Different insulin-secretory responses to calcium-channel blockers in islets of lean and obese (ob/ob) mice.

The purpose of these experiments was to determine whether the activity of the voltage-dependent Ca2+ channel was modulated in the same manner in islets of the ob/ob mouse as in islets of homozygous lean mice of the same strain. The effect of agents that are known to alter the concentrations and movements of intracellular Ca2+ were investigated in relation to glucose-stimulated insulin secretion and in relation to the effect of forskolin. In islets of obese mice, verapamil and nifedipine both inhibited glucose-induced insulin release, nifedipine being the more potent inhibitor. Forskolin-stimulated secretion was inhibited either not at all (verapamil) or much less (nifedipine) in islets of the ob/ob mouse compared with those of lean mice. At basal glucose concentrations, verapamil initiated insulin secretion in islets of the ob/ob mouse and acted synergistically with forskolin to evoke a secretory activity that was 3-fold greater than that evoked by 20 mM-glucose. Nifedipine also initiated secretion at basal glucose concentrations and acted synergistically with forskolin, but its effect was considerably smaller than that of verapamil. A comparison of the effect of forskolin in the presence of Ca2+-channel blockers and in the absence of Ca2+ suggests that, in the obese mouse, the operation of the voltage-dependent Ca2+ channel is impaired.     

Effects of dehydrouramil on protein phosphorylation and insulin secretion in rat islets of Langerhans.

Dehydrouramil hydrate hydrochloride (DHU), a stable analogue of alloxan, inhibited the phosphorylation of an endogenous protein of Mr 53,000 catalysed by a Ca2+-calmodulin-dependent protein kinase in extracts of islets of Langerhans. The concentration of DHU required for 50% inhibition was 0.09 mM. DHU did not inhibit islet cyclic AMP-dependent protein kinase and caused only slight inhibition of Ca2+-phospholipid-dependent protein kinase. Inhibition of Ca2+-calmodulin-dependent protein kinase was neither prevented nor reversed by dithiothreitol. DHU did not affect the ability of calmodulin to activate cyclic AMP phosphodiesterase. In intact islets, pre-exposure to DHU impaired the insulin-secretory response to glucose and blocked the potentiatory effect on insulin secretion of forskolin, an activator of adenylate cyclase, and of tetradecanoylphorbol acetate (TPA), an activator of Ca2+-phospholipid-dependent protein kinase. The increase in islet cyclic AMP elicited by forskolin was not affected by DHU. The data are consistent with the hypothesis that protein phosphorylation catalysed by a Ca2+-calmodulin-dependent protein kinase may play a central role in the regulation of insulin secretion.     

Glycogen metabolism and cyclic AMP levels in isolated islets of lean and genetically obese mice.

The levels of glycogen and cyclic AMP, incorporation of glucose into glycogen and activities of glycogen synthetase and phosphorylase were determined in pancreatic islets isolated from genetically obese mice and their lean litter-mates. Islets from obese mice had elevated glycogen levels, increased phosphorylase activity and an increased amount of glycogen synthetase in the physiologically more effective I-form, indicating an increased turnover of glycogen. There was no significant difference in cyclic AMP levels between islets of lean and obese mice, but inhibition of phosphodiesterase or stimulation of adenyl cyclase increased cyclic AMP levels more in obese than in lean mouse islets, indicating a more rapid turnover of cyclic AMP in the former. It is suggested that cyclic AMP stimulated phosphorolytic breakdown of glycogen may be one of the mechanisms responsible for the increased insulin secretory response to glucose observed in islets from genetically obese mice.     

The influence of K(+)-induced membrane depolarization on insulin secretion in islets of lean and obese (ob/ob) mice

The present study was undertaken to determine whether factors that affect K+ permeability produce differences in insulin secretion in the islets of obese versus lean mice. At basal glucose (3 mM), the obese islets secreted more insulin for a given increment in depolarizing K+ concentration and responded to a wider range of K+ concentrations (5-45 mM) than the lean islets (5-25 mM). In contrast, the membrane potential changes induced by increments in pK+ were not significantly different in the two types of islets. The islets of lean and obese mice treated with pertussis toxin showed a qualitatively similar response to glucose and to epinephrine, but only the control and pertussis toxin treated obese islets responded to K+ depolarization when deprived of calcium. Abnormal responses to quinine and apamin were identified in the islets of obese mice. These findings show that the abnormal insulin secretory response of the obese islet is due, at least in part, to a defect independent of glucose metabolism. This is best explained by an altered sensitivity of voltage-dependent events, most likely the result of differential effects of an intracellular element acting on ATP-sensitive and Ca2(+)-activated K+ channels, both of which are implicated in membrane repolarization.     

Regulation of insulin release from isolated islets of Langerhans of the rat in pregnancy. The role of adenosine 3':5'-cyclic monophosphate

1. The concentrations of cyclic AMP were compared in islets of Langerhans isolated from the pancreases of normal female and pregnant rats and were higher in islets in pregnancy. 2. There was also a significant increase in adenylate cyclase activity in homogenates of islets from pregnant rats compared with those from normal rats. 3. Increased cyclic AMP concentration in islets from pregnant rats was reflected in increased protein kinase activity. When the cyclic AMP-dependent protein kinase activity was increased by 3-isobutyl-1-methylxanthine this stimulated activity was significantly greater in pregnancy. 4. Insulin-secretion studies with islets from normal and pregnant rats showed that theophylline or 3-isobutyl-1-methylxanthine, which raise intracellular cyclic AMP concentrations, caused a significantly greater insulin secretion in pregnancy. 5. It was also found that in the presence of a glucose concentration too low to stimulate insulin secretion, the latter could be induced if the cyclic AMP concentrations were raised sufficiently with 3-isobutyl-1-methylxanthine. 6. It is suggested that the higher cyclic AMP concentrations observed in islets in pregnancy mediate the greater insulin-secretory capacity, as well as the greater sensitivity of these islets to low glucose concentrations.     

The effect of calcium on somatostatin inhibition of insulin release and cyclic AMP production in mouse pancreatic islets.

The effect of somatostatin on glucose-induced insulin secretion and cyclic AMP accumulation in isolated islets from obese, hyperglycemic ob/ob mice was studied in a microperifusion system. The normal biphasic pattern of insulin release as well as the inhibitory pattern of insulin release produced by somatostatin (0.5--1 microgram/ml) was matched by similar changes in the intracellular concentration of cyclic AMP. When islets were stimulated by glucose (3 mg/ml) plus 3-isobutyl-1-methylxanthine (0.1 mM), somatostatin (0.5 microgram/ml) failed to inhibit insulin secretion or cyclic AMP formation in the second phase whereas in the first phase both parameters were significantly reduced by somatostatin (0.5 microgram/ml). In batch-type incubations it was shown that addition of excess calcium (to 6 mM) reversed this inhibition. In the second phase calcium potentiated the (glucose + 3-isobutyl-1-methylxanthine)-stimulated insulin secretion without affecting the cyclic AMP production. This potentiation was inhibited by somatostatin (0.1 microgram/ml). Somatostatin (1 microgram/ml) inhibited adenylate cyclase activity in islet homogenates. No effect of somatostatin on islet glucose utilization could be demonstrated. The results indicate a dual action of somatostatin in the inhibition of insulin release, one involving the islet adenylate cyclase and one affecting the islet uptake of calcium.     

Inosine-stimulated insulin release and metabolism of inosine in isolated mouse pancreatic islets.

Inosine is a potent primary stimulus of insulin secretion from isolated mouse islets. The inosine-induced insulin secretion was totally depressed during starvation, but was completely restored by the addition of 5 mM-caffeine to the medium and partially restored by the addition of 5 mM-glucose. Mannoheptulose (3 mg/ml) potentiated the effect of 10 mM-inosine in islets from fed mice. The mechanism of the stimulatory effect of inosine was further investigated, and it was demonstrated that pancreatic islets contain a nucleoside phosphorylase capable of converting inosine into hypoxanthine and ribose 1-phosphate. Inosine at 10 mM concentration increased the lactate production and the content of ATP, glucose 6-phosphate (fructose 1,6-diphosphate + triose phosphates) and cyclic AMP in islets from fed mice. In islets from starved mice inosine-induced lactate production was decreased and no change in the concentration of cyclic AMP could be demonstrated, whereas the concentration of ATP and glucose 6-phosphate rose. Inosine (10 mM) induced a higher concentration of (fructose 1,6-diphosphate + triose phosphates) in islets from starved mice than in islets from fed mice suggesting that in starvation the activities of glyceraldehyde 3-phosphate dehydrogenase or other enzymes below this step in glycolysis are decreased. Formation of glucose from inosine was negligible. Inosine had no direct effect on adenylate cyclase activity in islet homogenates. The observed changes in insulin secretion and islet metabolism mimic what is seen when glucose and glyceraldehyde stimulate insulin secretion, and as neither ribose nor hypoxanthine-stimulated insulin release, the results are interpreted as supporting the substrate-site hypothesis for glucose-induced insulin secretion according to which glucose has to be metabolized in the beta-cells before secretion is initiated.     

The effect of calcium on somatostatin inhibition of insulin release and cyclic AMP production in mouse pancreatic islets

The effect of somatostatin on glucose-induced insulin secretion and cyclic AMP accumation in isolated islets from obese, hyperglycemic ob/ob mice was studied in a microperifusion system. The normal biphasic pattern of insulin release as well as the inhibitory pattern of insulin release produced by somatostatin (0.5–1 μg/ml) was matched by similar changes in the intracellular concentration of cyclic AMP. When islets were stimulated by glucose (3 mg/ml) plus 3-isobutyl-1-methylxanthine (0.1 mM), somatostatin (0.5 μg/ml) failed to inhibit insulin secretion or cyclic AMP formation in the second phase whereas in the first phase both parameters were significantly reduced by somatostatin (0.5 μg/ml). In batch-type incubations it was shown that addition of excess calcium (to 6 mM) reversed this inhibition. In the second phase calcium potentiated the (glucose + 3-isobutyl-1-methylxanthine)-stimulated insulin secretion without affecting the cyclic AMP production. This potentiation was inhibited by somatostatin (0.1 μg/ml). Somatostatin (1 μg/ml) inhibited adenylate cyclase activity in islet homogenates. No effect of somatostatin on islet glucose utilization could be demonstrated.The results indicate a dual action of somatostatin in the inhibition of insulin release, one involving the islet adenylate cyclase and one affecting the islet uptake of calcium.     

Factors influencing insulin and glucagon secretion in lean and genetically obese mice.

The control of insulin and glucagon secretion from isolated pancreatic islets of lean and genetically obese mice has been compared. The enlarged islets of obese mouse pancreas and islets of obese mouse pancreas and islets of obese mice maintained on a restricted diet manifested a greater response to glucose stimulation of insulin secretion than the lean mice islets. The glucagon content of the islets, the secretion of glucagon in a medium containing 150 mg% glucose and the stimulation of glucagon secretion by arginine did not differ significantly in the two groups. Adrenaline stimulated glucagon secretion in vitro from obese mice but not from lean mice. Antinsulin serum injections into obese mice increased the plasma glucagon levels about twofold and had no effect on glucagon levels in lean mice, although the level of hyperglycaemia was the same in both groups. It is suggested that the suppression of glucagon release by glucose requires a higher concentration of insulin in the obese mouse pancreas than in lean mice.     

Inhibition of Ca2+-independent phospholipase A2 results in insufficient insulin secretion and impaired glucose tolerance

Islet Ca2+-independent phospholipase A2 (iPLA2) is postulated to mediate insulin secretion by releasing arachidonic acid in response to insulin secretagogues. However, the significance of iPLA2 signaling in insulin secretion in vivo remains unexplored. Here we investigated the physiological role of iPLA2 in beta-cell lines, isolated islets, and mice. We showed that small interfering RNA-specific silencing of iPLA2 expression in INS-1 cells significantly reduced insulin-secretory responses of INS-1 cells to glucose. Immunohistochemical analysis revealed that mouse islet cells expressed significantly higher levels of iPLA2 than pancreatic exocrine acinar cells. Bromoenol lactone (BEL), a selective inhibitor of iPLA2, inhibited glucose-stimulated insulin secretion from isolated mouse islets; this inhibition was overcome by exogenous arachidonic acid. We also showed that iv BEL administration to mice resulted in sustained hyperglycemia and reduced insulin levels during glucose tolerance tests. Clamp experiments demonstrated that the impaired glucose tolerance was due to insufficient insulin secretion rather than decreased insulin sensitivity. Short-term administration of BEL to mice had no effect on fasting glucose levels and caused no apparent pathological changes of islets in pancreas sections. These results unambiguously demonstrate that iPLA2 signaling plays an important role in glucose-stimulated insulin secretion under physiological conditions.     

Dibutyryl cyclic AMP triggers Ca2+ influx and Ca2+-dependent electrical activity in pancreatic B cells

In the presence of 7 mM glucose, dibutyryl cyclic AMP induced electrical activity in otherwise silent mouse pancreatic B cells. This activity was blocked by cobalt or D600, two inhibitors of Ca2+ influx. Under similar conditions, dibutyryl cyclic AMP stimulated 45Ca2+ influx (5-min uptake) in islet cells; this effect was abolished by cobalt and partially inhibited by D600. The nucleotide also accelerated 86Rb+ efflux from preloaded islets, did not modify glucose utilization and markedly increased insulin release. Its effects on release were inhibited by cobalt, but not by D600. These results show that insulin release can occur without electrical activity in B cells and suggest that cyclic AMP not only mobilizes intracellular Ca, but also facilitates Ca2+ influx in insulin secreting cells.     

Augmented Insulinotropic Action of Arachidonic Acid through the Lipoxygenase Pathway in the Obese Zucker Rat

Objective: The metabolism of arachidonic acid (AA) has been shown to be altered in severe insulin resistance that is present in obese (fa/fa) Zucker rats. We examined the effects and mechanism of action of AA on basal and glucose‐stimulated insulin secretion in pancreatic islets isolated from obese (fa/fa) Zucker rats and their homozygous lean (Fa/Fa) littermates. Research Methods and Procedures: Islets were isolated from 10‐ to 12‐week‐old rats and incubated for 45 minutes in glucose concentrations ranging from 3.3 to 16.7 mM with or without inhibitors of the cyclooxygenase or lipoxygenase pathways. Medium insulin concentrations were measured by radioimmunoassay, and islet production of the 12‐lipoxygenase metabolite, 12‐hydroxyeicosatetraenoic acid (12‐HETE), was measured by enzyme immunoassay. Results: In islets from lean animals, AA stimulated insulin secretion at submaximally stimulatory glucose levels (< 11.1 mM) but not at 16.7 mM glucose. In contrast, in islets derived from obese rats, AA potentiated insulin secretion at all glucose concentrations. AA‐induced insulin secretion was augmented in islets from obese compared with lean rats at high concentrations of AA in the presence of 3.3 mM glucose. Furthermore, the inhibitor of 12‐lipoxygenase, esculetin (0.5 μM), inhibited AA‐stimulated insulin secretion in islets from obese but not lean rats. Finally, the islet production of the 12‐HETE was markedly enhanced in islets from obese rats, both in response to 16.7 mM glucose and to AA. Discussion: The insulin secretory response to AA is augmented in islets from obese Zucker rats by a mechanism related to enhanced activity of the 12‐lipoxygenase pathway. Therefore, augmented action of AA may be a mechanism underlying the adaptation of insulin secretion to the increased demand caused by insulin resistance in these animals.     

Glucose homeostasis, insulin secretion, and islet phospholipids in mice that overexpress iPLA2beta in pancreatic beta-cells and in iPLA2beta-null mice

Studies with genetically modified insulinoma cells suggest that group VIA phospholipase A(2) (iPLA(2)beta) participates in amplifying glucose-induced insulin secretion. INS-1 insulinoma cells that overexpress iPLA(2)beta, for example, exhibit amplified insulin-secretory responses to glucose and cAMP-elevating agents. To determine whether similar effects occur in whole animals, we prepared transgenic (TG) mice in which the rat insulin 1 promoter (RIP) drives iPLA(2)beta overexpression, and two characterized TG mouse lines exhibit similar phenotypes. Their pancreatic islet iPLA(2)beta expression is increased severalfold, as reflected by quantitative PCR of iPLA(2)beta mRNA, immunoblotting of iPLA(2)beta protein, and iPLA(2)beta enzymatic activity. Immunofluorescence microscopic studies of pancreatic sections confirm iPLA(2)beta overexpression in RIP-iPLA(2)beta-TG islet beta-cells without obviously perturbed islet morphology. Male RIP-iPLA(2)beta-TG mice exhibit lower blood glucose and higher plasma insulin concentrations than wild-type (WT) mice when fasting and develop lower blood glucose levels in glucose tolerance tests, but WT and TG blood glucose levels do not differ in insulin tolerance tests. Islets from male RIP-iPLA(2)beta-TG mice exhibit greater amplification of glucose-induced insulin secretion by a cAMP-elevating agent than WT islets. In contrast, islets from male iPLA(2)beta-null mice exhibit blunted insulin secretion, and those mice have impaired glucose tolerance. Arachidonate incorporation into and the phospholipid composition of RIP-iPLA(2)beta-TG islets are normal, but they exhibit reduced Kv2.1 delayed rectifier current and prolonged glucose-induced action potentials and elevations of cytosolic Ca(2+) concentration that suggest a molecular mechanism for the physiological role of iPLA(2)beta to amplify insulin secretion.     

Polyamines in pancreatic islets of obese-hyperglycemic (ob/ob) mice of different ages

To further evaluatethe role of polyamines in insulin production and cell replication indiabetic pancreatic islets, we have studied hyperplastic islets ofobese-hyperglycemic mice of different ages and normal islets of thesame strain. The aims of the study were to investigate the impact ofthe diabetic state and aging on polyamine contents and requirements inthese islets. Cultured islets from lean and obese animals containedsignificantly less polyamines than freshly isolated islets.Spermine-to-spermidine ratio was elevated in freshly isolated isletsfrom young obese mice compared with those from lean mice. In isletsfrom old obese animals, spermidine content was decreased, whereas thecontent of spermine was not different from that of young obese mice.The physiological significance of polyamines was investigated byexposing islets in tissue culture to inhibitors of polyamine synthesis. This treatment caused a partial polyamine depletion in whole islets butfailed to affect polyamine content of cell nuclei. Insulin content wasnot affected in polyamine-deficient islets of obese mice, irrespectiveof age, in contrast to decreased islet insulin content inpolyamine-depleted young lean animals. Polyamine depletion depressedDNA synthesis rate in obese mouse islets; in lean mice it actuallystimulated DNA synthesis. We concluded that important qualitative andquantitative differences exist between islets from obese-hyperglycemicand normal mice with respect to polyamine content and requirements ofpolyamines for regulation of insulin content and cell proliferation.The results suggest that spermine may be involved in mediating therapid islet cell proliferation noted early in obese-hyperglycemicsyndrome, but changes in spermine concentration do not seem to accountfor the decline in islet cell DNA synthesis in aged normoglycemic animals.

     

The effect of starvation on phosphodiesterase activity and the content of adenosine 3′:5′-cyclic monophosphate in isolated mouse pancreatic islets

1. The concentration of cyclic AMP and the activity of phosphodiesterase were measured in isolated pancreatic islets from fed or 48h-starved mice. 2. Two different phosphodiesterases were detected. Neither the maximum activity nor the K(m) values of these enzymes were changed by starvation. 3. The concentration of cyclic AMP in non-incubated islets was the same in islets from fed and starved mice. 4. Incubation with 3.3mm-glucose for 5-30min had no effect on the concentration of cyclic AMP, irrespective of the nutritional state of the mice. Incubation with 16.7mm-glucose for 5-30min raised the concentration of cyclic AMP by about 30% in islets from fed mice. This rise was prevented by addition of mannoheptulose (3mg/ml). Incubation with 16.7mm-glucose had no effect on the cyclic AMP content in islets from starved mice. 5. In islets from fed mice 10min incubation with 5mm-caffeine had no effect on the concentration of cyclic AMP in the presence of 3.3 or 16.7mm-glucose, whereas the cyclic AMP content was increased approx. 150% in islets from starved mice. 6. After 10min incubation with 1mm-3-isobutyl-1-methylxanthine in the presence of 3.3 or 16.7mm-glucose the concentration of cyclic AMP was raised by 250% in islets from fed mice and by 400% in islets from starved mice. 7. A threefold function of glucose in the insulin-secretory process is suggested, according to which the decreased islet glucose metabolism is the primary defect in the insulin-secretory mechanism during starvation.     

Abnormally decreased NO and augmented CO production in islets of the leptin-deficient ob/ob mouse might contribute to explain hyperinsulinemia and islet survival in leptin-resistant type 2 obese diabetes

The role of the gaseous messengers NO and CO for β-cell function and survival is controversial. We examined this issue in the hyperglycemic-hyperinsulinemic ob/ob mouse, an animal model of type 2 obese diabetes, by studying islets from obese vs lean mice regarding glucose-stimulated insulin release in relation to islet NO and CO production and the influence of modulating peptide hormones. Glucose-stimulated increase in ncNOS-activity in incubated lean islets was converted to a decrease in ob/ob islets associated with markedly increased insulin release. Both types of islets displayed iNOS activity appearing after ~60 min in high-glucose. In ob/ob islets the insulinotropic peptides glucagon, GLP-1 and GIP suppressed NOS activities and amplified glucose-stimulated insulin release. The insulinostatic peptide leptin induced the opposite effects. Suppression of islet CO production inhibited, while stimulation amplified glucose-stimulated insulin release. Nonincubated isolated islets from young and adult obese mice displayed very low ncNOS and negligible iNOS activity. In contrast, production of CO, a NOS inhibitor, was impressively raised. Glucose injections induced strong activities of islet NOS isoforms in lean but not in obese mice and confocal microscopy revealed iNOS expression only in lean islets. Islets from ob/ob mice existing in a hyperglycemic in vivo milieu maintain elevated insulin secretion and protection from glucotoxicity through a general suppression of islet NOS activities achieved by leptin deficiency, high CO production and insulinotropic cyclic-AMP-generating hormones. Such a beneficial effect on islet function and survival might have its clinical counterpart in human leptin-resistant type 2 obese diabetes with hyperinsulinemia.     

Long-term treatment with atrial natriuretic peptide inhibits ATP production and insulin secretion in rat pancreatic islets

Atrial natriuretic peptide (ANP) levels correlate with hyperglycemia in diabetes mellitus, but ANP effects on pancreatic islet β-cell insulin secretion are controversial. ANP was investigated for short- and long-term effects on insulin secretion and mechanisms regulating secretion in isolated rat pancreatic islets. A 3-h incubation with ANP did not affect basal or glucose-stimulated islet insulin secretion. However, 7-day culture of islets with 5.5 mM glucose and ANP (1 nM - 1 μM) markedly inhibited subsequent glucose (11 mM)-stimulated insulin secretion; total islet insulin content was not affected. Following ANP removal for 24 h, the islet insulin-secretory response to glucose was restored. The insulin-secretory response to other insulin secretagogues, including α-ketoisocaproic acid, forskolin, potassium chloride, and ionomycin were also markedly inhibited by chronic exposure to ANP. However, the combination of potassium chloride and α-ketoisocaproic acid was sufficient to overcome the inhibitory effects of ANP on insulin secretion. The glucose-stimulated increases in islet ATP levels and the ATP/ADP ratio were completely inhibited in ANP 7-day-treated islets vs. control; removal of ANP for 24 h partially restored the glucose response. ANP did not affect islet glycolysis. ANP significantly increased levels of islet activated hormone-sensitive lipase and the expression of uncoupling protein-2 and peroxisome proliferator-activated receptor-δ and -α. Although islet ANP-binding natriuretic peptide receptor-A levels were reduced to 60% of control after 7-day culture with ANP, the ANP-stimulated cGMP levels remained similar to control islet levels. Thus, long-term exposure to ANP inhibits glucose-stimulated insulin secretion and ATP generation in isolated islets.     

Islet cyclic AMP levels are not lowered during alpha 2-adrenergic inhibition of insulin release

Epinephrine-induced changes in insulin release and cyclic AMP levels were measured simultaneously in isolated rat islets. Forskolin was used to enhance islet cyclic AMP levels. Forskolin (30 microM) stimulated adenylate cyclase activity 10-fold in islet homogenates and raised cyclic AMP levels 5-fold in intact islets (both at low and high glucose). Insulin release was enhanced by forskolin only at high glucose. Epinephrine (0.1 microM) inhibited glucose- and forskolin-induced insulin release to basal rates. At the same time epinephrine potentiated forskolin-elevated cyclic AMP levels. In contrast epinephrine attenuated forskolin-stimulated adenylate cyclase activity in islet homogenates. At low glucose, both alpha 2- and beta-adrenergic blockade counteracted the epinephrine potentiation, each by 50%. At high glucose the effect was mainly beta-adrenergic in nature. The actions of epinephrine in the presence of a beta-blocker were mimicked by the alpha 2-agonist clonidine. Despite the variations in cyclic AMP levels stimulated insulin release was always inhibited by activation of alpha 2-receptors. Finally, insulin release stimulated by exogenous cyclic AMP was abolished by epinephrine. These results suggest that epinephrine inhibits insulin release at a step distal to the generation of cyclic AMP.     

Effects of adenosine A(1) receptor antagonism on insulin secretion from rat pancreatic islets

Adenosine is known to influence different kinds of cells, including beta-cells of the pancreas. However, the role of this nucleoside in the regulation of insulin secretion is not fully elucidated. In the present study, the effects of adenosine A(1) receptor antagonism on insulin secretion from isolated rat pancreatic islets were tested using DPCPX, a selective adenosine A(1) receptor antagonist. It was demonstrated that pancreatic islets stimulated with 6.7 and 16.7 mM glucose and exposed to DPCPX released significantly more insulin compared with islets incubated with glucose alone. The insulin-secretory response to glucose and low forskolin appeared to be substantially potentiated by DPCPX, but DPCPX was ineffective in the presence of glucose and high forskolin. Moreover, DPCPX failed to change insulin secretion stimulated by the combination of glucose and dibutyryl-cAMP, a non-hydrolysable cAMP analogue. Studies on pancreatic islets also revealed that the potentiating effect of DPCPX on glucose-induced insulin secretion was attenuated by H-89, a selective inhibitor of protein kinase A. It was also demonstrated that formazan formation, reflecting metabolic activity of cells, was enhanced in islets exposed to DPCPX. Moreover, DPCPX was found to increase islet cAMP content, whereas ATP was not significantly changed. These results indicate that adenosine A(1) receptor blockade in rat pancreatic islets potentiates insulin secretion induced by both physiological and supraphysiological glucose concentrations. This effect is proposed to be due to increased metabolic activity of cells and increased cAMP content.     

Altered liver glycogen metabolism in fed genetically obese mice.

The cyclic AMP and glycogen concentrations and the activities of phosphorylase kinase, phosphorylase a and glycogen synthase a were not different in livers from lean or ob/ob mice despite increased plasma glucose and insulin in the obese group. The liver water content was decreased by 10% in the obese mice. In hepatocytes isolated from lean mice and incubated with increasing glucose concentrations (14-112 mM), a sequential inactivation of phosphorylase and activation of glycogen synthase was observed. In hepatocytes from obese mice the inactivation of phosphorylase was not followed by an activation of synthase. The inactivation of phosphorylase occurred more rapidly and was followed by an activation of synthase in hepatocytes isolated from both groups of mice when in the incubation medium Na+ was replaced by K+ or when Ca2+ was omitted and 2.5 mM-EGTA included. The inactivation of phosphorylase and activation of synthase were not different in broken-liver-cell preparations from lean and obese animals. The re-activation of phosphorylase in liver filtrates in the presence of 0.1 microM-cyclic AMP and MgATP was inhibited by about 70% by EGTA and stimulated by Ca2+ and was always greater in preparations from ob/ob mice. The apparent paradox between the impairment of glycogen metabolism in isolated liver preparations and the situation in vivo in obese mice is discussed.