The PDZ/coiled-coil domain containing protein PIST modulates insulin secretion in MIN6 insulinoma cells by interacting with somatostatin receptor subtype 5

The multi-domain protein PIST (protein interacting specifically with Tc10) interacts with the SSTR5 (somatostatin receptor 5) and is responsible for its intracellular localization. Here, we show that PIST is expressed in pancreatic beta-cells and interacts with SSTR5 in these cells. PIST expression in MIN6 insulinoma cells is reduced by somatostatin (SST). After stimulation with SST, SSTR5 undergoes internalization together with PIST. MIN6 cells over-expressing PIST display enhanced glucose-stimulated insulin secretion and a decreased sensitivity to SST-induced inhibition of insulin secretion. These data suggest that PIST plays an important role in insulin secretion by regulating SSTR5 availability at the plasma membrane.     

SSTR2 mediates the somatostatin-induced increase in intracellular Ca(2+) concentration and insulin secretion in the presence of arginine vasopressin in clonal beta-cell HIT-T15

The effects of somatostatin (SRIF) are mediated through the seven transmembrane receptor family that signals via Gi/Go. To date, five distinct SRIF receptors have been characterized and designated SSTR1-5. We have characterized the SRIF receptor that mediates the increase in [Ca(2+)](i) and insulin secretion in HIT-T15 cells (Simian virus 40-transformed Syrian hamster islets) using high affinity, subtype selective agonists for SSTR1 (L-797,591), SSTR2 (L-779,976), SSTR3 (L-796,778), SSTR4 (L-803,087), SSTR5 (L-817,818) and PRL-2903, a specific SSTR2 antagonist. In the presence of arginine vasopressin (AVP), SRIF increased [Ca(2+)](i) and insulin secretion. Treatment with the SSTR2 agonist L-779,976 resulted in similar responses to SRIF. In addition, L-779,976 increased both [Ca(2+)](i) and insulin secretion in a dose-dependent manner. Treatment with L-779,976 alone did not alter [Ca(2+)](i) or basal insulin secretion. In the presence of AVP, all other SRIF receptor agonists failed to increase [Ca(2+)](i) and insulin secretion. The effects of SRIF and L-779,976 were abolished by the SSTR2 antagonist PRL-2903. Our results suggest that the mechanism underlying SRIF-induced insulin secretion in HIT-T15 cells be mediated through the SSTR2.     

Identification and characterization of subtype selective somatostatin receptor agonists.

High affinity, subtype selective non-peptide agonists of somatostatin receptor subtypes 1-5 were identified in combinatorial libraries constructed based on molecular modeling of known peptide agonists. Simultaneous traditional chemical synthesis yielded an additional series of somatostatin subtype-2 receptor (SSTR2) selective agonists. These compounds have been used to further define the physiological functions of the individual somatostatin receptor subtypes. In vitro experiments demonstrated the role of the SSTR2 in inhibition of glucagon release from mouse pancreatic alpha-cells and the somatostatin subtype-5 receptor (SSTR5) as a mediator of insulin secretion from pancreatic beta-cells. Both SSTR2 and SSTR5 regulated growth hormone release from the rat anterior pituitary gland. In vivo studies performed with SSTR2 receptor selective compounds demonstrated effective inhibition of pulsatile growth hormone release in rats. The SSTR2 selective compounds also lowered plasma glucose levels in normal and diabetic animal models. The availability of high affinity, subtype selective non-peptide agonists for each of the somatostatin receptors provides a direct approach to defining their physiological function both peripherally and in the central nervous system.     

Identification and characterization of Ca2+-phospholipid-dependent protein kinase in rat islets and hamster beta-cells.

We show that extracts of rat islets of Langerhans and of cloned hamster beta-cells (HIT-T15 cells) contain Ca2+-phospholipid-dependent protein kinase and endogenous protein substrates for the kinase. We purified Ca2+-phospholipid-dependent protein kinase from HIT-T15 beta-cells and report here its physical and kinetic properties.     

Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets.

To examine their role in insulin secretion, actin filaments (AFs) were disrupted by Clostridium botulinum C2 toxin that ADP-ribosylates G-actin. Ribosylation also prevents polymerization of G-actin to F-actin and inhibits AF assembly by capping the fast-growing end of F-actin. Pretreatment of HIT-T15 cells with the toxin inhibited stimulated insulin secretion in a time- and dose-dependent manner. The toxin did not affect cellular insulin content or nonstimulated secretion. In static incubation, toxin treatment caused 45-50% inhibition of secretion induced by nutrients alone (10 mM glucose + 5 mM glutamine + 5 mM leucine) or combined with bombesin (phospholipase C-activator) and 20% reduction of that potentiated by forskolin (stimulator of adenylyl cyclase). In perifusion, the stimulated secretion during the first phase was marginally diminished, whereas the second phase was inhibited by approximately 80%. Pretreatment of HIT cells with wartmannin, a myosin light chain kinase inhibitor, caused a similar pattern of inhibition of the biphasic insulin release as C2 toxin. Nutrient metabolism and bombesin-evoked rise in cytosolic free Ca2+ were not affected by C2 toxin, indicating that nutrient recognition and the coupling between receptor activation and second messenger generation was not changed. In the toxin-treated cells, the AF web beneath the plasma membrane and the diffuse cytoplasmic F-actin fibers disappeared, as shown both by staining with an antibody against G- and F-actin and by staining F-actin with fluorescent phallacidin. C2 toxin dose-dependently reduced cellular F-actin content. Stimulation of insulin secretion was not associated with changes in F-actin content and organization. Treatment of cells with cytochalasin E and B, which shorten AFs, inhibited the stimulated insulin release by 30-50% although differing in their effects on F-actin content. In contrast to HIT-T15 cells, insulin secretion was potentiated in isolated rat islets after disruption of microfilaments with C2 toxin, most notably during the first phase. This effect was, however, diminished, and the second phase became slightly inhibited when the islets were degranulated. These results indicate an important role for AFs in insulin secretion. In the poorly granulated HIT-T15 cells actin-myosin interactions may participate in the recruitment of secretory granules to the releasable pool. In native islet beta-cells the predominant function of AFs appears to be the limitation of the access of granules to the plasma membrane.     

Somatostatin receptor subtype-dependent regulation of nitric oxide release: involvement of different intracellular pathways

We reported previously that, in addition to direct effects, somatostatin (SST) affects tumor growth inhibiting the tumoral neoangiogenesis, via an interference with NO synthesis. Here, we analyzed the effects of SST on nitric oxide (NO) production induced by different agonists [basic fibroblast growth factor (bFGF), insulin, cholecystokinin (CCK)] and the intracellular signaling involved, using Chinese hamster ovary-k1 cells stably transfected with individual SSTR1-SSTR4. bFGF and insulin induced endothelial nitric oxide synthase activity via the generation of ceramide or the Akt-dependent phosphorylation of endothelial nitric oxide synthase, respectively. CCK regulates neuronal nitric oxide synthase activity in a Ca++-dependent manner. SST inhibited NO production stimulated by bFGF through SST receptor 1 (SSTR1), SSTR2, and SSTR3 and by CCK through SSTR2 and SSTR3. In all the cell lines, SST treatment did not modify NO synthesis induced by insulin. SSTR4 activation was not effective on any of the stimuli tested. The effects on bFGF-induced NO production were downstream from receptor phosphorylation and ceramide synthesis. SSTR2 and -3 on CCK activity were related to the inhibition of intracellular Ca++ mobilization, whereas the lack of effects on insulin was paralleled by the absence of SST activity on Akt phosphorylation. These data, identifying for the first time a selective receptor subtype-inhibitory role of SST on NO generation, may open new perspectives in the use of SST agonists to control tumoral angiogenesis.     

Agouti signaling protein stimulates islet amyloid polypeptide (amylin) secretion in pancreatic beta-cells

Ectopic overexpression of the murine agouti gene results in yellow coat color, obesity, hyperinsulinemia, and type II diabetes. We have shown the human homologue of agouti (agouti signaling protein; ASP) to regulate human adipocyte metabolism and lipid storage via a Ca(2+)-dependent mechanism. We have also demonstrated agouti expression in human pancreas, and that ASP stimulates insulin release via a similar Ca(2+)-dependent mechanism. Plasma amylin is also elevated in agouti mutant mice. Amylin is cosecreted with insulin from beta-cells, and overexpression of human amylin in beta-cells in yellow agouti mutant mice resulted in accelerated pancreatic amyloid deposition, severely impaired beta-cell function, and a diabetic phenotype. We report here that ASP stimulates amylin release in both the HIT-T15 beta-cell line and human pancreatic islets in the presence of a wide range of glucose concentrations (0-16.7 mmol/L), similar to its effect on insulin release; this effect was blocked by 30 mumol/L nitrendipine, confirming a Ca(2+)-dependent mechanism. Accordingly, ASP stimulation of amylin release may serve as a compensatory system to regulate blood glucose in yellow agouti mutants.     

Munc-18 associates with syntaxin and serves as a negative regulator of exocytosis in the pancreatic beta -cell

The Munc-18 protein (mammalian homologue of the unc-18 gene; also called nSec1 or rbSec1) has been identified as an essential component of the synaptic vesicle fusion protein complex. The cellular and subcellular localization and functional role of Munc-18 protein in pancreatic beta-cells was investigated. Subcellular fractionation of insulin-secreting HIT-T15 cells revealed a 67-kDa protein in both cytosol and membrane fractions. Immunohistochemistry showed punctate Munc-18 immunoreactivity in the cytoplasm of rat pancreatic islet cells. Direct double-labeling immunofluorescence histochemistry combined with confocal laser microscopy revealed the presence of Munc-18 immunoreactivity in insulin-, glucagon-, pancreatic polypeptide-, and somatostatin-containing cells. Syntaxin 1 immunoreactivity was detected in extracts of HIT-T15 cells, which were immunoprecipitated using Munc-18 antiserum, suggesting an intimate association of Munc-18 with syntaxin 1. Administration of Munc-18 peptide or Munc-18 antiserum to streptolysin O-permeabilized HIT-T15 cells resulted in significantly increased insulin release, but did not have any significant effect on voltage-gated Ca(2+) channel activity. The findings taken together show that the Munc-18 protein is present in insulin-secreting beta-cells and implicate Munc-18 as a negative regulator of the insulin secretory machinery via a mechanism that does not involve syntaxin-associated Ca(2+) channels.     

Tolbutamide and diazoxide modulate phospholipase C-linked Ca(2+) signaling and insulin secretion in beta-cells

Arginine vasopressin (AVP), bombesin, and ACh increase cytosolic free Ca(2+) and potentiate glucose-induced insulin release by activating receptors linked to phospholipase C (PLC). We examined whether tolbutamide and diazoxide, which close or open ATP-sensitive K(+) channels (K(ATP) channels), respectively, interact with PLC-linked Ca(2+) signals in HIT-T15 and mouse beta-cells and with PLC-linked insulin secretion from HIT-T15 cells. In the presence of glucose, the PLC-linked Ca(2+) signals were enhanced by tolbutamide (3-300 microM) and inhibited by diazoxide (10-100 microM). The effects of tolbutamide and diazoxide on PLC-linked Ca(2+) signaling were mimicked by BAY K 8644 and nifedipine, an activator and inhibitor of L-type voltage-sensitive Ca(2+) channels, respectively. Neither tolbutamide nor diazoxide affected PLC-linked mobilization of internal Ca(2+) or store-operated Ca(2+) influx through non-L-type Ca(2+) channels. In the absence of glucose, PLC-linked Ca(2+) signals were diminished or abolished; this effect could be partly antagonized by tolbutamide. In the presence of glucose, tolbutamide potentiated and diazoxide inhibited AVP- or bombesin-induced insulin secretion from HIT-T15 cells. Nifedipine (10 microM) blocked both the potentiating and inhibitory actions of tolbutamide and diazoxide on AVP-induced insulin release, respectively. In glucose-free medium, AVP-induced insulin release was reduced but was again potentiated by tolbutamide, whereas diazoxide caused no further inhibition. Thus tolbutamide and diazoxide regulate both PLC-linked Ca(2+) signaling and insulin secretion from pancreatic beta-cells by modulating K(ATP) channels, thereby determining voltage-sensitive Ca(2+) influx.     

Progesterone inhibits insulin secretion by a membrane delimited,non-genomic action

In rat islets, progesterone caused a prompt concentration-dependent inhibition of glucose-stimulated insulin release with an IC50 of 10 microM at 8.4mM glucose. The inhibition was specific since both testosterone and 17beta-estradiol had no such effect. The degree of inhibition was similar in islets from male and female rats. The inhibition was not blocked in PTX-treated islets thus ruling out the Gi/Go proteins as mediators of the inhibition. Progesterone inhibited both glucose- and BayK-8644-stimulated insulin secretion in HIT-T15 cells and the IC50 vs. 10 mM glucose was also 10 microM. There was no effect on intracellular cyclic AMP concentration in the presence 0.2 and 10 mM glucose. Progesterone decreased [Ca2+]i under all conditions tested. The decrease in [Ca2+]i was due to blockade of the L-type voltage-dependent Ca2+ channels. Under Ca(2+)-free conditions, progesterone did not inhibit the stimulation of insulin release due to the combination of glucose, phorbol ester and forskolin. Thus blockade of Ca2+ entry appears to be the sole mechanism by which progesterone inhibits insulin release. As progesterone covalently linked to albumin had a similar inhibitory effect as progesterone itself, it is concluded that the steroid acts at the outer surface of the beta-cell plasma membrane. These effects would be classified as either AI or AIIb in the Mannheim classification of nongenomically initiated steroid actions.     

Inhibition of glucose-stimulated insulin release by pseudo-alpha-DL-glucose as a glucokinase inhibitor

Pseudo-alpha- and pseudo-beta-DL-glucose, the isomers of 5-hydroxymethyl-1,2,3,4-cyclohexanetetrol with alpha-gluco and beta-gluco configurations, were used as synthetic analogs of glucose anomers to study the mechanism of glucose-stimulated insulin release by pancreatic islets. Neither isomer was phosphorylated by liver glucokinase nor stimulated insulin release from islets. Incubation of islets with pseudo-alpha-DL-glucose resulted in a considerable accumulation of the glucose analog, probably the D form, in islets. The alpha-isomer, but not the beta-isomer, inhibited both glucose-stimulated insulin release (44% inhibition at 20 mM) and islet glucokinase activity (36% inhibition at 20 mM) in a concentration-dependent manner and to a comparable degree. These results strongly suggest that the inhibition of glucose-stimulated insulin release by pseudo-alpha-DL-glucose is due to the inhibition of islet glucokinase by the glucose analog, providing additional evidence for the essential role of islet glucokinase in glucose-stimulated insulin release.     

Double-stranded RNA inhibits beta-cell function and induces islet damage by stimulating beta-cell production of nitric oxide

Viral infection has been implicated as a triggering event that may initiate beta-cell damage during the development of autoimmune diabetes. In this study, the effects of the viral replicative intermediate, double-stranded RNA (dsRNA) (in the form of synthetic polyinosinic-polycytidylic acid (poly IC)) on islet expression of inducible nitric oxide synthase (iNOS), production of nitric oxide, and islet function and viability were investigated. Treatment of rat islets with poly(IC) + interferon-gamma (IFN-gamma) stimulates the time- and concentration-dependent expression of iNOS and production of nitrite by rat islets. iNOS expression and nitrite production by rat islets in response to poly(IC) + IFN-gamma correlate with an inhibition of insulin secretion and islet degeneration, effects that are prevented by the iNOS inhibitor aminoguanidine (AG). We have previously shown that poly(IC) + IFN-gamma activates resident macrophages, stimulating iNOS expression, nitric oxide production and interleukin-1 (IL-1) release. In addition, in response to tumor necrosis factor-alpha (TNF-alpha) + lipopolysaccharide, activated resident macrophages mediate beta-cell damage via intraislet IL-1 release followed by IL-1-induced iNOS expression by beta-cells. The inhibitory and destructive effects of poly(IC) + IFN-gamma, however, do not appear to require resident macrophages. Treatment of macrophage-depleted rat islets for 40 h with poly(IC) + IFN-gamma results in the expression of iNOS, production of nitrite, and inhibition of insulin secretion. The destructive effects of dsRNA + IFN-gamma on islets appear to be mediated by a direct interaction with beta-cells. Poly IC + IFN-gamma stimulates iNOS expression and inhibits insulin secretion by primary beta-cells purified by fluorescence-activated cell sorting. In addition, AG prevents the inhibitory effects of poly(IC) + IFN-gamma on glucose-stimulated insulin secretion by beta-cells. These results indicate that dsRNA + IFN-gamma interacts directly with beta-cells stimulating iNOS expression and inhibiting insulin secretion in a nitric oxide-dependent manner. These findings provide biochemical evidence for a novel mechanism by which viral infection may directly mediate the initial destruction of beta-cells during the development of autoimmune diabetes.     

Regulatory effects of 5'-deoxypyridoxal on glutamate dehydrogenase activity and insulin secretion in pancreatic islets

It has been known that glutamate, generated by glutamate dehydrogenase (GDH), acts as an intracellular messenger in insulin exocytosis in pancreatic beta cells. Here we demonstrate the correlation of GDH activity and insulin release in rat pancreatic islets perfused with 5'-deoxypyridoxal. Perfusion of islets with 5'-deoxypyridoxal, an effective inhibitor of GDH, reduced the islet GDH activity at concentration-dependent manner. Treatment of 5'-deoxypyridoxal up to 2 mM did not affect the cell viability. There was reduction in V(max) values on average about 60%, whereas no changes in K(m) values for substrates and coenzymes were observed. The concentration of GDH on the Western blot analysis and the level of GDH mRNA remained unchanged. The concentration of glutamate decreased by 52%, whereas the concentration of 2-oxoglutarate increased up to 2.3-fold in the presence of 5'-deoxypyridoxal. 5'-Deoxypyridoxal had no effects on inhibition by GTP and activation by ADP or L-leucine of islet GDH. In parallel with the inhibition of GDH activity, perfusion of islets with 5'-deoxypyridoxal reduced insulin release up to 2.5-fold. Although precise mechanism for correlation between GDH activity and insulin release remains to be studied further, our results suggest a possibility that the inhibitory effect of 5'-deoxypyridoxal on islet GDH activity may correlate with its effect on insulin release.     

Stimulation of insulin secretion by transforming growth factor-beta

Effects of transforming growth factor-beta (TGF-beta) on insulin secretion were studied in rat pancreatic islets. When islets were incubated in a batch incubation system with various concentrations of TGF-beta in the presence of 2.8 mM glucose, TGF- beta increased insulin release in a concentration-dependent manner. Both TGF- beta 1 and TGF- beta 2 were equally effective. The stimulatory action of TGF- beta was greater in the presence of stimulatory concentration of glucose. In perifusion system, TGF- beta induced an immediate monotonic increase in insulin secretion. These results indicate that TGF- beta is a stimulator of insulin secretion.     

Tetracaine stimulates insulin secretion from the pancreatic beta-cell by release of intracellular calcium

The role of intracellular calcium stores in stimulus-secretion coupling in the pancreatic beta-cell is largely unknown. We report here that tetracaine stimulates insulin secretion from collagenase-isolated mouse islets of Langerhans in the absence of glucose or extracellular calcium. We also found that the anesthetic evokes a dose-dependent rise of the intracellular free-calcium concentration ([Ca2+]i) in cultured rat and mouse beta-cells. The tetracaine-specific [Ca2+]i rise also occurs in the absence of glucose, or in beta-cells depolarized by exposure to a Ca(2+)-deficient medium (< 1 microM) or elevated [K+]o. Furthermore, tetracaine (> or = 300 microM) depolarized the beta-cell membrane in mouse pancreatic islets, but inhibited Ca2+ entry through voltage-gated Ca2+ channels in HIT cells, an insulin-secreting cell line. From these data we conclude that tetracaine-enhancement of insulin release occurs by mechanisms that are independent of Ca2+ entry across the cell membrane. The tetracaine-induced [Ca2+]i rise in cultured rat beta-cells and insulin secretion from mouse islets is insensitive to dantrolene (20 microM), a drug that inhibits Ca2+ release evoked by cholinergic agonists in the pancreatic beta-cell, and thapsigargin (3 microM), a blocker of the endoplasmic reticulum (ER) Ca2+ pump. We conclude that the Ca2+ required for tetracaine-potentiated insulin secretion is released from intracellular Ca2+ stores other than the ER. Furthermore, tetracaine-induced Ca2+ release was unaffected by the mitochondrial electron transfer inhibitors NaN3 and rotenone. Taken together, these data show that a calcium source other than the ER and mitochondria can affect beta-cell insulin secretion.     

Secretory phospholipase A2 is released from pancreatic beta-cells and stimulates insulin secretion via inhibition of ATP-dependent K+ channels

The release of sPLA(2) from single mouse pancreatic beta-cells was monitored using a fluorescent substrate of the enzyme incorporated in the outer leaflet of the plasma membrane. Stimulation of beta-cells with agents that increased cytosolic free Ca(2+) concentration ([Ca(2+)](i)) induced a rapid release of sPLA(2) to the extracellular medium. Exogenous sPLA(2) strongly stimulated insulin secretion in mouse pancreatic islets at both basal and elevated glucose concentrations. The stimulation of insulin secretion by sPLA(2) was mediated via inhibition of ATP-dependent K(+) channels and an increase in [Ca(2+)](i). Measurements of cell capacitance in single beta-cells revealed that sPLA(2) did not modify depolarisation-induced exocytosis. Our data suggest that a positive feedback regulation of insulin secretion by co-released sPLA(2) is operational in pancreatic beta-cells and point to this enzyme as an autocrine regulator of insulin secretion.     

Pharmacological properties and functional role of Kslow current in mouse pancreatic beta-cells: SK channels contribute to Kslow tail current and modulate insulin secretion

The pharmacological properties of slow Ca(2+)-activated K(+) current (K(slow)) were investigated in mouse pancreatic beta-cells and islets to understand how K(slow) contributes to the control of islet bursting, [Ca(2+)](i) oscillations, and insulin secretion. K(slow) was insensitive to apamin or the K(ATP) channel inhibitor tolbutamide, but UCL 1684, a potent and selective nonpeptide SK channel blocker reduced the amplitude of K(slow) tail current in voltage-clamped mouse beta-cells. K(slow) was also selectively and reversibly inhibited by the class III antiarrythmic agent azimilide (AZ). In isolated beta-cells or islets, pharmacologic inhibition of K(slow) by UCL 1684 or AZ depolarized beta-cell silent phase potential, increased action potential firing, raised [Ca(2+)](i), and enhanced glucose-dependent insulin secretion. AZ inhibition of K(slow) also supported mediation by SK, rather than cardiac-like slow delayed rectifier channels since bath application of AZ to HEK 293 cells expressing SK3 cDNA reduced SK current. Further, AZ-sensitive K(slow) current was extant in beta-cells from KCNQ1 or KCNE1 null mice lacking cardiac slow delayed rectifier currents. These results strongly support a functional role for SK channel-mediated K(slow) current in beta-cells, and suggest that drugs that target SK channels may represent a new approach for increasing glucose-dependent insulin secretion. The apamin insensitivity of beta-cell SK current suggests that beta-cells express a unique SK splice variant or a novel heteromultimer consisting of different SK subunits.     

Characteristics of Ba2+-stimulated insulin release with special reference to pancreatic beta-cells sensitized by cyclic AMP

Ca2+-dependent processes are activated by Ba2+ in a variety of biological systems. When Ca2+ was replaced by equimolar amounts of Ba2+ there was a marked increase in insulin secretion from beta-cell-rich pancreatic islets microdissected from ob/ob-mice. At both 3 and 20 mM glucose Ba2+ stimulated insulin release in a concentration-dependent manner, being less stimulatory at high concentrations. The stimulatory effect of Ba2+ on insulin release is similar to that of Ca2+ in being more pronounced and reached at lower concentrations when the beta-cells were sensitized by cyclic AMP. However, both glucose oxidation and utilization were suppressed when Ca2+ was replaced by equimolar amounts of Ba2+. Ba2+-stimulated insulin release resembled physiological secretion initiated by Ca2+ in being inhibited by L-epinephrine, pentobarbital and a low oxygen tension.     

Activation of insulin-secreting cells by pyruvate and halogenated derivatives.

Addition of pyruvate to rat islets perifused in the presence of 5 mM-glucose elicited an immediate pronounced biphasic stimulation of insulin secretion. At lower concentrations of glucose (2.5 mM), only the initial, transient, phase of secretion was observed. Pyruvate inhibited 45Ca2+ efflux from islets at 2.5 mM-glucose and stimulated efflux at 5 mM-glucose. Pyruvate also decreased the rate of efflux of 86Rb+ from perifused islets. A marked stimulation of insulin secretion and 45Ca2+ efflux rate was observed in response to 3-fluoropyruvate and 3-bromopyruvate, compounds which inhibited oxidative metabolism of [14C]glucose and [14C]pyruvate in islets. The stimulatory effects of 3-fluoro- and 3-bromo-pyruvate were associated with enhanced 86Rb+ efflux. Withdrawal of pyruvate or halogenated analogues from the perfusate resulted in a secondary stimulation of insulin release, 45Ca2+ efflux and, to some extent, 86Rb+ efflux rates. Pyruvate, 3-fluoropyruvate and 3-bromopyruvate were all effective in promoting intracellular acidification and a rise in cytosolic Ca2+ concentration, as judged from fluorescence measurements in HIT-T15 cells loaded with 2',7'-biscarboxyethyl-5'(6')-carboxyfluorescein and Quin 2 respectively. It is proposed that oxidative metabolism of pyruvate is not a prerequisite for its stimulatory actions on pancreatic beta-cells. An alternative mechanism of activation by pyruvate and its halogenated derivatives is proposed, based on the possible electrogenic flux of these anions across the cell membrane.