Inhibition of connexin 36 hemichannels by glucose contributes to the stimulation of insulin secretion.

The existence of functional connexin36 (Cx36) hemichannels in ß-cells was investigated in pancreatic islets of rat and wild-type (Cx36(+/+)), monoallelic (Cx36(+/-)), and biallelic (Cx36(-/-)) knockout mice. Hemichannel opening by KCl depolarization was studied by measuring ATP release and changes of intracellular ATP (ADP). Cx36(+/+) islets lost ATP after depolarization with 70 mM KCl at 5 mM glucose; ATP loss was prevented by 8 and 20 mM glucose or 50 µM mefloquine (connexin inhibitor). ATP content was higher in Cx36(-/-) than Cx36(+/+) islets and was not decreased by KCl depolarization; Cx36(+/-) islets showed values between that of control and homozygous islets. Five minimolar extracellular ATP increased ATP content and ATP/ADP ratio and induced a biphasic insulin secretion in depolarized Cx36(+/+) and Cx36(+/-) but not Cx36(-/-) islets. Cx36 hemichannels expressed in oocytes opened upon depolarization of membrane potential, and their activation was inhibited by mefloquine and glucose (IC50 ∼8 mM). It is postulated that glucose-induced inhibition of Cx36 hemichannels in islet ß-cells might avoid depolarization-induced ATP loss, allowing an optimum increase of the ATP/ADP ratio by sugar metabolism and a biphasic stimulation of insulin secretion. Gradual suppression of glucose-induced insulin release in Cx36(+/-) and Cx36(-/-) islets confirms that Cx36 gap junction channels are necessary for a full secretory stimulation and might account for the glucose intolerance observed in mice with defective Cx36 expression. Mefloquine targeting of Cx36 on both gap junctions and hemichannels also suppresses glucose-stimulated secretion. By contrast, glucose stimulation of insulin secretion requires Cx36 hemichannels' closure but keeping gap junction channels opened.

Tacrolimus-induced diabetes in rats courses with suppressed insulin gene expression in pancreatic islets.

An animal model of post-transplant diabetes was induced in rats by treating them daily with 0.1 mg/kg body weight of tacrolimus (FK506) in two i.p. injections. Rats developed hyperglycaemia and glucose intolerance after 9 days of treatment. Pancreatic islets, isolated from treated rats on different days, showed a decreased capacity to secrete insulin in response to 20 mM glucose at days 7 and 14. This suppression of insulin secretion was preceded by a reduction of the islet insulin content on day 5 that was progressively decreasing until the end of the treatment (day 14). Islet content of insulin mRNAs, transcribed from rat insulin genes 1 and 2, was strongly suppressed, similar to the insulin content, at days 7 and 14. Islet mass was not strikingly modified by tacrolimus treatment: the DNA content was slightly decreased at the end (day 14) and the rate of islet cell apoptosis slightly increased. Tacrolimus-induced diabetes in the rat seems to be mainly provoked by a decreased insulin gene transcription with little or no alteration of islet mass. This explains that the observed suppression of all the islet and animal parameters studied was completely reversed 2 weeks after interrupting tacrolimus treatment.

Effect of maternal low-protein diet and taurine on the vulnerability of adult Wistar rat islets to cytokines.

AIMS/HYPOTHESIS: A maternal low-protein diet has been shown to induce an increased susceptibility of fetal islets to cytokines, but this effect can be avoided by maternal taurine supplementation. Here, we question whether these effects persist until adulthood in the offspring, despite the animal having a normal diet after weaning. METHODS: Pregnant Wistar rats received a diet of either 20% or 8% protein (control [C group] and recuperated [R group] respectively), which was or was not supplemented with taurine (control treated with taurine [CT group] and recuperated treated with taurine [RT group] respectively) during gestation and lactation. When the female offspring reached adulthood, an OGTT was performed. In a second stage, islets were isolated from these offspring, then pretreated or not with taurine, and subsequently treated with cytokines. RESULTS: Fasting glycaemia was higher (p<0.05) and insulinaemia was lower (p<0.01) in the R group than in the C group. Taurine supplementation decreased insulinaemia in the CT group and tended to increase it in the RT group. After the OGTT, glycaemia in R animals was not different from that in the C group, despite a blunted insulin response (p<0.05) which was restored by taurine. Supplementation in C-group mothers led to a weak glucose intolerance. In vitro, more apoptotic cells were observed in R islets after cytokines treatment (p<0.01). The addition of taurine to the culture medium in the R and C groups protected the islets from the cytokines (p<0.01). Maternal taurine supplementation decreased the sensitivity of islets in the RT group (p<0.01), but increased sensitivity in the CT group (p<0.01). CONCLUSIONS/INTERPRETATION: The increased vulnerability of islets to cytokines due to a restriction of protein during fetal development was still evident when the offspring reached adulthood. The low-protein diet also induced hyperglycaemia in the presence of lower insulinaemia. Taurine supplementation protected adult islets of the R group from cytokine toxicity and restored the insulinaemia. However, unnecessary supplementation of taurine could have detrimental effects.

An osmotic-sensitive taurine pool is localized in rat pancreatic islet cells containing glucagon and somatostatin.

Previous reports have dealt with the hypoglycemic properties of taurine and its effects on insulin secretion by adult and fetal isolated islets. We have studied the presence and cellular distribution of taurine in rat islets, the conditions to evoke its release, and its possible modulatory action on insulin secretion. We localized taurine by techniques of double immunolabeling in most glucagon-positive cells and in some somatostatin-positive cells, whereas insulin-positive cells were not labeled with the taurine antibody. Although high-glucose stimulation did not evoke any taurine release, a hyposmotic solution (17% osmolarity reduction) induced a specific phasic release of taurine and GABA (34 and 52% increase on their basal release rate). On the other hand, taurine (10 mmol/l) application slightly reduced the second phase of insulin secretion induced by glucose stimulation. In conclusion, taurine is highly concentrated in glucagon-containing cells of the islet periphery. It is not liberated by glucose stimulation but is strongly released under hyposmotic conditions. All of these data suggest that taurine plays an osmoregulatory role in alpha-cells.

Lactate production in pancreatic islets.

Lactate production, glucose utilization, glucose oxidation, and insulin release were studied in islets from rat and ob/ob mice. Lactate was determined with a highly sensitive method, based on esterification, subsequent separation, and quantitation with high-performance liquid chromatography. There was a significant lactate production in the absence of glucose, which increased with glucose concentrations up to 3 mmol/l, reaching its half-maximal rate in the presence of 0.2-1.0 mmol/l glucose in both species. Glucose utilization displayed a wider glucose concentration dependence, with a K0.5 value between 3 and 10 mmol/l glucose. The rates of glucose utilization and lactate production were similar at 3 mmol/l glucose in rat islets and at about 6 mmol/l glucose in ob/ob mice islets. Saturation of lactate production at low glucose concentrations is probably contributing to the observed preferential stimulation of oxidative metabolism at higher concentrations. D-Mannoheptulose caused a marked inhibition of glucose utilization and glucose oxidation at 20 mmol/l glucose in islets from rat or ob/ob mice, as would be expected from a competitive inhibition of glucokinase. By contrast, D-mannoheptulose reduced only marginally the islet metabolism at 3 mmol/l glucose, which is consistent with an effective mannoheptulose-induced inhibition of the glucokinase-dependent, minor part of glucose phosphorylation at this low glucose concentration.

Stimulation of islet protein kinase C translocation by palmitate requires metabolism of the fatty acid.

The secretory, metabolic, and signaling aspects of glucose/palmitate interaction on beta-cell function have been studied on rat islets. Palmitate potentiated the glucose-induced insulin response of perifused islets at suprathreshold (>3 mmol/l) sugar concentrations. This potentiating effect could be suppressed by 8-bromo-cGMP, which also blocks palmitate metabolism. Palmitate did not modify glucose utilization, but it slightly reduced glucose oxidation and concomitantly increased lactate production. The very low rate of palmitate oxidation (80-fold lower than that of 20 mmol/l glucose) might explain its lack of effect on glycolysis and hence that the glucose/fatty acid cycle is inoperative in islet cells. However, glucose determines the metabolic fate of exogenous palmitate, which is mainly diverted toward lipid synthesis at high sugar concentrations and might then generate lipid messengers for cell signaling. Palmitate did not increase glucose-induced production of inositol-1,4,5-trisphosphate, but it stimulated the translocation of protein kinase C activity from a cytosolic to a particulate fraction at 20 but not at 3 mmol/l glucose. This increased translocation was partially or completely blocked by hydroxycitrate or 8-bromo-cGMP, respectively, which are agents interfering with palmitate metabolism (inhibiting lipid synthesis). The metabolic interaction between glucose and palmitate might generate lipid messengers (diacylglycerol, phosphatidylserine) necessary for the activation of islet protein kinase C, which would in turn result in a potentiation of glucose-induced insulin secretion.

The stimulation of insulin secretion by D-glyceraldehyde correlates with its rate of oxidation in islet cells.

D-Glyceraldehyde's capacity to mimic the effect of D-glucose on insulin secretion has not yet been sufficiently substantiated. It has been recently proposed, however, that they might act through different mechanisms in insulin-secreting tumoral cells. Therefore, we have performed a dose-related study of both the secretory and metabolic effects of D-glyceraldehyde on islets, which have been compared with those produced by D-glucose. D-Glyceraldehyde's capacity to stimulate secretion was paralleled in a dose-dependent manner by its rate of oxidation to 14CO2. Partial inhibition of D-glyceraldehyde oxidation by beta-iodoacetamide resulted in a proportional decrease in the secretory response. L-Glyceraldehyde, which was apparently very poorly oxidized by islets, did not stimulate secretion. The ratio of the maximum insulin responses D-glyceraldehyde and D-glucose (57%) correlated with the ratio of their respective maximum rates of oxidation (68%). At sub-maximal concentrations there was a potentiation of the secretagogue effects of the hexose by the triose, which was not apparent at a maximum effective dose of glucose. It is concluded that D-glyceraldehyde mimics the secretory effect of glucose because, similarly to the hexose, it is metabolized through islet aerobic glycolysis. The lower potency of D-glyceraldehyde as an insulin secretagogue than D-glucose is determined by the lower capacity of islets to oxidize the triose compared with the hexose. D-Glyceraldehyde, unlike D-glucose, is metabolized in islets to D-lactate. Alternative routes for the metabolism of D-glyceraldehyde might explain some of the secretagogue differences between the triose and the hexose.

Low lactate dehydrogenase and high mitochondrial glycerol phosphate dehydrogenase in pancreatic beta-cells. Potential role in nutrient sensing.

Nutrient metabolism was examined with regard to insulin secretion in purified rat islet beta- and non-beta-cells, beta-cell lines, and hepatocytes. Lactate dehydrogenase (LDH) activity (nanomoles.min-1.mg protein-1) was remarkably low in the glucose-sensitive INS-1 cell line (15.7) and in beta-cells (22.3). Thus, beta-cell LDH was respectively 8-, 122-, 17-, and 136-fold lower than in islet non-beta, liver, HIT-T15, and RINm5F cells. Plasma membrane lactate transport activity was 3-10-fold lower in beta- or INS-1 cells than in the other cell types. Conversely, mitochondrial glycerol phosphate dehydrogenase was strongly expressed only in beta- and INS-1 cells. The significance of these findings to nutrient recognition was explored using INS-1 cells as a model of native beta-cells. Glucose-stimulated lactate output and glucose utilization were, respectively, 12- and 5-fold lower in INS-1 than in RINm5F cells. Each process was entirely blocked by respiratory chain inhibitors in INS-1 cells, whereas glucose utilization was barely affected and lactate output stimulated in RINm5F cells. Glucose oxidation represented 73% of total utilization in INS-1 cells, but only 9% in RINm5F cells. Absolute rates of glucose oxidation, and the extent of mitochondrial NAD(P) reduction, were similar in the two cell types, and glucose stimulated insulin secretion 1.9-fold in INS-1 and 1.4-fold in RINm5F cells. The mitochondrial substrates, monomethyl succinate, pyruvate, and leucine, each triggered secretion in INS-1 cells. The balance of LDH, plasma membrane lactate transport, and mitochondrial glycerol phosphate dehydrogenase activities therefore appear to be important in beta- and INS-1 cell glucose recognition to ensure that mitochondrial oxidation is the principle fate of pyruvate and NADH produced by glycolysis. The resultant close coupling of glycolysis with mitochondrial oxidation explains the absence in beta-cells of Crabtree and Pasteur effects.

Effects of calcium channel blockers on insulin secretion and 45Ca(2+)-uptake of rat islets stimulated by glucose or K(+)-depolarization.

Two calcium channel antagonists, verapamil and nifedipine, have been used to explore the dependence of secretion on voltage-gated influx of calcium. Both antagonists were able to suppress the secretory response to K(+)-depolarization as well as the stimulation of 45Ca(2+)-uptake. However, they inhibited only partially the stimulation of both secretion and 45Ca(2+)-uptake. However, they inhibited only partially the stimulation of both secretion and 45Ca(2+)-uptake induced by glucose, alone or with palmitate. The stimulation of 45Ca(2+)-uptake by K(+)-depolarization, unlike that induced by glucose, was not sensitive to norepinephrine, starvation or fatty acid oxidation inhibitors. Therefore, it is suggested that glucose either modifies the properties of the voltage-dependent calcium channel and/or accelerates the exchange of a particular intracellular pool of calcium.

Does cyclic guanosine monophosphate mediate noradrenaline-induced inhibition of islet insulin secretion stimulated by glucose and palmitate?

Noradrenaline inhibits in rat islets the stimulation of insulin secretion induced by glucose and its potentiation by palmitate, but the signalling system responsible remains unknown. We have tested the hypothesis that noradrenaline-induced inhibition is mediated by an elevation of cyclic GMP (cGMP) levels. The analogue 8-Br-cGMP decreases dose-dependently the potentiation by palmitate of glucose-induced insulin secretion, whereas it only slightly affects the proper effect of glucose. Similarly, it abolishes palmitate acceleration of glucose-induced 45Ca2+ uptake without modifying the sugar effect. Finally, 8-Br-cGMP completely inhibits the stimulation of the lipid synthesis de novo induced by palmitate, but not that caused by glucose alone. On the other hand, noradrenaline increases dose-dependently islet cGMP content, with alpha 2-adrenergic specificity. As noradrenaline-induced elevation of cGMP is sensitive to pertussis toxin, it probably results from alpha 2-adrenoceptor activation of islet guanylate cyclase through a guanine nucleotide regulatory protein. It is concluded that the elevated cGMP levels mediate noradrenaline inhibition of lipid synthesis de novo, and hence of acceleration by palmitate of 45Ca2+ uptake and insulin secretion in the presence of glucose.

Insulin, glucagon, somatostatin, and thyrotropin-releasing hormone content and secretion by perifused fetal rat islets during culture.

In the neonatal period of the rat, pancreatic thyrotropin-releasing hormone content decreases and the sensitivity of insulin secretion to glucose increases. In adult rat islets, TRH inhibits glucose-induced insulin release. The aim of this study was to investigate whether a high TRH content and release can be part of the explanation for the functional immaturity of neonatal islets. For that purpose, we have measured the tissue content and the secretion of immunoreactive insulin, glucagon, somatostatin and TRH in islets from 21.5-day-old rat fetuses cultured for up to one week. Insulin, glucagon and somatostatin content increased during one week of culture in the presence of 11.1 mmol/l glucose. The TRH content decreased during culture, but did not equal adult values. Insulin, glucagon and somatostatin responses to glucose were present after one week of culture. Glucose had no effect on TRH release in cultured fetal islets, but inhibited TRH release in adult islets. We conclude that glucose can stimulate insulin secretion without inhibiting TRH release, but that a decrease in islet TRH content and a sensitization of TRH secretion to glucose may be important in the full maturation of fetal pancreatic islets.

Trifluoperazine reproduces in rat islets the effects of calcium omission on insulin secretion and de novo lipid synthesis, without affecting 45Ca2(+)-uptake.

Calmodulin is thought to mediate at least some of the effects produced by the elevation of cytosolic calcium in response to a B-cell secretagogue. Trifluoperazine, an inhibitor of calcium-calmodulin interaction, has been used to test, comparatively with calcium-omission, whether the changes of lipid metabolism accompanying the stimulation of insulin release by glucose and palmitate are dependent on activation by the calcium binding protein. Low doses of trifluoperazine (1 and 5 mumol/l) reproduced quantitatively and qualitatively the effects of calcium omission on both insulin secretion and de novo lipid synthesis, without altering islet 45Ca2(+)-uptake. The apparent dependence on calcium-calmodulin of the "de novo" synthesis of neutral lipids, but not of acidic phospholipids, might reflect a possible regulation of islet phosphatidate phosphohydrolase by calcium.

Norepinephrine inhibits islet lipid metabolism, 45Ca2+ uptake, and insulin secretion.

We have previously shown that palmitate potentiates, in isolated islets, glucose-induced stimulation of insulin release, "de novo" lipid synthesis, and 45Ca2+ turnover in a correlative manner. Norepinephrine, a known inhibitor of the secretory response, has now been used to further investigate the relationships among the three phenomena. The amine decreased insulin secretion dose dependently in response to glucose and palmitate with alpha 2-adrenergic specificity. It also reduced similarly the oxidation of 1 mmol/l [U-14C]palmitate as well as the incorporation of 20 mmol/l D-[U-14C]glucose into islet phospholipids and neutral lipids through an alpha 2-adrenergic mechanism. These results indirectly suggest that alpha 2-adrenoceptor stimulation inhibits in islets both palmitate oxidation and esterification through an inactivation of long-chain acyl-CoA synthetase and other enzymes of glycerolipid synthesis. Islet uptake of 45Ca2+ was also decreased by norepinephrine with a similar sensitivity to that shown by insulin release and de novo lipid synthesis. Therefore, it is suggested that alpha 2-adrenoceptor-mediated reduction of the potentiation by palmitate of the secretory response to glucose depends on the inhibition of fatty acid metabolism and the resulting impairment of de novo lipid synthesis and 45Ca2+ turnover.

Palmitate dependence of insulin secretion, "de novo" phospholipid synthesis and 45Ca2+-turnover in glucose stimulated rat islets.

Palmitate ability to modify D-[U-14C]glucose incorporation into different lipids ("de novo" synthesis), as well as sugar-stimulation of insulin release and 45Ca2+-fluxes, was investigated in islets of fed and 48-h starved rats. The fatty-acid induced dose-dependent, correlative increments of insulin secretion, 45Ca2+-influx and the "de novo" synthesis of each phospholipid fraction analysed at 20 mmol/l (but not 3 mmol/l) glucose. Omission of calcium reduced drastically (p less than 0.001) insulin release and the "de novo" synthesis of neutral glycerolipids, leaving unaltered that of acidic phospholipids (phosphatidate and phosphoinositides). The increased synthesis of the latter is therefore not the consequence of stimulated secretion. It could initiate or contribute to maintain an increased turnover of islet phosphoinositides, thus generating some mediators of the calcium signalling system (inositol phosphates). Starvation led to a drastic reduction (p less than 0.001) of both insulin secretion, "de novo" synthesis of each lipid fraction, and 45Ca2+-influx in response to glucose and palmitate. The presence of a fatty-acid oxidation inhibitor (2-bromostearate or 2-tetradecylglycidate) prevented the effect of starvation on 45Ca2+-influx, as it has been shown to do on insulin secretion and palmitate incorporation into islet lipids. It is finally suggested that palmitate might amplify the insulin secretory response of islets to glucose, through the stimulation of the "de novo" synthesis of phosphoinositides and the subsequent generation of inositol phosphates, which would contribute to accelerated calcium turnover.

Islet secretion of immunoreactive thyrotropin-releasing hormone and the 'paracrine-like' effects of its exogenous administration.

In order to know more about the secretory pattern of islet TRH in response to glucose and its possible physiological relevance, the release of this hormone as well as that of insulin, glucagon, and somatostatin was radioimmunologically measured. Whereas the secretion of immunoreactive insulin and somatostatin by incubated rat islets is known to be dose-dependently stimulated by glucose, that of glucagon and TRH was inhibited by glucose. Similarly, palmitate dose-dependently inhibited islet glucagon and TRH release. Exogenous TRH exerted strong and dose-dependent effects on islet secretion of the other hormones at the same concentration range at which its hypophysiotropic effects are produced (10(-10) to 10(-8) mol/l). It inhibited the insulin response to glucose and blocked that of glucagon, whereas it enhanced glucose-induced stimulation of somatostatin. These results are suggestive of a possible paracrine inhibitory role of islet TRH, either directly exerted on the secretion of insulin and glucagon or partially mediated through the stimulation of somatostatin release.

Effects of L-leucine on palmitate metabolism and insulin release by isolated islets of fed and starved rats.

The occurrence of lipid metabolic changes associated with L-leucine (10 mM) stimulation of insulin release was investigated in isolated islets from either fed or starved rats. L-Leucine-stimulated secretion was potentiated by 3 mM glucose and/or 0.5 mM palmitate and was unaffected by 48 h of starvation. Islet palmitate oxidation showed a maximum rate at 3 mM glucose, and starvation increased it almost 2-fold. Regardless of the nutritional state, L-leucine strongly reduced the oxidation of palmitate and increased its incorporation into islet triacylglycerols and phospholipids at 3 mM glucose. This shift of fatty acid metabolism toward esterification might play a role in the mechanism of potentiation of the islet secretory response to L-leucine by glucose and palmitate.

Glucose stimulation of insulin secretion in islets of fed and starved rats and its dependence on lipid metabolism.

The influence of a physiologic range of palmitate concentrations (0, 0.25, 0.5, and 1.0 mmol/L) on glucose ability to modify insulin secretion, (U-14C) palmitate oxidation, and (U-14C) glucose incorporation into lipids has been studied in islets isolated from either fed or 48-hour starved rats. Palmitate potentiated the insulin response of fed islets to glucose in a particular dose-related manner. Glucose stimulated secretion was accompanied by a decreased palmitate oxidation and an increased (U-14C) glucose incorporation into di-, tri-acylglycerols, and predominantly into phospholipids. These metabolic parameters showed also a positive dependence on palmitate concentration. Starvation increased islet capacity to oxidize palmitate, rendered it insensitive to glucose inhibition, and inhibited both (U-14C) glucose incorporation into all lipid fractions and sugar induced insulin release. The stimulation of islet lipid synthesis by glucose seems to be limited by the exogenous supply of fatty acids and their rate of oxidation. As judged from (U-14C) glucose incorporation data, the rate of phospholipid biosynthesis showed a significant and positive correlation with insulin secretion. This metabolic pathway might provide islet cells with some lipid intermediates (diacylglycerol and/or specific phospholipids) that have been considered as possible mediators of the calcium messenger system.

Antigenic specificity of a new and potent somatostatin antiserum.

The specificity of a new somatostatin antiserum (Ab 6) has been investigated using eight different peptide analogues. For this purpose, somatostatin and all the analogues were tested, in an equimolar concentration range, for their ability to displace 125I-tyr1-somatostatin from its binding to antibodies. Analysis of the displacement curves shows that antiserum Ab 6 predominantly recognizes changes at the central aminoacid sequence of the somatostatin molecule. Its ligand specificity seems to be similar to that of other somatostatin antisera previously described (R-141 and A-101).