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.     

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.     

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.     

A possible role of adenylate cyclase in the longterm dietary regulation of insulin secretion from rat islets of Langerhans

1. Adenylate cyclase activity and patterns of insulin release in response to various concentrations of glucose were determined in islets of Langerhans isolated from starving, fed, or glucose-loaded rats. 2. Basal and glucagon-stimulated activities of adenylate cyclase were lower in islets from starved than from fed rats. The minimum glucose concentration required for stimulation of insulin secretion was higher, whereas the maximum secretory response to glucose was lower, in islets from starved than from fed rats. 3. Adenylate cyclase activity in islets of Langerhans obtained from fed rats loaded with glucose by intermittent intravenous or intraperitoneal injections over 5h was significantly higher than that seen in islets from normal fed rats. Islets obtained from glucose-loaded rats required a lower glucose concentration for stimulation of insulin secretion and attained a higher maximal response to glucose stimulation than those derived from fed rats. 4. Incubation in vitro of islets isolated from normal fed rats, for periods of 1 to 24h in the presence of high concentrations of glucose resulted in an activation of adenylate cyclase that occurred progressively from 2 to 7h and which was maintained during 24h of incubation. The increase of adenylate cyclase activity in isolated islets incubated for 4h in the presence of glucose was not prevented by addition of cycloheximide or actinomycin D. Galactose or 2-deoxyglucose was ineffective in increasing adenylate cyclase activity, and pyruvate (20mm) was less effective than glucose. 5. It is suggested that glucose or a glucose metabolite may exert long-term effects on islet cell adenylate cyclase.     

Studies on the role of inositol trisphosphate in the regulation of insulin secretion from isolated rat islets of Langerhans.

Glucose (20 mM) and carbachol (1 mM) produced a rapid increase in [3H]inositol trisphosphate (InsP3) formation in isolated rat islets of Langerhans prelabelled with myo-[3H]inositol. The magnitude of the increase in InsP3 formation was similar when either agent was used alone and was additive when they were used together. In islets prelabelled with 45Ca2+ and treated with carbachol (1 mM), the rise in InsP3 correlated with a rapid, transient, release of 45Ca2+ from the cells, consistent with mobilization of 45Ca2+ from an intracellular pool. Under these conditions, however, insulin secretion was not increased. In contrast, islets prelabelled with 45Ca2+ and exposed to 20mM-glucose exhibited a delayed and decreased 45Ca2+ efflux, but released 7-8-fold more insulin than did those exposed to carbachol. Depletion of extracellular Ca2+ failed to modify the increase in InsP3 elicited by either glucose or carbachol, whereas it selectively inhibited the efflux of 45Ca2+ induced by glucose in preloaded islets. Under these conditions, however, glucose was still able to induce a small stimulation of the first phase of insulin secretion. These results demonstrate that polyphosphoinositide metabolism, Ca2+ mobilization and insulin release can all be dissociated in islet cells, and suggest that glucose and carbachol regulate these parameters by different mechanisms.     

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.     

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.     

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.     

Potassium ions and the secretion of insulin by islets of Langerhans incubated in vitro

1. A method was devised for the isolation of islets of Langerhans from rabbit pancreas by collagenase digestion in order to study the influx and efflux of K(+) in islets during insulin secretion. 2. Glucose-induced insulin release was accompanied by an increased rate of uptake of (42)K(+) by the islets of Langerhans, though this was not the case for secretion in response to tolbutamide. Ouabain significantly inhibited the uptake of (42)K(+) by islet tissue. 3. No significant increase in the rate of efflux of (42)K(+) was demonstrated during active insulin secretion. 4. Slices of rabbit pancreas were incubated in media of different K(+) content, and rates of insulin release were determined. Alteration of the K(+) concentration of the medium between 3 and 8mm had no effect on the rate of insulin release by pancreas slices. However, decrease of the K(+) concentration to 1mm resulted in inhibition of secretion in response to both glucose and to tolbutamide. Conversely, an increase in K(+) concentration increased rates of insulin release in response to both these stimuli. 5. It is concluded that, though unphysiological concentrations of K(+) may influence the secretion of insulin, fluxes of K(+) in the islets do not appear to be important in the initiation of insulin secretion.     

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.     

Insulin receptor activation inhibits insulin secretion from human islets of Langerhans

There is no consensus on the role of insulin secreted from pancreatic β-cells in regulating its own secretion, either in rodent islets or in human islets. We have now investigated whether there is an autocrine signalling role for insulin in human islets by determining insulin receptor expression and assessing the effects of insulin receptor activation using a non-peptidyl insulin mimetic termed L-783,281. Human insulin receptor mRNA was detected by PCR amplification of human islet cDNA, and translation of the message in human islets was confirmed by Western blotting. Perifusion experiments revealed that both glucose-stimulated and basal insulin secretion were significantly inhibited following human islet insulin receptor activation with L-783,281, and that signalling through phosphatidylinositol 3-kinase (PI 3-kinase) was responsible, at least in part, for this inhibitory effect. These studies indicate that human islets express insulin receptors and that they are functionally coupled to a PI 3-kinase-dependent inhibition of insulin secretion.     

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.     

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.     

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 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.     

The role of protein kinase C in cholinergic stimulation of insulin secretion from rat islets of Langerhans.

The role of the Ca2+/phospholipid-dependent protein kinase C (PKC) in cholinergic potentiation of insulin release was investigated by measuring islet PKC activity and insulin secretion in response to carbachol (CCh), a cholinergic agonist. CCh caused a dose-dependent increase in insulin secretion from cultured rat islets at stimulatory glucose concentrations (greater than or equal to 7 mM), with maximal effects observed at 100 microM. Short-term exposure (5 min) of islets to 500 microM-CCh at 2 mM- or 20 mM-glucose resulted in redistribution of islet PKC activity from a predominantly cytosolic location to a membrane-associated form. Prolonged exposure (greater than 20 h) of islets to 200 nM-phorbol myristate acetate caused a virtual depletion of PKC activity associated with the islet cytosolic fraction. Under these conditions of PKC down-regulation, the potentiation of glucose-stimulated insulin secretion by CCh (500 microM) was significantly decreased, but not abolished. CCh stimulated the hydrolysis of inositol phospholipids in both normal and PKC-depleted islets, as assessed by the generation of radiolabelled inositol phosphates. These results suggest that the potentiation of glucose-induced insulin secretion by cholinergic agonists is partly mediated by activation of PKC as a consequence of phospholipid hydrolysis.     

Regulation of insulin secretion from islets of Langerhans rendered permeable by electric discharge

Hepatocytes were isolated from preweaned neonatal and adult rats and maintained in primary monolayer culture. Cells from preweaned newborns possessed no L-type pyruvate kinase, nor did they synthesize the enzyme. Incubation for 48-72 h in culture medium supplemented with 2 mM-fructose and 0.1 microM-insulin induced the synthesis of L-type pyruvate kinase, as judged by increased enzyme activity and the increased incorporation of [3H]leucine into immunoprecipitable L-type pyruvate kinase. Hepatocytes isolated from 48 h-starved adult rats incorporated less [3H]leucine into L-type pyruvate kinase than did cells isolated from high-carbohydrate-diet-fed rats. The rate of enzyme synthesis by cells from 48 h-starved rats was increased by the inclusion of fructose and insulin in the incubation medium, after a lag phase of 24-48 h. After 4 days in culture in the presence of fructose and insulin, hepatocytes from 48 h-starved rats synthesized L-type pyruvate kinase at similar rates to hepatocytes isolated from high-carbohydrate-diet-fed rats.     

Regulation of insulin and glucagon secretion from rat pancreatic islets in vitro by somatostatin analogues

Somatostatin is an inhibitor of hormone secretion through specific receptors (sst1-5). The aim of this study was to investigate the putative regulatory role of somatostatin analogues on the secretion of insulin and glucagon by rat pancreatic islets. After 48 h exposure only the non-selective agonists (somatostatin, octreotide and SOM-230) inhibited insulin accumulation. The inhibition of insulin secretion was accompanied by increased islet insulin contents. None of the analogues showed a consistent effect on the glucagon accumulation in the medium after 48 h. Since we observed a difference in the regulatory effect between the non-selective and selective analogues, combinations of selective analogues were studied. Combination of sst2+sst5 agonists inhibited the medium insulin accumulation, while combination of sst1+sst2 analogues caused a decrease in glucagon accumulation. After removal of somatostatin a rebound effect with increased insulin secretion were observed. This effect was reversed after 6 h. For SOM-230 insulin secretion continued to be suppressed even after the analogue was removed and returned to control values after 3 h. As for glucagon secretion there was an initial decline after culture with octreotide, while the other substances failed to induce any changes. In summary, non-selective somatostatin analogues or combinations of receptor selective analogues may cause inhibition of hormone secretion from rat pancreatic islets. For insulin and glucagon, combinations of sst2+sst5 and sst1+sst2, respectively may exert this effects. Thus, our data suggest that more than one sst must be involved to down-regulate islet glucagon and insulin secretion.