Multisite control of insulin release by glucose.

Glucose controls insulin release by beta-cells at two sites at least. By controlling the membrane potential, it controls the influx of Ca2+ and the rise in cytoplasmic Ca2+ which triggers exocytosis. At this level, the principal targets of glucose are the K(+)-ATP channels whose activity may be modulated by changes in the ATP/ADP ratio. A second, newly identified, mechanism of regulation is independent of changes in beta-cell membrane potential and of changes in Cai2+. It is not sufficient to induce insulin release, but serves to increase the response. This appears to be achieved through an amplification of the effectiveness of Cai2+ on the secretory process and may also depend on the changes in energy state of beta-cells.

Mechanisms by which glucose can control insulin release independently from its action on adenosine triphosphate-sensitive K+ channels in mouse B cells.

Glucose stimulation of insulin release involves closure of ATP-sensitive K+ channels (K(+)-ATP channels), depolarization, and Ca2+ influx in B cells. However, by using diazoxide to open K(+)-ATP channels, and 30 mM K to depolarize the membrane, we could demonstrate that another mechanism exists, by which glucose can control insulin release independently from changes in K(+)-ATP channel activity and in membrane potential (Gembal et al. 1992. J. Clin. Invest. 89:1288-1295). A similar approach was followed here to investigate, with mouse islets, the nature of this newly identified mechanism. The membrane potential-independent increase in insulin release produced by glucose required metabolism of the sugar and was mimicked by other metabolized secretagogues. It also required elevated levels of cytoplasmic Cai2+, but was not due to further changes in Cai2+. It could not be ascribed to acceleration of phosphoinositide metabolism, or to activation of protein kinases A or C. Thus, glucose did not increase inositol phosphate levels and hardly affected cAMP levels. Moreover, increasing inositol phosphates by vasopressin or cAMP by forskolin, and activating protein kinase C by phorbol esters did not mimic the action of glucose on release, and down-regulation of protein kinase C did not prevent these effects. On the other hand, it correlated with an increase in the ATP/ADP ratio in islet cells. We suggest that the membrane potential-independent control of insulin release exerted by glucose involves changes in the energy state of B cells.

[Transplantation of free beta islet cells as a method leading to normalization of glucose tolerance in rats with experimental diabetes]. / Transplantacja wolnych komórek beta wysp trzustki jako metoda wyrównania zaburzen tolerancji glukozy u szczurów z cukrzyca doswiadczalna.

The aim of present study was to investigate the effect of free beta cells transplantation on carbohydrate tolerance in rats with streptozotocin-induced diabetes. Free beta cells were isolated by mechanical disruption of isolated pancreatic islets in Ca and Mg free medium. 1500 and 3000 isolated islets were used for the isolation of 500 x 10(3)-1.099 x 10(3) and 1.398 x 10(3)-2.098 x 10(3) free cells, respectively. Between 78% and 98% of all isolated cells were the variable cells. After isogenic transplantation of free beta cells, blood glucose concentration in all animals was normalized within 14 days. The rate of glycemia normalization was related to the amount of viable free cells. The glucose assimilation coefficient after intravenous glucose administration in normal rats, diabetic rats and diabetic rats after transplantation of free cells was: 2.98 x 10(-2); 0.68 x 10(-2) and 2.04 x 10(-2) mg/dl/min, respectively. Allogenic transplantation of free beta cells caused only a transient decrease in blood glucose level.

Protective effect of allopurinol, alpha-tocopherol and chlorpromazine on rat pancreatic islets stored prior to transplantation.

Iso-transplantation of 800 isolated rat pancreatic islets into the portal vein of diabetic rats was performed. Isolated islets were kept in Hanks buffer for 12 hours prior to transplantation. Part of donors was pretreated with allopurinol, alpha-tocopherol and chlorpromazine. Transplantation of islets isolated from nonpretreated rats did not cause any significant changes in plasma glucose concentration of recipients, while transplantation of islets isolated from donors after pretreatment with the tested substances brought glucose level back to normal 3 days after transplantation. Our results indicate that the combination of free radical scavengers, antioxidants and membrane stabilizing drugs may be used to increase the effectiveness of islet transplantation in humans.

Binding of 3H-norepinephrine to receptors of pancreatic islets isolated from neonatal and adult rats.

Binding of 3H-norepinephrine to receptors and insulin release from pancreatic islets isolated from newborns and adult rats were investigated. In the presence of norepinephrine at the concentration of 2.4 x 10(-8) mol/l adults islets bound 0.37 +/- 0.11 fmol of norepinephrine per micrograms of islet protein while neonatal islets bound 0.25 +/- 0.08 fmol/micrograms. When norepinephrine concentration was raised to 10(-5) mol/l the amount of bound norepinephrine increased up to 22.66 +/- 8.7 fmol/micrograms and 14.20 +/- 5.36 fmol/micrograms for both examined groups, respectively. Addition of 5 x 10(-4) mol/l of phentolamine to the incubation medium induced 74% and 70% decrease of bound norepinephrine to adult and neonatal islets, respectively. At similar range of concentrations both norepinephrine and phentolamine modified the rate of insulin secretion and caused changes in binding of 3H-norepinephrine. There were no differences between the binding of norepinephrine to neonatal and adult islets. This may suggest that adrenergic system may play greater role in the regulation of insulin release from neonatal than from adult islets.

Evidence that glucose can control insulin release independently from its action on ATP-sensitive K+ channels in mouse B cells.

Glucose stimulation of insulin release involves closure of ATP-sensitive K+ channels, depolarization, and Ca2+ influx in B cells. Mouse islets were used to investigate whether glucose can still regulate insulin release when it cannot control ATP-sensitive K+ channels. Opening of these channels by diazoxide (100-250 mumol/liter) blocked the effects of glucose on B cell membrane potential (intracellular microelectrodes), free cytosolic Ca2+ (fura-2 method), and insulin release, but it did not prevent those of high K (30 mmol/liter). K-induced insulin release in the presence of diazoxide was, however, dose dependently increased by glucose, which was already effective at concentrations (2-6 mmol/liter) that are subthreshold under normal conditions (low K and no diazoxide). This effect was not accompanied by detectable changes in B cell membrane potential. Measurements of 45Ca fluxes and cytosolic Ca2+ indicated that glucose slightly increased Ca2+ influx during the first minutes of depolarization by K, but not in the steady state when its effect on insulin release was the largest. In conclusion, there exists a mechanism by which glucose can control insulin release independently from changes in K(+)-ATP channel activity, in membrane potential, and in cytosolic Ca2+. This mechanism may serve to amplify the secretory response to the triggering signal (closure of K(+)-ATP channels--depolarization--Ca2+ influx) induced by glucose.

Ca(2+)- and ATP-dependent reversible inactivation of pancreatic islet phosphoinositide-specific phospholipase C activity.

Phosphoinositide-specific phospholipase C (PI-PLC) activity in whole homogenates of mouse pancreatic islets decreased 60-85% when the homogenates were incubated at 37 degrees C for 1 h in the presence of down to micromolar concentrations of Ca2+. Ca(2+)-induced inactivation was augmented by calmodulin, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in the presence of ATP-Mg, and by Mg2+. Inactivation was inhibited when ATP was removed and completely abolished by trifluoperazine and EGTA. Inactivation was not affected by the non-phosphorylating ATP analogue, AMP-PCP, GMP-PNP, glucose, Zn2+ or a series of protease inhibitors. These observations suggest that PI-PLC in broken cell preparations of pancreatic islets may be inactivated via phosphorylation by Ca(2+)-calmodulin-stimulated protein kinase and/or protein kinase C. Inactivation of PI-PLC was reversible. Reactivation started after approx. 2 h incubation, when the concentration of ATP in the homogenate was below 0.15 x 10(-6) M. PI-PLC activity returned to values approx. 25% higher than the initial values. PI-PLC inactivation via phosphorylation by the mentioned protein kinases may constitute a feedback control on the phosphoinositide response, attenuating subsequent diacylglycerol formation and/or Ca2+ mobilization by inositol trisphosphate.

Characteristics of phosphoinositide-specific phospholipase C activity from mouse pancreatic islets.

In pancreatic islets the bulk of phosphoinositide-specific phospholipase C (PI-PLC) activity was cytosolic. The soluble enzyme was activated by submicromolar concentrations of Ca2+, independent of calmodulin. It was unaffected by glucose and a series of glycolytic intermediates, including glyceraldehyde 3-phosphate. These observations lend support to the hypothesis that glucose-stimulated inositol triphosphate production in islets may be secondary to and provoked by glucose-mediated Ca2+ influx. All four pyridine nucleotides stimulated PI-PLC. Phosphatidylinositol hydrolysis was also stimulated by dioleine and arachidonic acid, and by the polyamines, putrescine and spermine. Phosphatidylinositol hydrolysis was inhibited by chlorpromazine, tetracaine, ATP, 5'-AMP, inorganic pyrophosphate and by phosphatidylinositol 4,5-bisphosphate, phosphatidylcholine and phosphatidylserine--but not affected by phosphatidylethanolamine. The cyclic nucleotides, cAMP and cGMP had no effect on the enzyme, and GTP-gamma-S did not activate the enzyme event at very low Ca2+ concentrations. The diglyceride lipase inhibitor, RHC 80267, and the cyclooxygenase inhibitor, indomethacin, had no effect on PI-PLC activity.