ß-Cell preservation and regeneration in diabetes by modulation of ß-cell Ca²âº homeostasis.

Ca(2+) extrusion from the ß-cell is mediated by two processes the Na/Ca exchanger (NCX) and the plasma membrane Ca(2+) -ATPase (PMCA). Gain of function studies show that overexpression of NCX or PMCA leads to endoplasmic reticulum (ER) Ca(2+) depletion with subsequent ER stress, decrease in ß-cell proliferation and ß-cell death by apoptosis. Interestingly, chronic exposure to cytokines or high free fatty acid concentrations also induce ER Ca(2+) depletion and ß-cell death in diabetes. Loss of function studies show, on the contrary, that heterozygous inactivation of NCX1 (Ncx1(+/-)) leads to an increase in ß-cell function (insulin production and release), and a fivefold increase in both ß-cell mass and proliferation. The mutation also increases ß-cell resistance to hypoxia, and Ncx1(+/-) islets show a two to four times higher rate of diabetes cure than Ncx1(+/+) islets when transplanted in diabetic animals. Thus, down-regulation of the Na/Ca exchanger leads to various changes in ß-cell function that are opposite to the major abnormalities seen in diabetes. This provides a unique model for the prevention and treatment of ß-cell dysfunction in diabetes and following islet transplantation.

Preconditioning in hypoxic-ischemic neonate mice triggers Na+-Ca2+ exchanger-dependent neurogenesis.

To identify alternative interventions in neonatal hypoxic-ischemic encephalopathy, researchers' attention has been focused to the study of endogenous neuroprotective strategies. Based on the preconditioning concept that a subthreshold insult may protect from a subsequent harmful event, we aimed at identifying a new preconditioning protocol able to enhance Ca2+-dependent neurogenesis in a mouse model of neonatal hypoxia ischemia (HI). To this purpose, we also investigated the role of the preconditioning-linked protein controlling ionic homeostasis, Na+/Ca2+ exchanger (NCX). Hypoxic Preconditioning (HPC) was reproduced by exposing P7 mice to 20' hypoxia. HI was induced by isolating and cutting the right common carotid artery. A significant reduction in ischemic damage was observed in mice subjected to 20' hypoxia followed,3 days later, by 60' HI, thus suggesting that 20' hypoxia functions as preconditioning stimulus. HPC promoted neuroblasts proliferation in the dentate gyrus mirrored by an increase of NCX1 and NCX3-positive cells and an improvement of behavioral motor performances in HI mice. An attenuation of HPC neuroprotection as well as a reduction in the expression of neurogenesis markers, including p57 and NeuroD1, was observed in preconditioned mice lacking NCX1 or NCX3. In summary, PC in neonatal mice triggers a neurogenic process linked to ionic homeostasis maintenance, regulated by NCX1 and NCX3.

The suppressor of cytokine signalling 2 (SOCS2) is a key repressor of insulin secretion.

AIMS/HYPOTHESIS: Suppressor of cytokine signalling (SOCS) proteins are powerful inhibitors of pathways involved in survival and function of pancreatic beta cells. Whereas SOCS1 and SOCS3 have been involved in immune and inflammatory processes, respectively, in beta cells, nothing is known about SOCS2 implication in the pancreas. METHODS: Transgenic (tg) mice were generated that constitutively produced SOCS2 in beta cells (betaSOCS2) to define whether this protein is implicated in beta cell functioning and/or survival. RESULTS: Constitutive production of SOCS2 in beta cells leads to hyperglycaemia and glucose intolerance. This phenotype is not a consequence of decreased beta cell mass or inhibition of insulin synthesis. However, insulin secretion to various secretagogues is profoundly altered in intact animals and isolated islets. Interestingly, constitutive SOCS2 production dampens the rise in cytosolic free calcium concentration induced by glucose, while glucose metabolism is unchanged. Moreover, tg islets have a depletion in endoplasmic reticulum Ca(2+) stores, suggesting that SOCS2 interferes with calcium fluxes. Finally, in betaSOCS2 mice proinsulin maturation is impaired, leading to an altered structure of insulin secretory granules and augmented levels of proinsulin. The latter is likely to be due to decreased production of prohormone convertase 1 (PC1/3), which plays a key role in proinsulin cleavage. CONCLUSIONS/INTERPRETATIONS: SOCS2 was shown to be a potent regulator of proinsulin processing and insulin secretion in beta cells. While its constitutive production is insufficient to induce overt diabetes in this mouse model, it causes glucose intolerance. Thus, increased SOCS2 production could be an important event predisposing to beta cell failure.

The stimulus-secretion coupling of glucose-induced insulin release. Metabolic and functional effects of NH4+ in rat islets.

NH4+ caused a dose-related, rapid, and reversible inhibition of glucose-stimulated insulin release by isolated rat islets. It also inhibited glyceraldehyde-, Ba2+-, and sulfonylurea-stimulated insulun secretion. NH4+ failed to affect glucose utilization and oxidation, glucose-stimulated proinsulin biosynthesis, the concentration of ATP, AD, and AMP, and the intracellular pH. NH4+ also failed to affect the ability of theophylline and cytochalasin B to augment glucose-induced insulin release. However, in the presence and absence of glucose, accumulation of NH4+ in islet cells was associated with a fall in the concentration of NADH and HADPH and a concomitant alteration of 86Rb+ and 45Ca2+ (or 133Ba2+) handling. These findings suggest that reduced pyridine nucleotides, generated by the metabolism of endogenous of exogenous nutrients, may modulate ionophoretic processes in the islet cells and by doing so, affect the net uptake of Ca2+ and subsequent release of insulin.

Insulinotropic effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate in rat pancreatic islets.

In isolated rat pancreatic islets, the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA), when used in the 2.10(-9) to 2.10(-7) M range, was found to stimulate insulin release both in the absence and presence of glucose. The non-tumor-promoting agent 4-methylphorbol-12,13-didecanoate failed to stimulate insulin release. The insulinotropic capacity of TPA was enhanced by glucose in a dose-related fashion. In the absence of glucose, the TPA-stimulated release of insulin was a slowly induced and not rapidly reversible phenomenon. It was inhibited by antimycin A, by epinephrine, at low temperatures, and in the absence of extracellular Ca2+ or the presence of cytochalasin B, was unaffected by the organic calcium antagonist D600 or indomethacin, and was potentiated by theophylline. No obvious effect of TPA upon 86Rb or 32P efflux and 45Ca net uptake could be detected in the isolated islets. However, TPA caused a progressive increase in both 45Ca fractional outflow rate and cyclic adenosine 3':5'-monophosphate content in the islets. It is proposed that the insulinotropic action of TPA may be due, in part at least, to interference with the transport of calcium by native ionophores.

Activation, but not inhibition, by glucose of Ca2+-dependent K+ permeability in the rat pancreatic B-cell.

The effect of glucose on the Ca2+-activated K+ permeability in pancreatic islet cells was investigated by measuring the rate of 86Rb efflux, 45Ca efflux and insulin release from perifused rat pancreatic islets exposed to step-wise increased in glucose concentration. When the glucose concentration was raised from intermediate (8.3 or 11.1 mM) to higher values, a rapid and sustained increase in 86Rb outflow, 45Ca outflow and insulin release was observed. Likewise, in the presence of 8.3 or 16.7 mM glucose, tolbutamide increased 86Rb and 45Ca efflux, as well as insulin release. In the two series of experiments, a tight correlation was found between the magnitude of the changes in 86Rb and 45Ca outflow, respectively. It is concluded that, at variance with current ideas, glucose does not inhibit the response to cytosolic Ca2+ of the Ca2+-sensitive modality of K+ extrusion. On the contrary, as a result of its effect upon Ca2+ handling, glucose stimulates the Ca2+-activated K+ permeability.

Effect of extracellular sodium removal upon 86Rb outflow from pancreatic islet cells.

The present study was undertaken to characterize the effect of extracellular Na+ removal on 86Rb outflow from perifused rat pancreatic islets. Complete Na+ omission inhibited 86Rb outflow whether the islets were perifused in the presence or in the absence of extracellular Ca2+. Ouabain (1 mM) did not reduce the inhibitory effect of Na+ deprivation, whilst diphenylhydantoin (72.9 microM) mimicked the Na+-removal-induced fall in 86Rb outflow. Glucose (16.7 mM) lost its capacity to inhibit 86Rb outflow when the perifusate was deprived of extracellular Na+. These results indicate that Na+ omission reproduces the inhibitory effect of glucose on 86Rb outflow. The reduction in 86Rb outflow recorded after Na+ deprivation could be mediated by an intracellular acidification and/or a decrease in the intracellular Na+ activity. It is tempting to speculate that the capacity of glucose to reduce the B-cell Na+ content may participate in the process by which the sugar decreases K+ permeability.

Effect of the absence of bicarbonate upon intracellular pH and calcium fluxes in pancreatic islet cells.

The removal of extracellular HCO3- together with a decrease in pCO2, in order to maintain a normal extracellular pH, caused a sustained increase of intracellular pH in rat pancreatic islets. This increase was more marked in glucose-deprived than in glucose-stimulated islets, and was associated with a facilitation of 45Ca efflux from the glucose-deprived islets. Such a facilitation was slightly reduced in the absence of extracellular Ca2+ and abolished at low extracellular Na+ concentration. It failed to occur in glucose-stimulated islets, whether in the presence or absence of extracellular Ca2+. The removal of HCO3- and decrease in the pCO2 also reduced the magnitude of both the secondary rise in 45Ca efflux and stimulation of insulin release normally evoked by an increase in glucose concentration. These findings suggest that changes in intracellular pH affect both the outflow of Ca2+ from islet cells as mediated by Na+ -Ca2+ countertransport and the inflow of Ca2+ by gated Ca2+ channels. The experimental data are also compatible with the view that islet cells are equipped with an active process of bicarbonate-chloride exchange involved in the regulation of intracellular pH.

Na+-H+ exchange in the process of glucose-induced insulin release from the pancreatic B-cell. Effects of amiloride on 86Rb, 45Ca fluxes and insulin release.

The effect of amiloride, an inhibitor of Na+-H+ exchange, on intracellular pH (pHi), 86Rb outflow, 45Ca outflow and insulin release from pancreatic rat islets was examined. In the 0.1-1 mM range, amiloride transiently reduced pHi of glucose-deprived islets and allowed glucose to induce a sustained decrease in pHi of the islet cells. Amiloride reproduced the effect of glucose to decrease 86Rb and 45Ca outflow. In the presence of glucose (5.6 mM or more), amiloride (100 microM) acted synergistically with the sugar to reduce K+ outflow, and to stimulate 40Ca inflow and insulin release from perifused islets. These results add strong support to the view that the generation of protons through the metabolism of glucose represents an important step in the process of glucose-induced release. The stimulation by glucose of Na+-H+ exchange apparently masks and even overcomes the glucose-induced decrease in pHi otherwise expected from the increase in catabolic fluxes.

Sodium nitroprusside inhibits glucose-induced insulin release by activating ATP-sensitive K+ channels.

Sodium nitroprusside (SNP) has been reported to be a potent stimulator of cGMP formation in different tissues, including pancreatic islets. The present study aimed at comparing the effects of sodium nitroprusside and dibutyryl cGMP on 86Rb outflow, 45Ca outflow, short-term 45Ca uptake, cytosolic Ca2+ concentration and insulin release from rat pancreatic islet cells. The data indicate that cGMP potentiates whilst SNP inhibits the glucose-induced insulin release. This inhibitory effect appears to be mediated by the activation of ATP-sensitive K+ channels leading to a decrease in Ca2+ influx and subsequent reduction in cytosolic free Ca2+ concentration. Whatever the exact mechanism(s) underlying the capacity of sodium nitroprusside to enhance the K+ permeability of the B-cell membrane, the drug appears to be an inadequate pharmacological tool to characterize the involvement of cGMP in the insulin secretory process. The experimental results also suggest that cGMP potentiates glucose-induced insulin release without affecting ionic movements.

Regulation of calcium fluxes in rat pancreatic islets. Quinine mimics the dual effect of glucose on calcium movements.

The effects of quinine and 9-aminoacridine, two blockers of potassium conductance in islet cells, on 45Ca efflux and insulin release from perifused islets were investigated in order to elucidate the mechanisms by which glucose initially reduces 45Ca efflux and later stimulates calcium inflow in islet cells. In the absence of glucose, 100 microM quinine stimulated 45Ca net uptake, 45Ca outflow rate and insulin release. Quinine also dramatically enhanced the cationic and the secretory response to intermediate concentrations of glucose, but had little effect on 45Ca net uptake, 45Ca fractional outflow rate and insulin release at a high glucose concentration (16.7 mM). The ability of quinine to stimulate 45Ca efflux depended on the presence of extracellular calcium, suggesting that it reflects a stimulation of calcium entry in the islet cells. In the absence of extracellular calcium, quinine provoked a sustained decrease in 45Ca efflux. Such an inhibitory effect was not additive to that of glucose, and was reduced at low extracellular Na+ concentration. At a low concentration (5 microM), quinine, although reducing 86Rb efflux from the islets to the same extent as a non-insulinotropic glucose concentration (4.4 mM), failed to inhibit 45Ca efflux. In the presence of extracellular calcium, 9-aminoacridine produced an important but transient increase in 45Ca outflow rate and insulin release from islets perifused in the absence of glucose. In the absence of extracellular calcium, 9-aminoacridine, however, failed to reduced 45Ca efflux from perifused islets. It is concluded that quinine, by reducing K+ conductance, reproduces the effect of glucose to activate voltage-sensitive calcium channels and to stimulate the entry of calcium into the B-cell. However, the glucose-induced inhibition of calcium outflow rate, which may also participate in the intracellular accumulation of calcium, does not appear to be mediated by changes in K+ conductance.

Modalities of gliclazide-induced Ca2+ influx into the pancreatic B-cell.

The hypoglycemic sulfonylurea gliclazide stimulated 45Ca efflux and insulin release from prelabeled and perifused pancreatic rat islets, whether in the absence or presence of glucose (8.3 mM). The gliclazide-induced increase in 45Ca efflux is thought to reflect a stimulation of 40Ca influx into islet cells; it is suppressed in the absence of extracellular Ca2+ or in the presence of the organic CA2/-antagonist verapamil. In the absence of glucose, the ED50 for the inhibitory action of verapamil on gliclazide-stimulated 45Ca efflux was almost identical to that found when the process of 40Ca-45Ca exchange was stimulated by an increase in the extracellular concentration of K+, a procedure that leads to the depolarization of the plasma membrane. In the presence of glucose, however, the cationic response to gliclazide displayed an increased sensitivity toward the inhibitory action of verapamil. It is proposed that hypoglycemic sulfonylureas facilitate Ca2+ inflow into islet cells mainly through voltage-sensitive Ca2+ channels. In the presence of glucose, however, the stimulant action of hypoglycemic sulfonylureas upon Ca2+ entry into islet cells may entail a modality of Ca2+ transport not identical to that evoked by depolarization of the plasma membrane.

Contribution of Na/Ca exchange to Ca2+ outflow and entry in the rat pancreatic beta-cell: studies with antisense oligonucleotides.

To characterize the role played by Na/Ca exchange in the pancreatic beta-cell, phosphorothioated antisense oligonucleotides (AS-oligos) were used to knock down the exchanger in rat pancreatic beta-cells. Na/Ca exchange activity was evaluated by measuring cytosolic free Ca2+ concentration ([Ca2+]i) in single cells using fura-2. Exposure of beta-cells to 500 nmol/l of the AS-oligos for 24 h inhibited Na/Ca exchange activity by approximately 77%. In contrast, control oligonucleotides (scrambled and mismatched) did not affect Na/Ca exchange activity. In AS-oligo-treated cells, the increase in [Ca2+]i induced by membrane depolarization (K+ or the hypoglycemic sulfonylurea, tolbutamide) was reduced by 28 or 40%, respectively. Likewise, the rate of [Ca2+]i decrease after K+ or tolbutamide removal was reduced by 72 or 40%, respectively. AS-oligos treatment also abolished the nifedipine-resistant increase in [Ca2+]i induced by K+ and profoundly altered the oscillatory or sustained increases in [Ca2+]i induced by 11.1 mmol/l glucose. The present study shows that AS-oligos may specifically inhibit Na/Ca exchange in rat pancreatic beta-cells. In those cells, Na/Ca exchange appears to mediate Ca2+ entry in response to membrane depolarization and to be responsible for up to 70% of Ca2+ removal from the cytoplasm upon membrane repolarization.

The stimulus-secretion coupling of amino acid-induced insulin release. Secretory and oxidative response of pancreatic islets to L-asparagine.

L-Asparagine (2-10 mM) failed to affect insulin secretion from rat pancreatic islets incubated in the absence of exogenous nutrient or presence of D-glucose, but caused a dose-related and progressive enhancement of insulin release evoked by L-leucine, 2-aminobicyclo[2,2,1]heptane-2-carboxylate, or 2-ketoisocaproate. The secretory response to the combination of L-asparagine and L-leucine was augmented by theophylline and inhibited in the absence of extracellular Ca2+ or presence of either menadione or methylamine. L-Asparagine augmented leucine-stimulated 45Ca net uptake. The ATP content, rate of O2 uptake, and malate/pyruvate ratio were not significantly different in islets exposed to L-leucine alone or to both L-asparagine and L-leucine, respectively. In the sole presence of L-asparagine, however, the malate/oxalacetate ratio was decreased and the malate/pyruvate ratio increased, relative to basal values. It is proposed that the enhancing action of L-asparagine upon insulin release evoked by L-leucine might be due to an accelerated generation rate of cytosolic NADPH, rather than to any sizable increase in either islet respiration or steady-state cytosolic NADPH/NADP+ ratio.

The stimulus-secretion coupling of amino acid-induced insulin release: metabolism and cationic effects of leucine.

When isolated rat pancreatic islets are exposed to L-leucine (20 mM), the rate of NH4 production is close to the summed rates of L-[1-14C] leucine decarboxylation and alpha-ketoisocarproate production, whereas the rates of acetoacetate production and L-[U-14C]-leucine oxidation are compatible with conversion of each mole of the amino acid to one mole of acetoacetate and three moles of CO2. ATP content, ATP/ADP ratio, and adenylate charge are maintained at normal values by L-leucine, whereas the NADH/NAD+ ratio (but not the NADPH/NADP+ ratio) is significantly increased. The release of insulin evoked by L-leucine is potentiated by 2-ketoisovalerate, unaffected by L-valine, and inhibited by menadione. L-leucine mimicks the effect of D-glucose on 86Rb+ and 45Ca2+ handling by the islets. However, relative to its rate of oxidation, the insulinotropic effect of L-leucine is less marked than that of D-glucose. This may be due, in part at least, to a decrease in the oxidation of endogenous nutrients. It is concluded that the metabolic, cationic, and secretory effects of L-leucine in isolated islets are not incompatible with the fuel hypothesis for insulin release.

Mode of action of clonidine upon islet function: dissociated effects upon the time course and magnitude of insulin release.

Clonidine (0.08 to 80.0 ng/ml) caused a dose-related inhibition of glucose-stimulated insulin release, but failed to affect glucose oxidation, glucose-stimulated 45Ca net uptake, and adenylate cyclase activity in isolated rat islets. Phentolamine antagonized the effect of clonidine upon insulin release. Despite profound inhibition of insulin secretion, the drug failed to affect the time course for the changes evoked by glucose in either 45Ca fractional outflow rate from perfused islets or insulin release from the isolated perfused pancreas. The latter changes were multiphasic, revealing an initial secretory peak, a period of low secretory activity, and a second secretory elevation before establishing a period characterized by a steadily and slowly increasing insulin output. In the clonidine-treated islets, the secretory rate was not significantly different from the basal value during the period after the initial secretory response. Thus, despite continuous stimulation with glucose, insulin release appears as a discontinuous phenomenon, even when little insulin is secreted during the initial phase of stimulation.

Identification, expression pattern and potential activity of Na/Ca exchanger isoforms in rat pancreatic B-cells.

In the pancreatic B-cell, Na/Ca exchange displays a quite high capacity and participates in the control of cytosolic free Ca2+ concentration. The Na/Ca exchanger was recently cloned in various tissues. Two genes coding for two different exchangers (NCX1 and NCX2) have been identified and evidence for several isoforms for NCX1 shown. To characterize the isoform(s) expressed in pancreatic B-cells, a RT-PCR analysis was performed on mRNA from rat pancreatic islets, purified B-cells and insulinoma B-cells (RINm5F cells). PCR amplification did not yield the expected NCX2 DNA fragment but yielded 2 NCX1 bands, corresponding to NaCa3 and NaCa7, in the three preparations. NaCa3 and NaCa7 were equally expressed in pancreatic islets and purified B-cells. In RINm5F cells, NaCa3 expression did not differ from that in islet and purified B-cells but NaCa7 was 3 times less expressed. This lower expression was accompanied by a 3 times lower Na/Ca exchange activity in RINm5F cells compared to islet cells. Our data indicate the existence of 2 NCX1 isoforms but not of NCX2 in pancreatic B-cells. The difference in both the expression patterns of NCX1 isoforms and the activity of Na/Ca exchange in islet cells and RINm5F cells is compatible with a difference in activity between NaCa3 and NaCa7.

A new Na/Ca exchanger splicing pattern identified in situ leads to a functionally active 70kDa NH(2)-terminal protein.

The Na/Ca exchanger (NCX) is an ubiquitous transporter that plays an important role in regulating cellular Ca(2+) balance. On gel electrophoresis, the NCX1 protein migrates as two major bands of 120 and 70kDa. While the 120kDa is thought to represent the native protein, the nature of the 70kDa protein remains unclear. In this report, we describe a new NCX1 splicing pattern, identified during the cloning of NCX1 isoforms from human eye. The insertion of a newly identified sequence upstream exons B and D of the NCX1.3 isoform, generates a stop codon in frame with the NCX1 coding sequence, that should lead to a truncated Na/Ca exchanger (that we called NCX1.33) comprising only the N-terminal portion of the exchanger and a shortened intracellular loop. Insulin-secreting cells were stably transfected with NCX1.33. Overexpression was assessed at the mRNA and protein level, the truncated exchanger migrating as a70kDa band. Appropriate targeting to the plasma membrane was assessed by microfluorescence and by the increase in Na/Ca exchange activity. The results of the present study constitute a clear piece of evidence indicating that the Na/Ca exchanger 70kDa protein corresponds to the N-terminal portion of the exchanger, and is functionally active.

Heterogeneous changes in [Ca2+]i induced by glucose, tolbutamide and K+ in single rat pancreatic B cells.

The effects of glucose, tolbutamide and K+ on cytosolic free Ca2+ ([Ca2+]i) in single rat pancreatic B cells were examined using Fura-2 and dual wavelength microfluorimetry. At basal glucose concentration (2.8 mM), about half of the cells were found to display spontaneous Ca2+ oscillations. Glucose (greater than or equal to 11.1 mM), tolbutamide (greater than or equal to 50 microM) and K+ (50 mM) induced rises in [Ca2+]i that could be inhibited by the Ca2+ channel blocker D600. The pattern of response and the sensitivity to the secretagogues were characterized by a marked heterogeneity. The majority of the cells responded to glucose and tolbutamide by a progressive increase in [Ca2+]i onto which sinusoidal oscillations were superimposed. The periodicity of these oscillations was about 2.5/min. Occasionally, some cells displayed slow and major waves in Ca2+ levels (about 0.2/min). None of the cells responded to glucose by displaying an initial decrease in [Ca2+]i. Likewise, the sugar failed to decrease [Ca2+]i in the absence of extracellular Ca2+. The present study shows that, despite a large heterogeneity of the response, the majority of the pancreatic B cells respond to different secretagogues by displaying fast [Ca2+]i oscillations that are reminiscent of the bursts of electrical activity recorded in B cells.

Effects of glucose on 45Ca2+ outflow, cytosolic Ca2+ concentration and insulin release from freshly isolated and cultured adult rat islets.

The present study aimed at comparing the effects of glucose on ionic and secretory events in freshly isolated and 5-7 day cultured rat pancreatic islets. The capacity of glucose to provoke insulin release was severely reduced in islets maintained in culture. Whether in freshly isolated or cultured islets, glucose provoked a marked and sustained decrease in 45Ca2+ outflow from islets deprived of extracellular Ca2+. In the presence of extracellular Ca2+ throughout, the magnitude of the glucose-induced secondary rise in 45Ca2+ outflow was reduced in cultured islets. Glucose provoked a weaker increase in [Ca2+]i in islet cells obtained from cultured islets than in islet cells dissociated from freshly isolated pancreatic islets. On the other hand, the stimulatory effect of carbamylcholine on 45Ca2+ outflow was unaffected by tissue culture. Lastly, in islet cells obtained from cultured islets, the increase in [Ca2+]i evoked by K+ depolarization averaged half of that observed in control experiments. These results indicate that the reduced secretory potential of glucose in cultured pancreatic islets can be ascribed to the inability of the nutrient secretagogue to provoke a suitable increase in Ca2+ influx.