Virus-induced alterations in cyclic adenosine monophosphate generation in hamster islets of Langerhans.

Inoculation of golden Syrian hamsters with Venezuelan encephalitis (VE) virus results in a sustained diminution in glucose-stimulated insulin release that is correctable by cyclic (c) AMP analogs and phosphodiesterase inhibitors. This suggested the importance of directly measuring cAMP content in VE-infected and control islets in response to insulin secretagogues. The basal cAMP content of VE-infected islets (0.14 +/- 0.02 pmol/micrograms islet DNA) was approximately half that of control islets (0.27 +/- 0.02 pmol/micrograms islet DNA) (P less than 0.05). In the presence of 10 microM glucagon (and 3 mM glucose), the rate of cAMP generation in VE-infected islets was only half that of control islets. With 10 mM alpha-ketoisocaproic acid, the rates of cAMP generation were indistinguishable between control and experimental groups. In response to 20 mM glucose and 3-isobutyl-1-methylxanthine (IBMX) (a phosphodiesterase inhibitor), cAMP generation in VE-infected islets was 81% (NS) of the control rate. When a more specific phosphodiesterase inhibitor, RO 20-1724, was used with 20 mM glucose, cAMP generation in the infected islets was only 44% (P less than 0.001) of the control value. Insulin secretion over the perifusion period paralleled the cAMP levels. In the presence of 10 mM alpha-ketoisocaproic acid, there was no difference in insulin secretion between VE-infected and control islets, while there was a statistically significant (P less than 0.05) difference with 10 microM glucagon or 20 mM glucose (in 1 mM RO 20-1724). These data point to a defect in the cAMP generation system of VE-infected islets, although additional factors involved in insulin secretion may also be impaired by the virus.     

Tolbutamide perifusion of rat islets. Sequential changes in calcium, phosphorus, sodium, potassium, and chlorine in single beta cells.

Fluctuations of calcium, phosphorus, sodium, potassium, and chlorine in beta cells were followed during rat islet perifusion with tolbutamide and related to insulin secretion. In 24 paired experiments two chambers containing 100 islets were perifused with buffered medium containing 4.2 mM glucose alone or with added tolbutamide (200 micrograms/ml). Effluent was collected frequently for insulin determinations. At eight different time intervals from 0 to 20 min islets were acutely fixed, prepared for scanning electron microscopy and beta cells in islet tissue were identified. Element content in 480 single cells was measured by energy dispersive x-ray analysis. Tolbutamide elicited typical monophasic insulin release that exceeded control islet secretory rates from 2 to 6 min with a peak value at 3 min. This pattern was preceded by monophasic calcium accumulation in beta cells that abruptly rose 150% above control cells at 1 min and declined to base line by 4 min. The rapid ascent of calcium was associated with significant depressions of sodium and potassium content without alterations of cell phosphorus. Chlorine fell at 2 min and then rose greater than 50% above control cells at 4 min. After 6 min insulin secretion and element content remained near control levels. We conclude that monophasic calcium accumulation in beta cells is the earliest, most predictive event of islet insulin secretion after a tolbutamide stimulus. Oscillations of beta cell sodium and potassium reciprocally relate to calcium, and an elevation of chlorine content is a relatively late phenomenon in the stimulus-secretion coupling process.     

Maintenance of insulin release from pancreatic islets stored in the cold for up to 5 weeks.

Insulin content and release were measured from hand-dissected pancreatic islets from noninbred ob/ob mice after 1-5 wk storage in tissue culture medium 199 at various temperatures and glucose concentrations. After storage of islets for 1 wk at 37 degrees, 22 degrees, or 8 degrees C in 18 mM glucose medium and preincubation with 1 mM glucose, glucose-stimulated insulin release during the subsequent incubation was only 20-35% of that of fresh islets. The addition of a 4-h period at 37 degrees C with 18 mM glucose between the cold storage and perincubation restored glucose-stimulated insulin release from 8 degrees C stored islets to fresh-islet levels. Release throughout the 1-18 mM glucose range was strikingly parallel to that of fresh islets. Exposure of fresh islets to the same 4-h period increased basal release but did not affect maximal release. When islets were stored at 8 degrees C with 18 mM glucose for more than 1 wk, a short period at 37 degrees C every week was necessary for maintenance of release. After 5 wk of this procedure, glucose-stimulated insulin release was one-third that of fresh islets, or similar to that of islets stored for only 1 wk at 37 degrees C. Storage at 8 degrees C for 1 wk with 3 mM glucose, or continuously for 3 or 5 wk with 18 mM glucose, maintained islet insulin content, whereas release was lost. Thus, glucose-stimulated insulin release is best maintained by storage of pancreatic islets in tissue culture medium with a high concentration of glucose at 8 degrees C with short weekly periods at 37 degrees C.     

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.     

Increased islet apoptosis in Pdx1+/- mice

Mice with 50% Pdx1, a homeobox gene critical for pancreatic development, had worsening glucose tolerance with age and reduced insulin release in response to glucose, KCl, and arginine from the perfused pancreas. Surprisingly, insulin secretion in perifusion or static incubation experiments in response to glucose and other secretagogues was similar in islets isolated from Pdx1(+/-) mice compared with Pdx1(+/+) littermate controls. Glucose sensing and islet Ca(2+) responses were also normal. Depolarization-evoked exocytosis and Ca(2+) currents in single Pdx1(+/-) cells were not different from controls, arguing against a ubiquitous beta cell stimulus-secretion coupling defect. However, isolated Pdx1(+/-) islets and dispersed beta cells were significantly more susceptible to apoptosis at basal glucose concentrations than Pdx1(+/+) islets. Bcl(XL) and Bcl-2 expression were reduced in Pdx1(+/-) islets. In vivo, increased apoptosis was associated with abnormal islet architecture, positive TUNEL, active caspase-3, and lymphocyte infiltration. Although similar in young mice, both beta cell mass and islet number failed to increase with age and were approximately 50% less than controls by one year. These results suggest that an increase in apoptosis, with abnormal regulation of islet number and beta cell mass, represents a key mechanism whereby partial PDX1 deficiency leads to an organ-level defect in insulin secretion and diabetes.     

Effect of age on glucose-stimulated insulin release by the beta-cell of the rat.

To assess the effect of age on beta-cell insulin release, collagenase-isolated islets of Langerhans were obtained from rats aged 2--18 mo and incubated with increasing concentrations of glucose. Similar islets were analyzed for insulin content or subjected to morphometric measurements to identify both the number of beta-cells and the volume of beta-granules per islet. In parallel studies, the islet content of intact pancreata was also determined. The results showed that beta-cell number increased from 2,300 t0 5,000 cells as rats aged from 2 to 18 mo and islet insulin content doubled. However, glucose-stimulated insulin release decreased progressively with age, and this was especially striking when considered in terms of the increase in number of beta-cells/islet; e.g., mean (+/- SEM) insulin secretion (nanounits per minute per beta-cell) of islets incubated with 450 mg/dl of glucose was 1.3 (+/- 0.02), 1.0 (+/- 0.1), 0.4 (+/- 0.05), and 0.3 (+/- 0.01), respectively for 2-, 6-, 12-, and 18-mo-old rats. Thus, insulin secretion per beta-cell was decreased, despite increased stores of insulin per cell. These findings demonstrate that the aging process leads to a profound defect in glucose-stimulated insulin release from the beta-cell. Whether this is a global secretory defect, or solely a failure of the beta-cell to respond to glucose, remains to be defined.     

PKClambda regulates glucose-induced insulin secretion through modulation of gene expression in pancreatic beta cells

Altered regulation of insulin secretion by glucose is characteristic of individuals with type 2 diabetes mellitus, although the mechanisms that underlie this change remain unclear. We have now generated mice that lack the lambda isoform of PKC in pancreatic beta cells (betaPKClambda(-/-) mice) and show that these animals manifest impaired glucose tolerance and hypoinsulinemia. Furthermore, insulin secretion in response to high concentrations of glucose was impaired, whereas the basal rate of insulin release was increased, in islets isolated from betaPKClambda(-/-) mice. Neither the beta cell mass nor the islet insulin content of betaPKClambda(-/-) mice differed from that of control mice, however. The abundance of mRNAs for Glut2 and HNF3beta was reduced in islets of betaPKClambda(-/-) mice, and the expression of genes regulated by HNF3beta was also affected (that of Sur1 and Kir6.2 genes was reduced, whereas that of hexokinase 1 and hexokinase 2 genes was increased). Normalization of HNF3beta expression by infection of islets from betaPKClambda(-/-) mice with an adenoviral vector significantly reversed the defect in glucose-stimulated insulin secretion. These results indicate that PKClambda plays a prominent role in regulation of glucose-induced insulin secretion by modulating the expression of genes important for beta cell function.     

Direct glucocorticoid inhibition of insulin secretion. An in vitro study of dexamethasone effects in mouse islets.

The direct effects of glucocorticoids on pancreatic beta cell function were studied with normal mouse islets. Dexamethasone inhibited insulin secretion from cultured islets in a concentration-dependent manner: maximum of approximately 75% at 250 nM and IC50 at approximately 20 nM dexamethasone. This inhibition was of slow onset (0, 20, and 40% after 1, 2, and 3 h) and only slowly reversible. It was prevented by a blocker of nuclear glucocorticoid receptors, by pertussis toxin, by a phorbol ester, and by dibutyryl cAMP, but was unaffected by an increase in the fuel content of the culture medium. Dexamethasone treatment did not affect islet cAMP levels but slightly reduced inositol phosphate formation. After 18 h of culture with or without 1 microM dexamethasone, the islets were perifused and stimulated by a rise in the glucose concentration from 3 to 15 mM. Both phases of insulin secretion were similarly decreased in dexamethasone-treated islets as compared with control islets. This inhibition could not be ascribed to a lowering of insulin stores (higher in dexamethasone-treated islets), to an alteration of glucose metabolism (glucose oxidation and NAD(P)H changes were unaffected), or to a lesser rise of cytoplasmic Ca2+ in beta cells (only the frequency of the oscillations was modified). Dexamethasone also inhibited insulin secretion induced by arginine, tolbutamide, or high K+. In this case also the inhibition was observed despite a normal rise of cytoplasmic Ca2+. In conclusion, dexamethasone inhibits insulin secretion through a genomic action in beta cells that leads to a decrease in the efficacy of cytoplasmic Ca2+ on the exocytotic process.     

The Roles of Intracellular and Extracellular Ca++ in Glucose-Stimulated Biphasic Insulin Release by Rat Islets

Verapamil, an agent known rapidly to block calcium uptake into islets of Langerhans, has been used to study the roles of intra- and extracellular calcium in the two phases of glucose-induced insulin release. Rates of calcium uptake and insulin release during the first phase were measured simultaneously over 5 min in rat islets after maintenance in tissue culture for 2 days. Rates of (45)Ca(++) efflux and insulin release during the first and second phases were also measured simultaneously under perifusion conditions. For this, islets were loaded with (45)Ca(++) during the entire maintenance period to complete isotopic equilibrium. Under static incubation conditions 5 muM Verapamil had no effect upon Ca(++) uptake or insulin release in the presence of 2.8 mM glucose. By contrast, glucose-stimulated calcium influx was totally abolished without there being any significant effect upon first phase insulin release. Thus first phase insulin release is independent of increased uptake of extracellular calcium. The lack of effect of 5 muM Verapamil blockade on first phase insulin release was confirmed, under perifusion conditions, and was in marked contrast to the observed 55% inhibition of second phase release. (45)Ca(++) efflux was inhibited during both phases of the insulin release response.The results show that increased calcium uptake in response to glucose is not involved in the mechanism of first phase insulin release but is required for the full development and maintenance of the second phase release. It seems possible that intracellular calcium is the major regulatory control for first phase insulin release and that intracellular calcium and increased uptake of extracellular calcium contribute almost equally to the second phase of glucose-induced release.     

Novel mechanism of chronic exposure of oleic acid-induced insulin release impairment in rat pancreatic beta-cells

A sustained, high circulating level of free fatty acids (FFAs) is an important risk factor for the development of insulin resistance, islet beta-cell dysfunction, and pathogenesis of type 2 diabetes. Here, we report a novel mechanism of chronic exposure of oleic acid (OA)-induced rat insulin release impairment. Following a 4-day exposure to 0.1 mM OA, there was no significant difference in basal insulin release when comparing OA-treated and untreated islets in the presence of 2.8 mM glucose, whereas 16.7 mM glucose-stimulated insulin release increased 2-fold in control, but not in OA-treated, islets. Perforated patch-clamp recordings showed that untreated beta-cells exhibited a resting potential of -62.1 +/- 0.9 mV and were electrically silent, whereas OA-treated beta-cells showed more positive resting potentials and spontaneous action potential firing. Cell-attached single-channel recordings revealed spontaneous opening of ATP-sensitive potassium (K(ATP)) channels in control, but not in OA-treated, beta-cells. Inside-out excised patch recordings showed similar activity in both OA-treated and untreated beta-cells in the absence of ATP on the inside of the cellular membrane, whereas in the presence of ATP, K(ATP) channel activity was significantly reduced in OA-treated beta-cells. Electron microscopy demonstrated that chronic exposure to OA resulted in the accumulation of triglycerides in beta-cell cytoplasm and reduced both the number of insulin-containing granules and insulin content. Collectively, chronic exposure to OA closed K(ATP) channels by increasing the sensitivity of K(ATP) channels to ATP, which in turn led to the continuous excitation of beta-cells, depletion of insulin storage, and impairment of glucose-stimulated insulin release.     

Proinsulin-like component of circulating insulin in the basal state and in patients and hamsters with islet cell tumors

The proinsulin-like component comprised approximately 20% of total circulating basal immunoreactive insulin in 15 patients without islet cell tumors. 15 min after oral glucose, the concentration of the proinsulin-like component was unchanged and its percentage of the total immunoreactive insulin decreased with the acute release of the insulin component. By 2 hr after oral glucose, the concentration of the proinsulin-like component increased and the insulin component concentration decreased so that the percentage of the proinsulin-like component was essentially the same as in the basal state.In five patients with islet cell tumors and fasting hypoglycemia, basal proinsulin-like component ranged from 26 to 79% of the total immunoreactive insulin. While basal proinsulin-like component was higher in the islet cell tumor patients, the fluctuations after stimulation were qualitatively similar to the nontumor patients. Acute stimulation with glucose, tolbutamide, leucine, and streptozotocin mainly released the insulin component resulting in a fall in the per cent proinsulin-like component with a subsequent increase in percentage of this component as the total insulin concentration returns towards basal levels. Three islet-cell tumor patients with less than 46% proinsulin-like component had favorable therapeutic responses to diazoxide whereas one patient with over 80% proinsulin-like component was completely refractory.Syrian hamsters bearing islet cell tumors provided an excellent model for islet cell tumors in man. These animals have a high proportion of a proinsulin-like component in plasma; stimulation of tumor slices in vitro with tolbutamide and glucagon releases mainly the insulin component similar to the observations in man.These studies suggest that the mechanisms regulating the release of the proinsulin-like and of the insulin components are different.     

p-Hydroxymercuribenzoic acid inhibits arachidonic acid esterification into two distinct pools and stimulates insulin release in intact rat islets

Activation of an islet phospholipase A2 may contribute to glucose-induced insulin release. In order to simulate the accumulation of the resultant hydrolytic products (arachidonic acid, AA; its lipoxygenase-derived oxygenation product 12-hydroxyeicosatetraenoic acid; and lysophospholipids) without many of the other concomitants of beta cell activation, we studied the effects on intact rat islets of p-hydroxymercuribenzoic acid (PHMB), which inhibits the reacylation of lysophospholipids with AA in other cell types. PHMB inhibited in a dose-responsive fashion (-90% at 500 microM) the incorporation of [3H]AA into a "basal" pool or pools whose release and reuptake mechanisms appeared to be largely energy- and Ca++-independent (resistant to inhibition by mannoheptulose, antimycin A or CoCl2); reciprocally, islets prelabeled with [3H]AA accumulated an increased amount of [3H]-12-hydroxyeicosatetraenoic acid (twice basal at 200 microM PHMB and three times basal at 500 microM) when reacylation of any [3H]AA released basally at 1.7 mM glucose was inhibited. PHMB also blocked (by up to 99% at 500 microM) the incorporation of [3H]AA into a functionally defined, glucose-stimulated compartment of fatty acid (tightly coupled to the islet 12-lipoxygenase) whose release and reuptake required metabolic energy and Ca++. It was also demonstrated that PHMB inhibited the esterification of [3H]AA (at low or high glucose concentrations) into specific phospholipids in islet membranes. In parallel with these alterations in lipid metabolism, PHMB caused rapid, potent and reversible increments in insulin release with a threshold concentration (about 25 microM) identical to that inhibiting AA fluxes. PHMB both initiated release (at 1.7 mM glucose) and potentiated the effects of islet fuels (16.7 mM glucose or 15 mM alpha-ketoisocaproic acid). Thus, pharmacologic manipulation of the AA reuptake mechanism is a new approach to unmask potential roles in insulin release of phospholipid hydrolysis products from different lipid pools and in the absence or presence of phospholipase A2 activation.     

Pulsatile insulin release from pancreatic islets with nonoscillatory elevation of cytoplasmic Ca2+.

The relationship between insulin release and cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in isolated pancreatic islets from ob/ob mice. Although [Ca2+]i was low and stable in the presence of 3 mM glucose, basal insulin release exhibited low amplitude pulsatility, with a frequency of 0.32 +/- 0.04 min-1. Depolarization by raising K+ from 5.9 to 30.9 mM or by the addition of 1 mM tolbutamide caused a pronounced initial insulin pulse followed by declining pulses, but there was no change in frequency. This decline in amplitude of the insulin pulses was prevented in similar experiments performed in the presence of 11 mM glucose. Corresponding measurements of [Ca2+]i in islets exposed to tolbutamide or the high K+ concentration revealed stable elevations without oscillations. Although the [Ca2+]i level is an important determinant for the rate of secretion, the results indicate that pulsatile insulin release does not always depend on [Ca2+]i oscillations. It is suggested that cyclic generation of ATP may fuel pulsatile release under conditions when [Ca2+]i remains stable.     

Synthesis and release of proinsulin and insulin by isolated rat islets of Langerhans

Isolated rat islets of Langerhans were incubated for 60, 120, and 180 min and the incorporation of leucine-(3)H into proinsulin and insulin moieties was followed. Synthesis and release of these hormones could be followed by separate extractions of islets and incubation media.RELEASE OF NEWLY SYNTHESIZED PROINSULIN AND INSULIN OCCURRED UNDER THE FOLLOWING CONDITIONS: (a) incubation for greater than 60 min; (b) glucose concentrations above 5.3 mmoles/liter; (c) incubation with 5 mM dibutyryl cyclic AMP or theophylline in 5.3 mM glucose (potentiated by 16 mM glucose); and (d) incubation with 5 mM tolbutamide and 16 mM glucose.Synthesis of proinsulin and insulin was enhanced by time of incubation, high glucose concentrations, by dibutyryl cyclic AMP or theophylline, and by tolbutamide only at 16 mM glucose. Synthesis was totally inhibited by tolbutamide at 5.3 mM glucose.     

Prolonged exposure of human pancreatic islets to high glucose concentrations in vitro impairs the beta-cell function.

The aim of the present study was to clarify whether prolonged in vitro exposure of human pancreatic islets to high glucose concentrations impairs the function of these cells. For this purpose, islets isolated from adult cadaveric organ donors were cultured for seven days in RPMI 1640 medium supplemented with 10% fetal calf serum and containing either 5.6, 11, or 28 mM glucose. There was no glucose-induced decrease in islet DNA content or signs of morphological damage. However, islets cultured at 11 or 28 mM glucose showed a 45 or 60% decrease in insulin content, as compared to islets cultured at 5.6 mM glucose. Moreover, when such islets were submitted to a 60-min stimulation with a low (1.7 mM) followed by a high (16.7 mM) concentration of glucose, the islets cultured at 5.6 mM glucose showed a higher insulin response to glucose than those of the two other groups. Islets cultured at the two higher glucose concentrations showed increased rates of insulin release in the presence of low glucose, and a failure to enhance further the release in response to an elevated glucose level. Islets cultured at 28 mM glucose showed an absolute decrease in insulin release after stimulation with 16.7 mM glucose, as compared to islets cultured at 5.6 mM glucose. The rates of glucose oxidation, proinsulin biosynthesis, and total protein biosynthesis were similar in islets cultured at 5.6 or 11 mM glucose, but they were decreased in islets cultured at 28 mM glucose. These combined results suggest that lasting exposure to high glucose concentrations impairs the function of human pancreatic islets.     

Gluco-incretins control insulin secretion at multiple levels as revealed in mice lacking GLP-1 and GIP receptors

The role of the gluco-incretin hormones GIP and GLP-1 in the control of beta cell function was studied by analyzing mice with inactivation of each of these hormone receptor genes, or both. Our results demonstrate that glucose intolerance was additively increased during oral glucose absorption when both receptors were inactivated. After intraperitoneal injections, glucose intolerance was more severe in double- as compared to single-receptor KO mice, and euglycemic clamps revealed normal insulin sensitivity, suggesting a defect in insulin secretion. When assessed in vivo or in perfused pancreas, insulin secretion showed a lack of first phase in Glp-1R(-/-) but not in Gipr(-/-) mice. In perifusion experiments, however, first-phase insulin secretion was present in both types of islets. In double-KO islets, kinetics of insulin secretion was normal, but its amplitude was reduced by about 50% because of a defect distal to plasma membrane depolarization. Thus, gluco-incretin hormones control insulin secretion (a) by an acute insulinotropic effect on beta cells after oral glucose absorption (b) through the regulation, by GLP-1, of in vivo first-phase insulin secretion, probably by an action on extra-islet glucose sensors, and (c) by preserving the function of the secretory pathway, as evidenced by a beta cell autonomous secretion defect when both receptors are inactivated.     

Transgenic mice overexpressing insulin-like growth factor-II in beta cells develop type 2 diabetes

During embryonic development, insulin-like growth factor-II (IGF-II) participates in the regulation of islet growth and differentiation. We generated transgenic mice (C57BL6/SJL) expressing IGF-II in beta cells under control of the rat Insulin I promoter in order to study the role of islet hyperplasia and hyperinsulinemia in the development of type 2 diabetes. In contrast to islets from control mice, islets from transgenic mice displayed high levels of IGF-II mRNA and protein. Pancreases from transgenic mice showed an increase in beta-cell mass (about 3-fold) and in insulin mRNA levels. However, the organization of cells within transgenic islets was disrupted, with glucagon-producing cells randomly distributed throughout the core. We also observed enhanced glucose-stimulated insulin secretion and glucose utilization in islets from transgenic mice. These mice displayed hyperinsulinemia, mild hyperglycemia, and altered glucose and insulin tolerance tests, and about 30% of these animals developed overt diabetes when fed a high-fat diet. Furthermore, transgenic mice obtained from the N1 backcross to C57KsJ mice showed high islet hyperplasia and insulin resistance, but they also developed fatty liver and obesity. These results indicate that local overexpression of IGF-II in islets might lead to type 2 diabetes and that islet hyperplasia and hypersecretion of insulin might occur early in the pathogenesis of this disease.     

Glucose-Stimulated 45Calcium Efflux from Isolated Rat Pancreatic Islets

Kinetics of (45)Ca efflux and insulin release were studied in collagenase-isolated rat islets during 2-h perifusions with calcium-depleted (0.05 mM) bicarbonate-phosphate buffer containing 2.2 mM glucose. Addition of glucose (16.7 mM) suppressed (45)Ca efflux by 30%. Removal of glucose caused an "off response" of insulin release. The perifusion of a normal concentration of Ca (2.3 mM) greatly stimulated (45)Ca efflux, indicating Ca <--> (45)Ca exchange. When Ca and glucose were superimposed, the effects on (45)Ca efflux and insulin release depended upon the order of presentation of the stimuli: when Ca was added to an ongoing 16.7-mM glucose perifusion, biphasic patterns of (45)Ca and insulin release were seen; when glucose was superimposed on a Ca perifusion, an inhibition of the Ca-stimulated (45)Ca efflux occurred, and a reduced but clearly biphasic insulin response was seen. The subsequent insulin off response after with-drawal of the glucose was also reduced.Mathematical "peeling" of (45)Ca efflux curves from unstimulated islets suggests that there are at least two, and probably three, different intracellular Ca compartments (not including the extracellular sucrose space). At the beginning of perifusion, these three compartments (I, II, III) contain 25, 56, and 19% of the intracellular (45)Ca, and their rates of efflux are 6.7, 1.2, and 0.1%/min, respectively. Glucose appears to suppress efflux from the largest compartment (II); Ca appears to exchange with (45)Ca from a more inert compartment (III). The relationship between insulin and (45)Ca release is not stoichiometric.     

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.