed by diazoxide (340 micro mol/l) or the metabolic inhibitor azide (3 mmol/l), suggesting an involvement of K(ATP) channels. Large tolbutamide-sensitive washout currents developed in standard whole-cell recordings, confirming the presence of K(ATP) channels. RT-PCR detected the K(ATP) channel subunits Kir6.2 and SUR1 and glucokinase. GLP-1 secretion was also stimulated by glucose over the concentration range 0-25 mmol/l and by tolbutamide. Our results suggest that glucose triggers GLP-1 release through closure of K(ATP) channels and action potential generation.