ADP-ribosylation of isolated rat islets of Langerhans

A rapid and reproducible radioimmunoassay method was developed for rat atrial natriuretic factor (ANF)-IV. The method is also applicable to human atrial peptide. ANF was detected in rat hypothalamus (5.03 pmoles/g tissue), right (86.8 pmoles/mg tissue) and left atria (52.5 pmoles/mg tissue), and plasma (156 fmoles/ml). After high salt intake immunoreactive ANF in atria and plasma increased significantly, while a significant decrease was observed in hypothalamus. Gel chromatography revealed high and low molecular weight ANF in atria and hypothalamus while only a low molecular weight form was found in plasma.     

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.     

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.     

Effects of monensin on metabolism of isolated rat islets of Langerhans.

Monensin, a univalent ionophore, is a carboxylic acid produced by Streptomyces cinnamonensis. It will complex various alkali-metal ions, but most readily binds Na+. Because of interest in the possible role of Na+ in the regulation of insulin secretion, we examined its effects on several aspects of the metabolism of isolated rat islets of Langerhans. The ionophore inhibited glucose-stimulated insulin release in a concentration-dependent manner, completely inhibiting secretion evoked by 20 mM-glucose at concentrations as low as 0.1 microM in static incubations. In perifusion experiments, both phases of insulin release were equally affected. Monensin (0.1 microM) had no significant effect on glucose oxidation as measured by the generation of 14CO2 from [14C]glucose. Monensin increased the rate of 22Na+ efflux from preloaded islets and net 22Na+ uptake over 30 min, in the absence of changes in islet volume or extracellular space. The ionophore increased the Rb+/K+ permeability of islet cells, as shown by its inhibition of 86Rb+ retention and stimulation of 86Rb+ efflux. At 0.1 microM, monensin abolished glucose-stimulated 45Ca2+ uptake by islets during 5 min incubations, and stimulated 45Ca2+ efflux from preloaded islets perifused with Ca2+-free medium, even in the complete absence of extracellular Na+. Studies of the uptake of 14C-labelled 5,5-dimethyloxazolidine-2,4-dione showed that 0.1 microM-monensin increased net intracellular pH from 7.05 to 7.13. 7 Monensin has widespread, complex, effects on the secretory responses and ion handling by the B cells, which are difficult to interpret in terms solely of actions as a Na+ ionophore.     

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.     

Insulin-releasing effect of quinaldic acid and its relatives on isolated Langerhans islets

Quinaldic acid and 8-hydroxyquinaldic acid, end-metabolites of kynurenine (1,2), were shown to cause release of insulin from isolated Langerhans islets of rats. In this respect, both kynurenic acid and xanthurenic acid were found to be somewhat less active than quinaldic acid, but L-kynurenine, 3-hydroxyl-L-kynurenine, quinolinic acid and L-tryptophan did not cause the insulin release.Possible correlation between disorder in tryptophan metabolisms directed toward the excessive formation of quinaldic acid and 8-hydroxyquinaldic acid and pathogenesis of diabetic state was discussed.     

Effect of glucose on polyphosphoinositide metabolism in isolated rat islets of Langerhans.

The metabolism of inositol-containing phospholipids during insulin secretion was studied in rat islets of Langerhans preincubated with [3H]inositol to label their phospholipids. Glucose (20 mM) caused a rapid breakdown of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate and an accumulation of inositol trisphosphate and inositol bisphosphate. This effect was maximal at 60s, did not require the presence of extracellular Ca2+, and was abolished by mannoheptulose (15 mM), but not by noradrenaline (1 microM). Mannose (20 mM) and DL-glyceraldehyde (10 mM) produced similar effects to those of glucose, but galactose (20 mM) and KCl (30 mM) were without effect. These results are compatible with the hypothesis that an early event in the stimulus-secretion coupling mechanism in the pancreatic B-cell is the rapid breakdown of polyphosphoinositides catalysed by phospholipase C. Moreover, they suggest that the breakdown of polyphosphoinositides is linked to sugar metabolism in the B-cell. This observation is important, since it demonstrates that events in a cell other than plasma-membrane receptor occupancy can promote polyphosphoinositide hydrolysis.     

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.     

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 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.     

The effect of metabolites,papaverine, and probenecid on cyclic AMP efflux from isolated rat islets of Langerhans

The process of cyclic AMP efflux from rat islets of Langerhans has been studied. The dynamics of glucose-induced cyclic AMP efflux closely resembled the pattern of glucose-induced insulin release. Thus, both processes were dose-dependent for glucose having the same threshold concentrations (4–8 mmol/l glucose), with the time course of cyclic AMP efflux and insulin release from 0–60 min being very similar. Galactose did not affect insulin release, cyclic AMP efflux and intra-islet cyclic AMP accumulation. On the other hand, inosine, N-acetylglucosamine, α-ketoisocaproic acid, L-leucine and xylitol all promoted insulin release and cyclic AMP efflux. Except for L-leucine, all these substances enhanced the intracellular accumulation of cyclic AMP. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, greatly augmented all these parameters in the presence of glucose whereas in the absence of glucose, insulin release was not enhanced, while both cyclic AMP efflux and cyclic AMP accumulation were elevated. The drug, probenecid, did not alter either insulin release or intra-islet cyclic AMP levels, while cyclic AMP efflux was markedly reduced (though not abolished). Papaverine inhibited both insulin release and cyclic AMP efflux, but was found to augment the intra-islet cyclic AMP levels. The efflux of cyclic AMP correlates more closely with insulin release than with the cyclic AMP accumulation in most instances. The efflux is independent of either insulin secretory granule extrusion or intracellular fluctuations of the nucleotide, though it is not yet known whether cyclic AMP efflux may have some regulatory significance in insulin release.     

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.     

Cyclic AMP-dependent stimulation of somatostatin secretion by isolated rat islets of Langerhans.

Immunoreactive somatostatin is released from islets of Langerhans, isolated from rat pancreas by collagenase digestion, when incubated in an $\text{in vitro}$ system. The rate of somatostatin secretion is independent of extracellular glucose concentration, but is stimulated by addition of 8-Br-cyclic AMP or theophylline.