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.

Metabolic characteristics of pancreatic beta-cells exposed to calcium-transporting ionophores.

The effects of the ionophores A-23187 and X-537 A on glucose metabolism, ATP content and sucrose permeability in pancreatic islets microdissected from obese-hyperglycemic mice were studied. The formation of 14CO2 from 10 mM D-[U-14C] GLUCOSE WAS INHIBITED BY OMISSION OF Ca2+ from the medium. A-23187 (10 muM) induced a further decrease of 14CO2 formation whereas X-537 A (10 muM) had no effect. At 20 mM glucose both A-23187 (48 muM) and X-537 A (43 muM) decreased the 14CO2 formation in the absence of Ca2+ whereas only X-537 A inhibited in the presence of Ca2+. X-537 A (43 muM) also decreased the formation of 3H2O from 20 mM D-[5-3H] glucose. The islet content of ATP was not changed after incubation in media deficient in either Mg2+ or Ca2+. However, omission of both Mg2+ and Ca2+ resulted in about 50% decrease of the ATP content. A-23187 and X-537 A induced dose-dependent decreases of the islet ATP content. X-537 A was much more potent than A-23187. Both ionophores induced stronger depression of the ATP content when Ca2+ was omitted. X-537 A (43 muM) but not A-23187 (48 muM) increased the beta-cell membrane permeability as indicated by an increased sucrose space in relation to the urea space of islets. Such an effect was not obtained with X-537 A at 1 muM or by omission of Ca2+. It is suggested that the marked metabolic effects of the ionophores reflect an impaired mitochondrial metabolism. These metabolic changes should be considered in interpretations of ionophore action on insulin secretion.

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.

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.

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.

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.

Different effects of the ionophore A-23187 and D-glucose on 45Ca2+ fluxes in isolated islets of ob/ob-mice.

Fluxes of 45Ca2+ were studied in beta-cell rich islets of non-inbred ob/ob-mice, using LaCl3 to wash out extra-cellular and superficially bound 45Ca2+. The ionophore A-23187 (10 microM) increased the 45Ca2+ uptake in islets both at 3 and 20 mM D-glucose, the effect being more pronounced after 10 min than after 120 min of incubation. In incubations for 120 min, 20 mM D-glucose induced a higher uptake of 45Ca2+ than did A-23187. The ionophore enhanced the unidirectional efflux of 45Ca2+ from preloaded islets. Pretreatment of islets with 20 mM D-glucose in non-radioactive medium inhibited the subsequent D-glucose-induced 45Ca2+ uptake. Similar pretreatment with A-23187 increased the subsequent ionophore-induced 45Ca2+ uptake. The results suggest that A-23187 acts by catalyzing Ca2+ fluxes across the beta-cell plasma membrane. The different effects of D-glucose and A-23187 on 45Ca2+ fluxes suggest that the two agents act through different mechanisms in the beta-cells.

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.

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.

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.

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.

Starvation-induced secretory changes of insulin, somatostatin, and glucagon and their modification by 2-bromostearate.

The hypothesis was made of an increased oxidation of fatty acids (FFA) and a decrease of their esterification rate contributing to the islet secretory defect during starvation. 2-Bromostearate (BrS), a FFA-oxidation inhibitor, was therefore tested on the islet secretion of insulin, glucagon and somatostatin stimulated by glucose or palmitate under fasted or fed conditions. Starvation for 48 h blocked both the glucose-induced stimulation and inhibition of insulin and somatostatin and the glucagon secretion. BrS completely restored the insulin response and stimulated both somatostatin and glucagon-basal release, the latter inhibition by glucose being partially recovered. Palmitate transient stimulation of insulin and somatostatin and inhibition of glucagon release was turned into a sustained increase in all three cases by addition of BrS. The potentiation by BrS of palmitate secretory effects in "fed" islets and of hormone release in "fasted" islets, apparently suggest that inhibition of FFA-oxidation may play a role in the regulation of islet secretion.

Starvation-induced changes of palmitate metabolism and insulin secretion in isolated rat islets stimulated by glucose.

The influence of 48 h starvation on glucose-induced changes of palmitate metabolism and insulin release in isolated rat islets was investigated. (1) Islet insulin response to 20 mM-glucose was abolished after 48 h starvation, and it was restored by 0.25 mM-2-bromostearate, an inhibitor of fatty acid oxidation. (2) The increase in glucose concentration from 3 to 20 mM was accompanied by a 50% decrease in the oxidation rate of 0.5 mM-[U-14C]palmitate in control (fed) islets, and a concomitant increase (100%) in its incorporation into triacylglycerol and phospholipid fractions. (3) Starvation induced a higher basal (3 mM-glucose) rate of palmitate oxidation, which was resistant to inhibition by 20 mM-glucose. The latter also failed to increase palmitate incorporation into islet triacylglycerols and phospholipids. (4) 2-Bromostearate (0.25 mM) strongly inhibited the high oxidation rate of palmitate in islets of starved rats, and allowed a normal stimulation of its incorporation rate into islet lipids by 20mM-glucose. (5) The results suggest that starvation restricts islet esterification of fatty acids by inducing a higher rate of their oxidative degradation that is insensitive to regulation by glucose.

Effects of valinomycin on Rb+ fluxes, ATP content and insulin release in pancreatic islets.

Valinomycin, 0.5-500 nM, was tested for its effects on pancreatic islets microdissected fron non-inbred ob/ob-mice. Valinomycin decreased the islet accumulation Rb+ and the content of ATP in a dose-dependent manner; efflux of Rb+ from pre-loaded islets was not noticeably changed. Rb+ accumulation and ATP content correlated markedly; on the model of linear regression, less than 10% of the change Rb+ accumulation in valinomycin-treated islets was statistically attributable to factors other than ATP. Valinomycin did not cause a prompt inhibition of glucose-stimulated insulin release that could reflect hyperpolarization due to increased K+ permeability. The following conclusions are drawn: 1) The plasma membranes of beta-cells resemble those of neurons in having such a high ion permeability as to be relatively little influenced by valinomycin; 2) Islet accumulation of Rb+ is due to a vectorial catalyst in theplasma membrane rather than to uptake by mitochondria; 3) Rb+ accumulation in islets is ATP-dependent.

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).

Effects of salicylate on insulin and glucagon secretion by the isolated and perfused rat pancreas.

The effects of sodium salicylate, a prostaglandin synthesis inhibitor, on glucose-induced secretion of insulin and glucagon by the isolated perfused rat pancreas have been studied. Sodium salicylate inhibited both basal (2.8 mM glucose) and stimulated (16.7 mM glucose) insulin release in a dose dependent manner (1, 5 and 10 mM). This inhibition is not interpretable in terms of a simple inhibition of cyclooxygenase by sodium salicylate. Basal glucagon release was not changed by 1 mM sodium salicylate but the latter partially blocked its inhibition by 16.7 mM glucose. Higher doses of sodium salicylate (5 and 10 mM) inhibited basal glucagon secretion without affecting its response to 16.7 mM glucose. These findings suggest a predominant stimulatory action of endogenous prostaglandins on glucagon release.

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.