Eberhard G. Siegel

Stimulation of Adenylate Cyclase by Ca2+ and Calmodulin in Rat Islets of Langerhans: Explanation for the Glucose-induced Increase in Cyclic AMP Levels

The effect of Ca2+ and calmodulin has been studied on adenylate cyclase activity in homogenates of rat islets of Langerhans. EGTA had a stimulatory effect on the enzyme in accord with the known inhibitory effect of Ca2+. In contrast, the addition of Ca2+ together with calmodulin is stimulatory and demonstrates the existence of a Ca2+-dependent adenylate cyclase in islets of Langerhans. It is suggested that the glucose-induced increase in cyclic AMP concentrations in intact islets is a secondary consequence of the glucose-induced increase in cytosol free-Ca2+ concentrations which, with calmodulin, causes an increase in the activity of adenylate cyclase.

Possible role for calmodulin in insulin release. Studies with trifluoperazine in rat pancreatic islets.

The role of calmodulin in insulin secretion from rat pancreatic islets has been examined by the use of trifluoperazine, an inhibitor of calmodulin-Ca++-directed functions. It was found that 30 microM trifluoperazine caused 50% inhibition, and 100 microM, up to 73% inhibition of 16.7 mM glucose-stimulated insulin release. 100 microM trifluoperazine caused a similar inhibition of 10 mM glyceraldehyde-stimulated release. Therefore, the site of action of trifluoperazine in glucose stimulus-secretion coupling appears to be after the trioses. As trifluoperazine had no effect upon insulin release stimulated by 1 mM 3-isobutyl-1-methylxanthine, the inhibitory effect of trifluoperazine appears to be rather specific. Further, the process of exocytosis per se is not affected. It was also found that although trifluoperazine inhibited the effect of glucose to stimulate insulin release, it did not affect the synergism between glucose and 3-isobutyl-1-methylxanthine to potentiate insulin release. It may be concluded that trifluoperazine selectively inhibits one part of the mechanism by which glucose stimulates insulin release. Calmodulin plays a role in the stimulation of insulin release by glucose at a site between metabolism of trioses and elevation of cytosol Ca++, but is not involved in the final process of exocytosis.

Dependency of cyclic AMP-induced insulin release on intra- and extracellular calcium in rat islets of Langerhans.

Calcium and cyclic AMP are important in the stimulation of insulin release. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) raises islet cAMP levels and causes insulin release at nonstimulatory glucose concentrations. In isolated rat pancreatic islets maintained for 2 d in tissue culture, the effects of IBMX on insulin release and 45Ca++ fluxes were compared with those of glucose. During perifusion at 1 mM Ca++, 16.7 mM glucose elicited a biphasic insulin release, whereas 1 mM IBMX in the presence of 2.8 mM glucose caused a monophasic release. Decreasing extracellular Ca++ a monophasic release. Decreasing extracellular Ca++ to 0.1 mM during stimulation reduced the glucose effect by 80% but did not alter IBMX-induced release. Both glucose and IBMX stimulated 45Ca++ uptake (5 min). 45Ca++ efflux from islets loaded to isotopic equilibrium (46 h) was increased by both substances. IBMX stimulation of insulin release, of 45Ca++ uptake, and of efflux were not inhibited by blockade of Ca++ uptake with verapamil, whereas glucose-induced changes are known to be inhibited. Because IBMX-induced insulin release remained unaltered at 0.1 mM calcium, it appears that cAMP-stimulated insulin release is controlled by intracellular calcium. This is supported by perifusion experiments at 0 Ca++ when IBMX stimulated net Ca++ efflux. In addition, glucose-stimulated insulin release was potentiated by IBMX. These results suggest that cAMP induced insulin release is mediated by increases in cytosolic Ca++ and that cAMP causes dislocation of Ca++ from intracellular stores.

Evidence for the Involvement of Na/Ca Exchange in Glucose-induced Insulin Release from Rat Pancreatic Islets

Glucose-induced inhibition of Ca(++) extrusion from the beta-cell may contribute to the rise in cytosol Ca(++) that leads to insulin release. To study whether interference with Na/Ca exchange is involved in this inhibition the effects of glucose were compared to those of ouabain. This substance inhibits Na/K ATPase, decreases the transmembrane Na(+) gradient in islets, and thus interferes with Na/Ca exchange. Collagenase isolated rat islets were maintained for 2 d in tissue culture with a trace amount of (45)Ca(++). Insulin release and (45)Ca(++) efflux were then measured during perifusion. In Ca(++)-deprived medium (to avoid changes in tissue specific radioactivity) 16.7 mM glucose inhibited (45)Ca(++) efflux. Initially 1 mM ouabain inhibited (45)Ca(++) efflux in a similar fashion, the onset being even faster than that of glucose. The effects of 16.7 mM glucose and ouabain were not additive, indicating that both substances may interfere with Na/Ca exchange. In the presence of Ca(++), 16.7 mM glucose induced biphasic insulin release. Ouabain alone caused a gradual increase of insulin release. Again, the effects of ouabain and 16.7 mM glucose were not additive. In contrast, at a submaximal glucose concentration (7 mM) ouabain enhanced both phases of release. An important role for Na/Ca exchange is suggested from experiments in which Ca(++) was removed at the time of glucose-stimulation (16.7 mM). The resulting marked inhibition of insulin release was completely overcome during first phase by ouabain added at the time of Ca(++) removal; second phase was restored to 60%. This could be due to the rapid inhibitory action of ouabain on Ca(++) efflux thereby preventing loss of cellular calcium critical for glucose to induce insulin release. It appears, therefore, that interference with Na/Ca exchange is an important event in the stimulation of insulin release by glucose.

Beneficial effects of low-carbohydrate-high-protein diets in long-term diabetic rats

Abstract Groups of streptozotocin-diabetic rats (65 mg/kg subcutaneously) were fed ad lib. on three different diets over 12 mo: a low-carbohydrate (CHO)-high-protein (6%:63% of calories) diet; a moderate-CHO-high-protein (27%:50%) diet; and a standard control diet (68% as CHO, 20% as protein). The 6%-CHO diet resulted in an initial decrease in nonfasting blood glucose from 550 to 280 mg/dl, followed by a further gradual improvement ending with blood glucose levels below 160 mg/dl and reduction of glycosuria to physiologic amounts. In the group fed the 27%-CHO diet, there was a similar initial decrease in blood glucose, but blood glucose values below 160 mg/dl after 12 mo were attained in only 3 of 8 rats. All rats fed the standard 68%-CHO diet maintained blood glucose levels between 500 and 650 mg/dl throughout. The late recovery in the low-CHO fed groups was associated with increased pancreatic insulin content. Animals fed low-CHO diets showed a better weight gain and improved general condition. The onset of cataracts as observed with the hand slit-lamp was regularly delayed in the animals on the 6%- and 27%-CHO diet; in some instances, it was entirely prevented. The threshold concentration of blood glucose seemingly required for onset of cataracts was about 300 mg/dl. Nerve sorbitol and fructose levels were clearly higher in hyperglycemic animals and correlated well with the severity of hyperglycemia. Thus, low-CHO-high-protein diets, which markedly improve hyperglycemia, may also improve at least some of the secondary changes usually seen in severely hyperglycemic rats.

Sites of Action of Trifluoperazine in the Inhibition of Glucose-Stimulated Insulin Release

Trifluoperazine, an inhibitor of calcium-calmodulin functions, was used in an attempt to understand the involvement of calcium-calmodulin in glucose-stimulated insulin release. Isolated rat pancreatic islets were used after a two-day period of maintenance in tissue culture. 45Ca2+ uptake and insulin release were measured during 5-min incubations. Dynamic insulin release and 45Ca2+ efflux were assessed during perifusion of the islets preloaded with 45Ca2+ during the culture period. Both phases of insulin release in response to 16.7 mM glucose were inhibited by approximately 60% in the presence of 10 μM trifluoperazine when the latter was added 35 min prior to high glucose. Stimulation of 45Ca2+ efflux by glucose was abolished. Glucose-stimulated 45Ca2+ uptake was inhibited by 43%. These results were compared with those of experiments in which depolarizing concentrations of potassium (24 mM) were used. Trifluoperazine inhibited K+-stimulated insulin release and 45Ca2+ uptake to a similar extent as that seen with glucose. Trifluoperazine did not appear to interfere with the inhibitory effect of glucose on 45Ca2+ efflux seen in the absence of extracellular Ca2+. Moreover, in Ca2+ deprived medium (with no possibility for Ca2+ uptake) insulin release in response to glucose + ouabain or in response to veratridine was also inhibited by trifluoperazine. It can be speculated that calmodulin is involved in the process by which glucose and potassium stimulate Ca2+ uptake, i.e., by activation of voltage-dependent Ca2+ channels in the plasma membrane. In addition, it appears that calmodulin is involved in the process by which glucose and veratridine act on stored calcium to raise the cytosolic Ca2+ concentration. Finally, trifluoperazine did not inhibit insulin release mediated by cyclic AMP (3-isobutyl-1-methyl-xanthine). Two conclusions may be drawn from this finding: (1) calmodulin may not be involved in the process of exocytosis per se and (2) cyclic AMP and glucose seem to mobilize stored calcium by different mechanisms.

Rapid Changes in Calcium Content of Rat Pancreatic Islets in Response to Glucose

To study whether glucose affects total islet Ca content, 45Ca content was measured in isolated pancreatic islets, loaded with 45Ca++, during up to 48 h in tissue culture conditions. In islets maintained in 2.8 mM or 8.3 mM glucose the 45Ca content did not increase further after 24 h, indicating that, at this time point, islets are in isotopie equilibrium. Islets maintained in 8.3 mM glucose had a significantly higher Ca content of 9 pmol/islet than the 5 pmol of islets maintained in 2.8 mM glucose. An increase in the glucose concentration from 2.8 mM to 8.3 mM after 24 h, in the continued presence of 45Ca++, caused a significant and rapid augmentation of the Ca content (+ 2.5 pmol/islet/5 min). Conversely, a decrease of the glucose concentration from 8.3 mM to 2.8 mM resulted in a rapid loss of cellular Ca. Thus, glucose rapidly affects the total Ca content of islets, the turnover of Ca amounting to approximately 10% of the total content per minute. Islets maintained in 8.3 mM glucose for 24 h exhibited a greater insulin release in response to 16.7 mM glucose than islets maintained for 24 h in 2.8 mM glucose. The increased response, however, was not correlative with the difference in Ca content.

Involvement of Ca2+ in the Impaired Glucose-induced Insulin Release from Islets Cultured at Low Glucose

Islet culture at a low glucose concentration results in a progressive impairment of glucose-induced insulin release. The role of Ca2+ in this defect was studied by comparing rat islets cultured for 6 days either at 8.3 mM (control) or 2.8 mM glucose. For measurement of 45Ca content and 45Ca2+ efflux, islets were kept in the presence of 45Ca2+ throughout. In islets cultured at 8.3 mM glucose, stimulation with 16.7 mM glucose during perifusion caused a typical biphasic pattern of insulin release paralleled by an increase in the rate of 45Ca2+ efflux. Both effects of glucose were markedly reduced in islets kept at 2.8 mM glucose, despite a similar insulin content. Islet 45Ca content was reduced. Both 45Ca content and insulin release were restored when islets were kept for an additional 24 h at 8.3 mM glucose. Insulin release induced by 3-isobutyl-1-methylxanthine (IBMX) or α-ketoisocaproic acid was not impaired, demonstrating that there is no generalized release defect. In contrast, glyceraldehyde- or K+-induced release was decreased. In islets maintained at 2.8 mM glucose, the stimulatory effect of glucose on Ca2+ uptake and the inhibitory effect on Ca2+ efflux (in the absence of Ca2+) were found to be operative. A defect may therefore lie distal to the Ca2+ uptake step involving either the mechanism by which glucose uses cellular Ca or another step yet to be identified.

Phenytoin Inhibition of Insulin Release: Studies on the Involvement of Ca2+ Fluxes in Rat Pancreatic Islets

The mechanism by which phenytoin inhibits insulin release was studied. Insulin release and 45Ca2+ efflux were measured during perifusion of collagenase isolated rat islets after 2-day maintenance in tissue culture in the presence of a trace amount of 45Ca2+. Islets maintained in the absence of the isotope were used to measure 45Ca2+ uptake over 5 min. Glucose (16.7 mM) induced a biphasic release of insulin, which was accompanied by a biphasic increase in the rate of 45Ca2+ efflux above basal. Phenytoin (80 μM) added during second phase rapidly inhibited insulin release. In contrast, phenytoin added together with glucose failed to affect first phase, but reduced second phase release by 64%. Phenytoin failed to affect basal 45Ca2+ efflux in the presence or absence of Ca2+, nor did it interfere with the inhibitory effect of high glucose on Ca2+ efflux. The drug did not affect basal Ca2+ uptake, but significantly reduced glucose-induced Ca2+ uptake. Glucose utilization was not inhibited by phenytoin. It is suggested that phenytoin inhibits glucose-stimulated insulin release by interfering with Ca2+ uptake via voltage-dependent Ca2+ channels. The pattern of inhibition of insulin release appears to favor this conclusion as the second phase is more dependent on the stimulation of Ca2+ uptake than is the first phase.

Glucose-induced first phase insulin release in the absence of extracellular Ca2+ in rat islets

Islets of Langerhans respond to a rapid and constant increase of the glucose concentration with biphasic release of insulin, a pattern that is observed in the portal vein in humans [l ] and in isolated pancreatic preparations [2-41. The presence of extracellular Ca” has been thought essential for both phases of insulin release [2,5,6]. However, recent studies have suggested that the first phase is dependent upon the utilization of cellular calcium, whereas the second phase depends both on cellular calcium and increased uptake of Ca2+ from the extracellular fluid [3,7], Those experiments were performed using either inhibitors of Ca2* uptake [3] or islets with increased calcium stores [7]. In islets with normal calcium stores, a preserved first phase insulin release in the absence of extracellular Ca2+ has not been demonstrated. This study was performed to examine the differential sensitivity of the two phases to Cap deprivation. It was found that first phase insulin release could occur in the absence of extracellular Ca2+, when Ca2* was removed from the medium before the glucose-induced rise of insulin release.