The role of adenosine 3':5'-cyclic monophosphate in the regulation of insulin release by isolated rat islets of Langerhans.
ABSTRACT: 1. Concentrations of cyclic AMP (adenosine 3':5'-cyclic monophosphate) and rates of insulin release were measured in islets of Langerhans isolated from rat pancreas and incubated for various times in the presence of glucose, 3-isobutyl-1-methylxanthine, caffeine, theophylline, adrenaline and diazoxide. 2. Caffeine and theophylline produced small but significant increases in both cyclic AMP and release of insulin when they were incubated in the presence of 10mm-glucose. 3. 3-Isobutyl-1-methylxanthine produced a marked increase in the intracellular concentration of cyclic AMP in the presence of 5mm- and 10mm-glucose. However, insulin release was stimulated only in the presence of 10mm-glucose. 4. In response to rising concentrations of extracellular glucose (5-20mm) there was no detectable increase in the intracellular concentration of cyclic AMP even though there was a marked increase in the rate of insulin release. 5. In response to 10mm-glucose insulin release occurred in two phases and 3-isobutyl-1-methylxanthine potentiated the effect of glucose on both phases. The intracellular concentration of cyclic AMP remained constant with glucose and rose within 10min to its maximum value with 3-isobutyl-1-methylxanthine. 6. Adrenaline and diazoxide inhibited insulin release and lowered the intracellular concentration of cyclic AMP when islets were incubated with glucose or 3-isobutyl-1-methylxanthine. 7. It is suggested that glucose does not stimulate insulin release by increasing the concentration of cyclic AMP in islet cells. However, the concentration of cyclic AMP in islet cells may modulate the effect of glucose on the release process.
Project description:1. Protein kinase activity was measured in islets of Langerhans that had been incubated in the presence of agents known to affect insulin release. 2. Glucagon, theophylline, caffeine and 3-isobutyl-1-methylxanthine, agents that raise cyclic AMP concentrations in islet cells and stimulate insulin release, increased protein kinase activity. Adrenaline and diazoxide, agents that decrease cyclic AMP concentrations and inhibit insulin secretion, decreased the activity. 3. The increase in protein kinase activity produced by different concentrations of 3-isobutyl-1-methylxanthine was apparently related to the increase in intracellular concentrations of cyclic AMP. 4. The sulphonylureas, tolbutamide and glibenclamide, agents that increase insulin release, also increased the protein kinase activity; however, leucine, arginine and xylitol, which also stimulate insulin release, were without effect on the kinase activity. 5. Increasing the glucose concentration of the incubation medium from 2 to 20mm had no effect on protein kinase activity. Further, the ability of 3-isobutyl-1-methylxanthine to increase the protein kinase activity was not affected by the glucose concentration of the incubation medium. 6. These results suggest that agents which affect insulin secretion by altering cyclic AMP concentrations may exert their effects on hormone release by altering the activity of a cyclic AMP-dependent protein kinase in islet cells.
Project description: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.
Project description:A protein-binding radioassay for cyclic AMP was modified to detect less than 0.025pmol of the nucleotide. The method was applied to the measurement of cyclic AMP in small numbers of mouse pancreatic islets (as little as 25mug of tissue) by use of barium acetate-H(2)SO(4) for deproteinization. The concentration of cyclic AMP in mouse islets incubated in media containing 3.3 or 20mm-glucose was 0.016pmol/10 islets (approx. 1mum in intracellular water). Glucose concentration (3.3 or 20mm) had no detectable effect on islet concentrations of cyclic AMP with periods of incubation or perifusion ranging from 0.5 to 60min, although insulin release rate was rapidly increased by 20mm-glucose. Caffeine (5mm) or 3-isobutyl-1-methylxanthine (1mm), which are known inhibitors of islet cyclic AMP phosphodiesterase, produced marked and rapid increases in islet cyclic AMP concentration at 3.3 or 20mm-glucose, but only enhanced the insulin release rate at the higher glucose concentration. The role of cyclic AMP in insulin release induced by glucose is discussed.
Project description:Adenosine (1.0-100 mum). N(6)-phenylisopropyladenosine (0.1-10 mum) and 2-deoxyadenosine (10 mm) all produced a dose-dependent inhibition of glucose-stimulated insulin release. The inhibition of glucose-stimulated insulin release by adenosine and N(6)-phenylisopropyladenosine was abolished by 3-isobutyl-1-methylxanthine (0.1 mm), whereas 2-deoxyadenosine inhibited insulin release even in the presence of 3-isobutyl-1-methylxanthine. These adenosine nucleosides also inhibited the release of insulin induced by 4-methyl-2-oxopentanoate (20 mm), dl-glyceraldehyde (30 mm) and l-leucine (20 mm). Adenosine (10 mum). N(6)-phenylisopropyladenosine (10 mum) and 2-deoxyadenosine (10 mm) did not inhibit insulin biosynthesis or [U-(14)C]glucose oxidation at concentrations of the nucleosides that gave maximal inhibition of insulin release. However, adenosine, 2-deoxyadenosine and N(6)-phenylisopropyladenosine produced marked inhibition of the glucose-stimulated increases seen in islet cyclic AMP accumulation. Similar to its effects on insulin release, 3-isobutyl-1-methylxanthine (0.1 mm) antagonized the inhibitory effects of cyclic AMP accumulation produced by adenosine and N(6)-phenylisopropyladenosine, but had no effect on the inhibition of cyclic AMP accumulation seen with 2-deoxyadenosine. These results show that adenosine and its specifically modified analogues, 2-deoxyadenosine and N(6)-phenylisopropyladenosine, are strong inhibitors of insulin release from rat islets, a function that appears to be the consequence of their ability to inhibit the accumulation of cyclic AMP. It is proposed that the B cells, in common with many other tissues, may possess two different sites at which adenosine nucleosides interact to produce their biological effects; these are the so-called ;P' and ;R' sites first described by Londos & Wolff [(1977) Proc. Natl. Acad. Sci. U.S.A.74, 5482-5486].
Project description:The release of growth hormone from heifer anterior pituitary slices and the cyclic AMP content of the slices were increased by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, both increases being related to inhibitor concentration over the range 0.1-1.0mm. Neither Ba(2+) (6.9 or 2.3mm), K(+) (72mm), nor p-chloromercuribenzoate (20mum) had any effect on pituitary cyclic AMP content over a 20min period. 3-Isobutyl-1-methylxanthine potentiated the release of growth hormone in response to Ba(2+) (2.3mm) and K(+) (24mm), but the degree of potentiation did not depend on inhibitor concentration in the same way as did tissue cyclic AMP content. 3-Isobutyl-1-methylxanthine decreased the concentration of K(+) required to give maximum stimulation of growth-hormone release, but did not significantly increase the maximum response to Ba(2+). Growth-hormone release in the presence of prostaglandin E(2) (1mum) was increased by 3-isobutyl-1-methylxanthine and was inhibited by the prostaglandin antagonist, 7-oxa-13-prostynoic acid, although this antagonist increased the pituitary cyclic AMP concentration and potentiated the prostaglandin E(2)-induced rise in cyclic AMP content. The stimulation of growth-hormone release by p-chloromercuribenzoate was not potentiated by 3-isobutyl-1-methylxanthine. The data suggest that Ba(+) and K(+) act at the same point in the secretory process as 3-isobutyl-1-methylxanthine, although by a different mechanism, and that p-chloromercuribenzoate has a different point of action.
Project description:1. An assay has been developed with sufficient sensitivity for determination of the adenosine 3':5'-cyclic monophosphate diesterase activity in islets of Langerhans, and has been used to investigate the response of the enzyme to various agents which are known to affect insulin release. 2. The subcellular distribution of the enzyme in islets of Langerhans prepared from guinea-pig pancreas was investigated and over 70% of the activity present in the original homogenate was recovered in the supernatant fraction. 3. Gel filtration of the activity present in the supernatant fraction on Sephadex G-200 gave a single peak of activity with an apparent molecular weight of 200000. The phosphodiesterase activity in the peak fraction showed two apparent K(m) values for adenosine 3':5'-cyclic monophosphate (cyclic AMP) of 3mum and 30mum, suggesting the presence of two activities. The pH optimum of the activity with the low K(m) value was 8.7. 4. Theophylline, caffeine, 3-isobutyl-1-methylxanthine (SC-2964), glibenclamide, tolbutamide, xylitol and leucine were inhibitors of the activity with the low K(m) value; imidazole and arginine stimulated the activity, and glucose and diazoxide were without significant effect. 5. It is suggested that the agents theophylline, caffeine, SC-2964, glibenclamide, tolbutamide, leucine and imidazole may alter the intracellular concentration of cyclic AMP in islets of Langerhans by affecting the cyclic AMP phosphodiesterase activity in islet cells and in this way may affect insulin release.
Project description:The possible involvement of calmodulin in insulin release was evaluated by studying the effects on intact islets of trifluoperazine and pimozide, two antipsychotic agents known to bind strongly to calmodulin in cell-free systems. Trifluoperazine (10-100mum) produced a dose- and time-dependent inhibition of the two phases of glucose-stimulated insulin release. The effect was not reversible by simple washing of the drug, but could be prevented by cytochalasin B or theophylline. Trifluoperazine also inhibited the release induced by glyceraldehyde, oxoisocaproate, tolbutamide or barium, but not that stimulated by 10mm-theophylline or 1mm-3-isobutyl-1-methylxanthine. Pimozide (0.5-10mum) also produced a dose-dependent inhibition of insulin release triggered by glucose, leucine or barium, but did not affect the release induced by methylxanthines. Glucose utilization by islet cells was not modified by trifluoperazine (25mum), which slightly increased cyclic AMP concentration in islets incubated without glucose. The drug did not prevent the increase in cyclic AMP concentration observed after 10min of glucose stimulation, but suppressed it after 60min. Basal or glucose-stimulated Ca(2+) influx (5min) was unaffected by 25mum-trifluoperazine, whereas Ca(2+)net uptake (60min) was inhibited by 20%. Glucose-stimulated Ca(2+) uptake was almost unaffected by pimozide. In a Ca(2+)-free medium, trifluoperazine decreased Ca(2+) efflux from the islets and did not prevent the further decrease by glucose; in the presence of Ca(2+), the drug again decreased Ca(2+) efflux and inhibited the stimulation normally produced by glucose. In the absence of glucose, trifluoperazine lowered the rate of Rb(+) efflux from the islets, decreased Rb(+) influx (10min), but did not affect Rb(+) net uptake (60min). It did not interfere with the ability of glucose to decrease Rb(+) efflux rate further and to increase Rb(+) net uptake. The results show thus that trifluoperazine does not alter the initial key events of the stimulus-secretion coupling. Its inhibition of insulin release suggests a role of calmodulin at late stages of the secretory process.
Project description:1. The effects of changes in the cytoplasmic [NADH]/[NAD+] ratio on the efficacy of glucagon to alter rates of metabolism in isolated rat hepatocytes were examined. 2. Under reduced conditions (with 10mM-lactate), 10nM-glucagon stimulated both gluconeogenesis and urea synthesis in isolated hepatocytes from 48h-starved rats; under oxidized conditions (with 10mM-pyruvate), 10nM-glucagon had no effect on either of these rates. 3. The ability of glucagon to alter the concentration of 3':5'-cyclic AMP and the rates of glucose output, glycogen breakdown and glycolysis in cells from fed rats were each affected by a change in the extracellular [lactate]/[pyruvate] ratio; minimal effects of glucagon occurred at low [lactate]/[pyruvate] ratios. 4. Dose-response curves for glucagon-mediated changes in cyclic AMP concentration and glucose output indicated that under oxidized conditions the ability of glucagon to alter each parameter was decreased without affecting the concentration of hormone at which half-maximal effects occurred. 5. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.05 mM) significantly reversed the inhibitory effects of pyruvate on glucagon-stimulated glucose output. 6. For exogenously added cyclic [3H]AMP(0.1 mM), oxidized conditions decreased the stimulatory effect on glucose output as well as the intracellular concentration of cyclic AMP attained, but did not alter the amount of cyclic [3H]AMP taken up. 7. The effects of lactate, pyruvate, NAD+ and NADH on cyclic AMP phosphodiesterase activities of rat hepatocytes were examined. 8. NADH (0.01--1 MM) inhibited the low-Km enzyme, particularly that which was associated with the plasma membrane. 9. The inhibition of membrane-bound cyclic AMP phosphodiesterase by NADH was specific, reversible and resulted in a decrease in the maximal velocity of the enzyme. 10. It is proposed that regulation of the membrane-bound low-Km cyclic AMP phosphodiesterase by nicotinamide nucleotides provides the molecular basis for the effect of redox state on the hormonal control of hepatocyte metabolism by glucagon.
Project description:Effects on insulin release, cyclic AMP content and protein phosphorylation of agents modifying cyclic AMP levels have been tested in intact rat islets of Langerhans. Insulin release induced by glucose was potentiated by dibutyryl cyclic AMP, glucagon, cholera toxin and 3-isobutyl-1-methylxanthine (IBMX); the calmodulin antagonist trifluoperazine reversed these potentiatory effects. Inhibition by trifluoperazine of IBMX-potentiated release was, however, confined to concentrations of IBMX below 50 microM; higher concentrations, up to 1 mM, were resistant to inhibition by trifluoperazine. IBMX-potentiated insulin release was also inhibited by 2-deoxyadenosine, an inhibitor of adenylate cyclase. In the absence of glucose, IBMX at concentrations up to 1 mM did not stimulate insulin release and in the presence of 3.3 mM-glucose IBMX was effective only at a concentration of 1 mM; under the latter conditions trifluoperazine again did not inhibit insulin secretion. The maximum effect on insulin release was achieved with 25 microM-IBMX. Islet [cyclic AMP] was increased by IBMX, with the maximum rise occurring with 100 microM-IBMX. The increase in [cyclic AMP] elicited by IBMX was more rapid than that induced by cholera toxin. Trifluoperazine did not significantly affect islet cyclic AMP levels under any of the conditions tested. When islets were incubated with [32P]Pi, radioactivity was incorporated into islet ATP predominantly in the gamma-position. The rate of equilibration of label was dependent on medium Pi and glucose concentration and at optimal concentrations of these 100% equilibration of internal [32P]ATP with external [32P]Pi required a period of 3h. Radioactivity was incorporated into islet protein and, in response to an increase in islet [cyclic AMP], the major effect was on a protein of Mr 15 000 on sodium dodecyl sulphate/polyacrylamide gels. The extent of phosphorylation of the Mr-15 000 protein was correlated with the level of cyclic AMP: phosphorylation in response to IBMX was inhibited by 2-deoxyadenosine but not by trifluoperazine. Fractionation of islets suggested that the Mr-15 000 protein was of nuclear origin: the protein co-migrated with histone H3 on acetic acid/urea/Triton gels. In the islet cytosol a number of proteins were phosphorylated in response to elevation of islet [cyclic AMP]: the major species had Mr values of 18 000, 25 000, 34 000, 38 000 and 48 000. Culture of islets with IBMX increased the rate of [3H]-thymidine incorporation.(ABSTRACT TRUNCATED AT 400 WORDS)
Project description: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.