Regulation of insulin secretion by cAMP in rat islets of Langerhans permeabilised by high-voltage discharge

Adenosine 3',5-cyclic monophosphate (cAMP) was shown to stimulate insulin secretion from electrically permeabilised islets of Langerhans incubated in Ca2+/EGTA buffers. cAMP-induced insulin secretion occurred in the presence of either sub-stimulatory (50 nM) or stimulatory (greater than 100 nM) concentrations of Ca2+. Similar effects on secretion were obtained in response to 8-bromo-cAMP (8-Br-cAMP) or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine. Forskolin (0.2-20 microM) increased adenylate cyclase activity and enhanced insulin secretion from the permeabilised islets. These results suggest that, in electrically permeabilised islets, cAMP-induced insulin secretion is not dependent on changes in cytosolic Ca2+.     

Effects of phenoxybenzamine on insulin secretion from isolated rat islets of Langerhans

In isolated rat islets the ₂-adrenergic antagonist phenoxybenzamine was found to be only partially effective at relieving the inhibition of glucose-induced insulin secretion mediated by noradrenaline. Further experiment revealed a direct inhibitory effects of phenoxybenzamine itself on the secretory response to glucose. At concentrations above 1 M the antagonist inhibited insulin secretion in a dose-dependent manner, with greater than 50% inhibition at 50 M. The inhibition of secretion developed rapidly in perifused islets, and was not altered when islets were also incubated with idazoxan or benextramine, suggesting that it did not reflect binding of phenoxybenzamine to the ₂-receptor. Paradoxically phenoxybenzamine significantly increased the basal secretion rate in the presence of 4 mM glucose. The results demonstrate that phenoxybenzamine can exert direct effects on insulin secretion which are unrelated to its -antagonist properties.     

Stimulation of insulin secretion from isolated rat islets of Langerhans by melittin

Melittin , an amphipathic polypeptide, stimulated the secretion of insulin from rat islets of Langerhans incubated in vitro . The secretory response was dose-dependent and saturable with half the maximal response elicited by a melittin concentration of 4 g/ml. The response was rapid in onset, an increase in secretion occurring within 2 rain of exposure of the islets to melittin (2 g/ml). An enhanced secretory rate could be maintained for at least 40 rain in the presence of melittin but declined steadily when the agent was removed. Stimulation of secretion by melittin occurred in the absence of glucose and in the presence of both 4 mM and 8 mM glucose but not in the presence of 20 mM glucose. The effect of melittin on secretion was dependent on the presence of extracellular calcium but was not inhibited by norepinephrine. The data suggest that melittin may be a valuable agent for further study of the role played by the B-cell plasma membrane in the regulation of insulin secretion.     

Effect of L-asparaginase on insulin secretion from isolated rat islets of Langerhans.

The effects of L-asparaginase were evaluated on glucose-induced insulin release from isolated rat islets of Langerhans. Islets were obtained by enzymatic digestion of pancreas from Sprague-Dawley rats. The study of L-asparaginase effects on insulin secretion was performed in a static incubation of islets. Insulin secretion was measured at 60 min of incubation with different secretagogues with and without L-asparaginase. L-Asparaginase at concentrations from 310 to 5,000 U/ml could inhibit the glucose-induced insulin secretion in a dose-dependent manner. This effect was not recovered after incubation in the absence of the drug for another 2 h. The half-maximal inhibitory effect of the enzyme on insulin secretion was observed at L-asparaginase concentrations of 1,000 U/ml. Tolbutamide (200 microM) and ketoisocaproic acid (20 mM) did not induce insulin secretion in the presence of moderately high L-asparaginase concentrations. L-Asparaginase did not inhibit glucose-induced insulin secretion in the presence of isobutyl-methyl-xanthine (IBMX) (20 microM) or forskolin (20 microM). L-Asparaginase promoted a decrease in total c-AMP in isolated rat islets at concentrations from 500 to 1,500 U/ml when they were stimulated by glucose. If islets were treated with IBMX or forskolin, L-asparaginase did not inhibit the glucose-induced total c-AMP levels in islets.     

Catecholamine inhibition of Ca2+-induced insulin secretion from electrically permeabilised islets of Langerhans

Noradrenaline (1-10 microM) inhibited Ca2+-induced insulin secretion from electrically permeabilised islets of Langerhans with an efficacy similar to that for inhibition of glucose-induced insulin secretion from intact islets. The inhibition of insulin secretion from permeabilised islets was blocked by the alpha 2-adrenoreceptor antagonist, yohimbine. Adenosine 3',5'-cyclic monophosphate (cAMP) did not relieve the noradrenaline inhibition of Ca2+-induced secretion from the permeabilised islets, although noradrenaline did not affect the secretory responses to cAMP at substimulatory (50 nM) concentrations of Ca2+. These results suggest that catecholamines do not inhibit insulin secretion solely by reducing B-cell adenylate cyclase activity, and imply that one site of action of noradrenaline is at a late stage in the secretory process.     

Adenosine and the regulation of insulin secretion by isolated rat islets of Langerhans.

The effect of adenosine in insulin secretion and adenylate cyclase activity of rat islets of Langerhans was investigated. Adenosine inhibited insulin secretion stimulated by glucose, glucagon, prostaglandin E2, tolbutamine and theophylline. Adenosine decreased basal adenylate cyclase activity of the islets as well as that stimulated by glucagon prostaglandin E2 and GTP, although fluoride-stimulated activity was not affected. Neither insulin secretion nor adenylate cyclase activity of the islets was affected by adenine, AMP or ADP. The inhibitory effect of adenosine on adenylate cyclase activity was not altered by either phenoxybenzamine (alpha-adrenergic blocker) or propranolol (beta-adrenergic blocker), suggesting that the effect is not mediated through the adrenergic receptors of the islet cells. These results suggest that the intracellular concentration of adenosine in the beta-cell may play a role in regulating insulin secretion and that this effect may be mediated via alterations in the activity of adenylate cyclase in the beta-cell.     

Effects of taxol and nocodazole on insulin secretion from isolated rat islets of Langerhans

Taxol, a promotor of microtubule polymerization, and nocodazole, which induces microtubule depolymerization, used at concentrations known to be specific for these effects in other cell types, were each shown to inhibit glucose-stimulated insulin secretion from isolated rat islets of Langerhans. These findings suggest that the dynamic regulation of microtubule polymerization-depolymerization in pancreatic B ceils may be important for insulin secretion via the microtubule-microfilamentous system.     

Oxygen and temperature dependence of stimulated insulin secretion in isolated rat islets of Langerhans

The effects of lowered O2 tension on insulin secretion and changes in cellular energy parameters were investigated in isolated rat pancreatic islets perifused with buffers equilibrated with 21, 9, 5, and 1% oxygen and containing 5 mM glucose. Decreasing the external [O2] reduced the amount of insulin released in response to 16 mM glucose, 20 mM alpha-ketoisocaproic acid, and 40 mM KCl. Secretion elicited by high glucose or KCl had declined significantly at 9% oxygen, whereas that caused by alpha-ketoisocaproic acid became inhibited below 5% O2. Lowering the oxygen tension also decreased the ability of islets to respond with a rise in [ATP]/[ADP] upon stimulation with metabolic secretagogues. This reduction in the evoked increase in the nucleotide ratios paralleled the inhibition of stimulated insulin secretion. Addition of 2 mM amytal markedly decreased the islet energy level and eliminated the secretory response to 16 mM glucose. The results suggest that enhancement of B-cell energy production and a consequent rise in [ATP] (or [ATP]/[ADP]) are a necessary event for the hormone release elicited by high glucose and alpha-ketoisocaproic acid. A decrease in temperature inhibited insulin secretion with all three secretagogues tested. The energies of activation were similar for high glucose and KCl-induced secretion, about 20 kcal/mol, but were higher for alpha-ketoisocaproic acid, about 35 kcal/mol. At 28 degrees C, the [ATP]/[ADP] was larger than that at 38 degrees C (8 versus 5) and was not increased further upon addition of 16 mM glucose. It is suggested that a decrease in the rate of energy production at lowered temperatures may contribute to the inhibition of insulin release caused by metabolic secretagogues.     

Dissociation between intracellular calcium mobilization and insulin secretion in isolated rat islets of Langerhans

The neuropeptide bombesin provoked a dose-dependent stimulation of 45Ca2+ efflux from pre-loaded islets of Langerhans. This response occurred rapidly, was not sustained and did not depend on the presence of extracellular calcium, suggesting that it resulted from the mobilization of intracellular calcium stores. Under conditions when large increases in 45Ca2+ efflux were observed, bombesin completely failed to stimulate the rate of insulin secretion. Similar results were also obtained with the muscarinic cholinergic agonist, carbachol. The data suggest that the release of calcium from intracellular pools is not sufficient to induce an increase in insulin secretion in normal islet cells.     

Pentitols and insulin release by isolated rat islets of Langerhans

1. Insulin secretion was studied in isolated islets of Langerhans obtained by collagenase digestion of rat pancreas. In addition to responding to glucose and mannose as do whole pancreas and pancreas slices in vitro, isolated rat islets also secrete insulin in response to xylitol, ribitol and ribose, but not to sorbitol, mannitol, arabitol, xylose or arabinose. 2. Xylitol and ribitol readily reduce NAD(+) when added to a preparation of ultrasonically treated islets. 3. Adrenaline (1mum) inhibits the effects of glucose and xylitol on insulin release. Mannoheptulose and 2-deoxy-glucose, however, inhibit the response to glucose but not that to xylitol. 4. The intracellular concentration of glucose 6-phosphate is increased when islets are incubated with glucose but not with xylitol, suggesting that xylitol does not promote insulin release by conversion into glucose 6-phosphate. 5. Theophylline (5mm) potentiates the effect of 20mm-glucose on insulin release from isolated rat islets of Langerhans, but has no effect on xylitol-mediated release. These results indicate that xylitol does not stimulate insulin release by alterations in the intracellular concentrations of cyclic AMP. 6. A possible role for the metabolism of hexoses via the pentose phosphate pathway in the stimulation of insulin release is discussed.     

Cyclic AMP-dependent protein phosphorylation and insulin secretion in intact islets of Langerhans.

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)     

The effect of phalloidin on insulin secretion from islets of Langerhans isolated from rat pancreas

Phalloidin, which stabilizes F-actin, has no effect on insulin secretion from intact islets, but penetrates and increases secretion from islets previously made permeable using a high voltage discharge technique. Use of this highly specific drug strongly suggests a role for microfilaments composed of F-actin in the insulin secretory process.     

Studies on the mechanism by which melittin stimulates insulin secretion from isolated rat islets of Langerhans

Incubation of isolated rat islets of Langerhans with melittin resulted in a dose-dependent stimulation of insulin secretion with half the maximal response occurring at 4 micrograms/ml melittin. The effect of melittin on insulin secretion was dependent on extracellular calcium, was inhibited by the phospholipase A2 inhibitor quinacrine and by the lipoxygenase inhibitor nordihydroguaiaretic acid. Stimulation of insulin secretion by melittin was associated with a calcium-dependent loss of [3H]arachidonic acid from phospholipids in islet cells prelabelled with [3H]arachidonic acid. Analysis of the islet phospholipids involved in this response revealed that the [3H]arachidonic acid was released predominantly from phosphatidylcholine. These results suggest that melittin may stimulate insulin secretion by activating phospholipase A2 in islet cells, causing the release of arachidonic acid from membrane phospholipid. The results are consistent with suggestions that the subsequent metabolism of arachidonic acid via the lipoxygenase pathway may be involved in regulating the insulin secretory response.     

Effects of guanosine on insulin secretion and adenylyl and guanylyl cyclase activities of isolated rat islets of Langerhans

The effect of guanosine on insulin secretion adenylyl and guanylyl cyclase activities of isolated rat islets of Langerhans was investigated. Guanosine (1–100 μM) inhibited glucose, tolbutamide, theophylline and prostaglandin E₂-stimulated insulin secretion although it failed to affect glucagon stimulated secretion. Prostaglandin E₂-stimulated adenylyl cyclase of islets was inhibited by guanosine although guanosine had no effect on basal, fluoride, glucagon or GTP-stimulated activity. Guanosine markedly decreased basal guanylyl cyclase activity of islets.These results suggest that guanosine may affect insulin release by inhibiting adenylyl and guanylyl cyclase activities in the ß-cell thereby decreasing the intracellular concentrations of cyclic nucleotides.This effect may be important in modulating the secretory response of the islets to a variety of hormonal agents.     

Effects of the calcium-channel agonist CGP 28392 on insulin secretion from isolated rat islets of Langerhans.

The rate of insulin secretion from isolated rat islets of Langerhans was affected by a number of dihydropyridine derivatives known to interact with voltage-sensitive Ca2+ channels in excitable cells. The channel antagonists nifedipine and nitrendipine were potent inhibitors of glucose-induced insulin secretion in response to both 8 mM- and 20 mM-glucose, although they did not lower the basal secretion rate observed in the presence of 4 mM-glucose. The Ca2+-channel agonist, CGP 28392, also failed to alter the basal rate of insulin secretion. In the presence of 8 mM-glucose, however, 1 microM-CGP 28392 enhanced the insulin-secretion rate to a value approximately double that with 8 mM-glucose alone. This effect was dose-dependent, with half the maximal response elicited by 0.1 microM-CGP 28392, and full enhancement at 10 microM. The response was rapid in onset, with an increase in insulin secretion evident within 2 min of CGP 28392 infusion in perifused islets. Stimulation of insulin secretion by CGP 28392 was correlated with a rapid enhancement of glucose-stimulated 45Ca2+ uptake into islets cells, and with a transiently increased rate of 45Ca2+ efflux from pre-loaded islets. Stimulation of insulin secretion by CGP 28392 was abolished in the presence of noradrenaline, although under these conditions the rapid stimulation of 45Ca2+ influx induced by CGP 28392 was only partially inhibited. In contrast with these results, when islets were incubated in the presence of 20 mM-glucose, CGP 28392 caused a dose-dependent inhibition of insulin secretion. Half-maximal inhibition required approx. 0.2 microM-CGP 28392, with maximal effects observed at 10 microM. Under these conditions, however, the extent of insulin secretion was still only decreased by about 50%, to a value which was similar to that seen in the presence of 8 mM-glucose and CGP 28392. These results suggest that dihydropyridine derivatives can alter the activity of voltage-dependent Ca2+ channels in islet cells, and are consistent with the possibility that gating of these channels plays an important role in regulating the rate of insulin secretion after glucose stimulation.     

Co-secretion of carboxypeptidase H and insulin from isolated rat islets of Langerhans.

The release of carboxypeptidase H activity from isolated rat islets was determined and compared to the secretion of immunoreactive insulin. Analysis of pancreatic islet cells sorted into beta and non-beta types indicated that approx. 80% of islet carboxypeptidase H activity is present in the beta cell. The release of both insulin and carboxypeptidase H was stimulated markedly by increasing the glucose concentration in the medium from 2.8 to 28 mM. The fractional release was in accordance with the observed cellular distribution of both proteins. The secretory response was biphasic with time, with an initial rapid transient phase of release within 5 min, followed by a more sustained response. The concentration-dependencies of glucose stimulation of release of insulin and carboxypeptidase H were similar, with a threshold for stimulation around 5.6 mM-glucose and maximal stimulatory response at 16.7-28 mM-glucose. The release of both proteins was inhibited by 20 mM-mannoheptulose, removal of Ca2+ from the medium and addition of 1 microM-noradrenaline. The combination of 10 mM-4-methyl-2-oxopentanoate and 10 mM-glutamine stimulated the release of carboxypeptidase H and insulin, as did 3-isobutyl-1-methylxanthine and 350 microM-tolbutamide in the presence of glucose. It is evident that carboxypeptidase H is released from the pancreatic beta-cell by an exocytotic process from the same intracellular compartment as insulin. The release of carboxypeptidase H by a constitutive process was at best equivalent to 0.4%/h, or less than 2% of the maximal rate of release via the regulated pathway. It is concluded that carboxypeptidase H can be used as a sensitive index of beta-cell secretion and an alternative marker to the insulin-related peptides.     

Adenylate cyclase activity in isolated rat islets of Langerhans Effects of agents which alter rates of insulin secretion

Adenylate cyclase activity was estimated inhomogenates of rat islets of Langerhans. by measurement of the conversion of [α-³²P]ATP to adenosine cyclic 3′,5′-[³²P]monophosphate. Islet cell adenyulate cyclase activity was stimulated by the addition to the homogenates of glucagon, fluoride, prostaglandins E₁ or E₂ GTP or CTP although not by UTP, TTP, GDP, or GMP. Adrenaline, noradrenaline and isoproterenol were each found to inhibit the activity, the order of potency at a concentration of 10^?4 M being adrenaline > noradrenaline > isoproterenol. The effects of these agents were not altered by β-blackade with propanolol but could be preventived by α-blockade with phenoxybenzamine. The following agents, present at concentrations previously shown to increase rates of insulin secretion from rat islets of Langerhans, were ineffective in altering adenylate cyclase activity when tested in the presence or absence of 0.1 mM GTP: glucose, glibenclamide, xylitol leucine, arginine, or potassium. These results suggest that the activity of adenylate cyclase in the B cells of rat islets of Langerhans may play an important role in mediating the direct effects of hormones and adrenergic agents on insulin release, although the short term effects of substrates such as glucose or amino acids on the release process do not appear to be mediated through alterations in the activity of this enzyme.