Anomeric dissociation between glucokinase activity and glycolysis in pancreatic islets

In pancreatic islet homogenates incubated in the presence of a high glucose concentration (40 mM), the beta-anomer of D-glucose is phosphorylated at a higher rate than the alpha-anomer, whether in the absence or presence of exogenous glucose 6-phosphate. However, in intact islets also exposed to 40 mM D-glucose, the production of 3H2O from D-[5-3H] glucose, the oxidation of D-[U-14C] glucose and the glucose-induced increment in either lactate production or 45Ca net uptake, as well as the release of insulin from isolated perfused pancreases, are not higher with beta- than alpha-D-glucose. It is concluded that the rate of glucose utilization by islet cells is not regulated solely by the activity of hexokinase and/or glucokinase.     

Is glucokinase responsible for the anomeric specificity of glycolysis in pancreatic islets?

At a low concentration of D-glucose (3.3 mM), the phosphorylation rate of this hexose in rat pancreatic islet homogenates incubated at 8 degrees C is higher with the beta- than with the alpha-anomer, as expected from the anomeric specificity of hexokinase. In the presence of a high concentration of glucose 6-phosphate (3.0 mM), which inhibits hexokinase but not glucokinase, the phosphorylation rates of the two anomers are not significantly different from one another. Nevertheless, in intact islets exposed at 8 degrees C to the same low concentration of D-glucose, the alpha-anomer augments, more than the beta-anomer, the production of lactic acid and net uptake of 45Ca. At the same concentration (3.3 mM), the alpha-anomer is also more potent than the beta-anomer in enhancing insulin release from perfused pancreases stimulated at 37 degrees C by L-leucine or by the combination of Ba2+ and theophylline. It is concluded that the participation of glucokinase is not essential for the anomeric specificity of glycolysis and insulin release in rat pancreatic islets.     

The conditions under which rat islets are labelled with [3H]inositol alter the subsequent responses of these islets to a high glucose concentration.

Isolated rat islets were incubated with myo-[2-3H]inositol for 2 h to label their phosphoinositide (PI) pools. Labelling was carried out under three separate conditions: in media containing low (2.75 mM) glucose, high (13.75 mM) glucose, or low (2.75 mM) glucose plus sulphated cholecystokinin (CCK-8S; 200 nM). After labelling, the islets were perifused and the insulin-secretory response to 20 mM-glucose was measured. PI hydrolysis in these same islets was assessed by measurements of both [3H]inositol efflux and the accumulation of labelled inositol phosphates. The following major observations were made. After prelabelling for 2 h in low glucose, perifusion with 20 mM-glucose resulted in a biphasic insulin-secretory response, an increase in [3H]inositol efflux and a parallel increase in the accumulation of labelled inositol phosphates. After prelabelling in high (13.75 mM) glucose, peak first-phase insulin secretion induced by 20 mM-glucose increased 2-2.5-fold, whereas the second phase of insulin release, as well as [3H]inositol efflux and inositol phosphate accumulation, were significantly decreased. The simultaneous infusion of the diacylglycerol kinase inhibitor 1-mono-oleoylglycerol (50 microM), along with 20 mM-glucose, restored the second-phase insulin-secretory response from these islets. After labelling in low (2.75 mM) glucose plus CCK-8S, the initial phases of the insulin-secretory and [3H]inositol-efflux responses to 20 mM-glucose were blunted and the sustained phases of both responses were markedly decreased. Inositol phosphate accumulation was also impaired. Labelling islets in high (13.75 mM) glucose or low (2.75 mM) glucose plus CCK-8S suppresses, in a parallel fashion, glucose-induced increases in PI hydrolysis and in second-phase insulin release. These findings suggest that desensitization of the insulin-secretory response is a consequence of impaired information flow in the inositol lipid cycle.     

Autocrine regulation of glucose metabolism in pancreatic islets

We evaluated the possible autocrine modulatory effect of insulin on glucose metabolism and glucose-induced insulin secretion in islets isolated from normal hamsters. We measured 14CO2 and 3H2O production from d-[U-14C]glucose and d-[5-3H]glucose, respectively, in islets incubated with 0.6, 3.3, 8.3, and 16.7 mM glucose alone or with 5 or 15 mU/ml insulin, anti-insulin guinea pig serum (1:500), 25 microM nifedipine, or 150 nM wortmannin. Insulin release was measured (radioimmunoassay) in islets incubated with 3.3 or 16.7 mM glucose with or without 75, 150, and 300 nM wortmannin. Insulin significantly enhanced 14CO2 and 3H2O production with 3.3 mM glucose but not with 0.6, 8.3, or 16.7 mM glucose. Addition of anti-insulin serum to the medium with 8.3 and 16.7 mM glucose decreased 14CO2 and 3H2O production significantly. A similar decrease was obtained in islets incubated with 8.3 and 16.7 mM glucose and wortmannin or nifedipine. This latter effect was reversed by adding 15 mU/ml insulin to the medium. Glucose metabolism was almost abolished when islets were incubated in a Ca2+-deprived medium, but this effect was not reversed by insulin. No changes were found in 14CO2 and 3H2O production by islets incubated with 3.3 mM glucose and anti-insulin serum, wortmannin, or nifedipine in the media. Addition of wortmannin significantly decreased insulin release induced by 16.7 mM glucose in a dose-dependent manner. Our results suggest that insulin exerts a physiological autocrine stimulatory effect on glucose metabolism in intact islets as well as on glucose-induced insulin release. Such an effect, however, depends on the glucose concentration in the incubation medium.     

A role for calcium in the breakdown of inositol phospholipids in intact and digitonin-permeabilized pancreatic islets.

Glucose (20 mM) and 4-methyl-2-oxopentanoate (10 mM) both caused a pronounced stimulation of insulin release and of [3H]inositol phosphate production in rat pancreatic islets prelabelled with myo-[3H]inositol. Secretory responses to these nutrients were markedly impaired by lowering the Ca2+ concentration of the incubation medium to 10(-4)M or less, whereas stimulated inositol phosphate production was sensitive to Ca2+ within the range 10(-6)-10(-4)M. Inositol phosphate formation in response to carbamoylcholine was also found to be dependent on the presence of 10(-5)M-Ca2+ or above. Raising the concentration of K+ in the medium resulted in a progressive, Ca2+-dependent stimulation of inositol phosphate production in islets, although no significant stimulation of insulin release was observed. In islets prelabelled with myo[3H]inositol, then permeabilized by exposure to digitonin, [3H]inositol phosphate production could be triggered by raising the Ca2+ concentration from 10(-7) to 10(-5)M. This effect was dependent on the concentration of ATP and the presence of Li+, and involved detectable increases in the levels of InsP3 and InsP2 as well as InsP. A potentiation of inositol phosphate production by carbamoylcholine was observed in permeabilized islets at lower Ca2+ concentrations, although nutrient stimuli were ineffective. No significant effects were observed with guanine nucleotides or with neomycin, although NADH produced a modest increase and adriamycin a small inhibition of inositol phosphate production in permeabilized islets. These results strongly suggest that Ca2+ ions play an important role in the stimulation of inositol lipid metabolism in islets in response to nutrient secretagogues, and that inositide breakdown may actually be triggered by Ca2+ entry into the islet cells.     

Effect of hexoses and mannoheptulose on cyclic AMP accumulation and insulin secretion in rat pancreatic islets.

The effects of various sugars on the simultaneous release of insulin and accumulation of cyclic AMP were studied in collagenase isolated rat pancreatic islets. D-Glucose stimulated the formation of cyclic AMP at 3 and 60 min of incubation, whether measured by a label incorporation technique, or by the protein kinase binding assay of Gilman. Only D-glucose and D-mannose were able to stimulate insulin release and cyclic [3H]AMP accumulation in the absence of other substrate. D-fructose had a stimulatory effect in the presence of 3.3 mM D-glucose only at a high concentration (33.8 mM), and enhanced the effects of 8.3 mM glucose when added at the concentration of 8.3 mM. D-Galactose was effective only together with 8.3 mM D-glucose. The order of potency of these hexoses, both regarding insulin secretion and cyclic [3H]AMP accumulation, was glucose-mannose-fructose-galactose. L-Glucose and 3-O-methylglucose had no effects at 60 min when incubated together with 8.3 mM D-glucose, whereas at 3 min, 3-O-methylglucose induced a small stimulation of the cyclic [3H]AMP response. D-mannoheptulose and D-glucosamine inhibited the insulin and cyclic [3H]AMP responses to 27.7 mM glucose. Mannoheptulose suppressed completely the glucose effect on cyclic nucleotide accumulation within 90 s. Although under all incubation conditions, the threshold stimulatory or inhibitory concentration of a given agent was identical for insulin release and cyclic [3H]AMP accumulation, these two variables showed quantitative differences in incubations of 60 min, the magnitude of the changes in insulin secretion being larger than that for the cyclic nucleotide. It is suggested that modulation of islet cyclic AMP level is an important step in the transmission of the effect of various sugars on insulin release; however, glucose and possibly other sugars may also enhance insulin release by additional mechanisms not involving the adenylate cyclase-cyclic AMP system of the beta-cell.     

Evidence for glucose-responsive and -unresponsive pools of phospholipid in pancreatic islets

The effect of glucose on the metabolism of phospholipids in pancreatic islets was studied with three radioactive phospholipid precursors, [32P]orthophosphate, [3H]myoinositol, and [3H]arachidonic acid, to determine the conditions necessary for studying the breakdown of prelabeled phospholipids. Islets were incubated in the presence of a radioactive precursor for 60 or 90 min and in the presence of either 3.3 or 16.7 mM glucose to prelabel phospholipids. To study the breakdown of prelabeled phospholipid, the unincorporated precursor was removed and the islets were reincubated for 15 or 20 min under conditions that either did or did not stimulate insulin release. Prelabeling in the presence of a noninsulinotropic concentration of glucose (3.3 mM) supported the incorporation of precursors into almost all islet phospholipids studied. Prelabeling in an insulinotropic concentration of glucose (16.7 mM) increased the incorporation of precursors into a number of phospholipids even more; and reincubation in 16.7 mM glucose caused a rapid loss of radioactivity from specific phospholipids (phosphatidylinositol and/or phosphatidylcholine, depending on the precursor). This breakdown was observed only when islets had been prelabeled in 16.7 mM glucose. The amount of radioactivity lost from phospholipid corresponded roughly to the additional amount incorporated during the prelabeling in the high concentration of glucose. Radioactivity in phospholipids in islets prelabeled in 3.3 mM glucose or in nonsecretagogue metabolic fuels, such as malate plus pyruvate, did not decrease when the islets were subsequently exposed to 16.7 mM glucose, nor did it decrease in 3.3 mM glucose when these islets had been prelabeled in 16.7 mM glucose. Glyceraldehyde, an insulin secretagogue, but not galactose or L-glucose which are not insulin secretagogues, stimulated phospholipid breakdown in islets that had been prelabeled in 16.7 mM glucose. Depriving islets of extracellular calcium, a condition that inhibits insulin release, inhibited phospholipid breakdown. The results suggest that pancreatic islets contain a glucose-responsive and a glucose-unresponsive phospholipid pool. The glucose-responsive pool becomes labeled and undergoes rapid turnover only under stimulatory conditions and may play a role in the stimulus-secretion coupling of insulin release.     

Acceleration by fructose of the ATP-sensitive K(+) channel-independent pathway of glucose-induced insulin secretion.

The mechanism with which fructose augments glucose-induced insulin secretion is still unclear. The present study was aimed at examining whether the ketohexose potentiates the ATP-sensitive K(+) channel-independent pathway of glucose-induced insulin secretion and, if so, how this happens. When isolated rat islets were depolarized by incubating them with 50 mM KCl in the presence of 150 microM diazoxide (an opener of ATP-sensitive K(+) channels), 10 mM glucose plus 20 mM fructose elicited significantly higher insulin secretion than 10 mM glucose alone, whereas 20 mM fructose alone did not stimulate insulin secretion. The fructose 1,6-bisphosphate and inositol trisphosphate contents were markedly higher in islets incubated with glucose plus fructose than in islets incubated with glucose alone. The results demonstrate that fructose has the ability to potentiate the ATP-sensitive K(+) channel-independent pathway of glucose-induced insulin secretion. The increase in fructose 1,6-bisphosphate content induced by the co-presence of fructose with glucose, resulting in the rise in inositol trisphosphate content, is likely to be one of the signals involved in the fructose potentiation of glucose-induced insulin secretion.     

A new method for the simultaneous estimation of phosphate efflux and influx during glucose stimulation of isolated pancreatic islets

Isolated rat pancreatic islets were prelabeled with [33Pi] and then incubated with basal (2.8 mM) or stimulatory (16.7 mM) glucose in the presence of [32Pi]. Subsequent changes in islet [33P] and [32P] were utilized as respective indices of net efflux and influx. During the initial eight min, (the period usually spanning the first phase of stimulated insulin secretion) efflux was significantly greater with 16.7 than 2.8 mM glucose whereas the lesser amount of phosphate influx did not differ in the two systems. During the subsequent seven min (a time usually associated with the onset of the second phase of stimulated insulin secretion), efflux was dampened in the presence of 16.7 mM glucose and Pi influx significantly exceeded the 2.8 mM glucose values. Thus, acute stimulation with glucose effects an initial phosphate depletion in pancreatic islets as efflux exceeds influx and repletion occurs thereafter as efflux is attenuated and influx is enhanced. These oscillations in islet phosphate may contribute to the biphasic pattern of glucose-stimulated insulin release.     

Expression of glucagon-like peptide-1 (GLP-1) receptor and the effect of GLP-1-(7-36) amide on insulin release by pancreatic islets during rat ontogenic development.

The expression of glucagon-like peptide-1 (GLP-1) receptor and the effects of GLP-1-(7-36) amide (t-GLP-1) on glucose metabolism and insulin release by pancreatic islets during rat development were studied. GLP-1 receptor mRNA was found in significant amounts in pancreatic islets from all age groups studied, GLP-1 receptor expression being maximal when pancreatic islets were incubated at physiological glucose concentration (5.5 mM), but decreasing significantly when incubated with either 1.67 or 16.7 mM glucose. Glucose utilization and oxidation by pancreatic islets from fetal and adult rats rose as a function of glucose concentration, always being higher in fetal than in adult islets. The addition of t-GLP-1 to the incubation medium did not modify glucose metabolism but gastric inhibitory polypeptide and glucagon significantly increased glucose utilization by fetal and adult pancreatic islets at 16.7 mM glucose. At this concentration, glucose produced a significant increase in insulin release by the pancreatic islets from 10-day-old and 20-day-old suckling rats and adult rats, whereas those from fetuses showed only a significant increase when glucose was raised from 1.67 to 5.5 mM. t-GLP-1 elicited an increase in insulin release by pancreatic islets from all the experimental groups when the higher glucose concentrations were used. Our findings indicate that GLP-1 receptors and the effect of t-GLP-1 on insulin release are already present in the fetus, and they therefore exclude the possibility that alterations in the action of t-GLP-1 are responsible for the unresponsiveness of pancreatic beta cells to glucose in the fetus, but stimulation of t-GLP-1 release by food ingestion in newborns may partially confer glucose competence on beta cells.     

Effect of hexoses and mannoheptulose on cyclic AMP accumulation and insulin secretion in rat pancreatic islets

The effects of various sugars on the simultaneous release of insulin and accumulation of cyclic AMP were studied in collagenase isolated rat pancreatic islets. d-Glucose stimulated the formation of cyclic AMP at 3 and 60 min of incubation, whether measured by a label incorporation technique, or by the protein kinase binding assay of Gilman. Only d-glucose and d-mannose were able to stimulate insulin release and cyclic [³H]AMP accumulation in the absence of other substrate. d-fructose had a stimulatory effect in the presence of 3.3 mM d-glucose only at a high concentration (38.8 mM), and enhanced the effects of 8.3 mM glucose when added at the concentration of 8.3 mM. d-Galactose was effective only together with 8.3 mM d-glucose. The order of potency of these hexoses, both regarding insulin secretion and cyclic [³H]AMP accumulation, was glucose-mannose-fructose-galactose.l-Glucose and 3-O-methylglucose had no effects at 60 min when incubated together with 8.3 mM d-glucose, whereas at 3 min, 3-O-methylglucose induced a small stimulation of the cyclic [³H]AMP response.d-mannoheptulose and d-glucosamine inhibited the insulin and cyclic [³H]-AMP responses to 27.7 mM glucose. Mannoheptulose suppressed completely the glucose effect on cyclic nucleotide accumulation within 90 s.Although under all incubation conditions, the threshold stimulatory or inhibitory concentration of a given agent was identical for insulin release and cyclic [³H]AMP accumulation, these two variables showed quantitative differences in incubations of 60 min, the magnitude of the changes in insulin secretion being larger than that for the cyclic nucleotide. It is suggested that modulation of islet cyclic AMP level is an important step in the transmission of the effect of various sugars on insulin release; however, glucose and possibly other sugars may also enhance insulin release by additional mechanisms not involving the adenylate cyclase-cyclic AMP system of the β-cell.     

Glucose-, calcium- and concentration-dependence of acetylcholine stimulation of insulin release and ionic fluxes in mouse islets.

Mouse islets were used to define the glucose-dependence and extracellular Ca2+ requirement of muscarinic stimulation of pancreatic beta-cells. In the presence of a stimulatory concentration of glucose (10 mM) and of Ca2+, acetylcholine (0.1-100 microM) accelerated 3H efflux from islets preloaded with myo-[3H]inositol. It also stimulated 45Ca2+ influx and efflux, 86Rb+ efflux and insulin release. In the absence of Ca2+, only 10-100 microM-acetylcholine mobilized enough intracellular Ca2+ to trigger an early but brief peak of insulin release. At a non-stimulatory concentration of glucose (3 mM), 1 microM- and 100 microM-acetylcholine increased 45Ca2+ and 86Rb+ efflux in the presence and absence of extracellular Ca2+. However, only 100 microM-acetylcholine marginally increased 45Ca2+ influx and caused a small, delayed, stimulation of insulin release, which was abolished by omission of Ca2+. At a maximally effective concentration of glucose (30 mM), 1 microM- and 100 microM-acetylcholine increased 45Ca2+ influx and efflux only slightly, but markedly amplified insulin release. Again, only 100 microM-acetylcholine mobilized enough Ca2+ to trigger a peak of insulin release in the absence of Ca2+. The results thus show that only high concentrations of acetylcholine (greater than or equal to 10 microM) can induce release at low glucose or in a Ca2+-free medium. beta-Cells exhibit their highest sensitivity to acetylcholine in the presence of Ca2+ and stimulatory glucose. Under these physiological conditions, the large amplification of insulin release appears to be the result of combined effects of the neurotransmitter on Ca2+ influx, on intracellular Ca2+ stores and on the efficiency with which Ca2+ activates the releasing machinery.     

Decreased islet sensitivity to insulin in hamsters with dietary-induced insulin resistance

In the present study, we evaluated the autocrine modulatory effect of insulin on glucose metabolism and glucose-induced insulin secretion in islets isolated from hamsters with insulin resistance (IR) induced by administration of a sucrose-rich diet (SRD) during 5 weeks. We used an approach of two metabolic pathways (glucose oxidation and utilization) based on the measurement of 14CO2 and 3H2O production from D-[U-14C]-glucose and D-[5-(3)H]-glucose, respectively, in isolated islets incubated with 3.3 and 16.7 mM glucose alone, or with 5 or 15 mU/ml insulin, anti-insulin guinea-pig serum (1:500), 25 microM nifedipine, or 150 nM wortmannin. Insulin release was measured by radioimmunoassay in islets incubated with 3.3 or 16.7 mM glucose, with or without 75, 150, and 300 nM wortmannin. Results showed that the stimulatory effect of insulin upon 14CO2 and 3H2O production in control islets was not observed in SRD islets. Addition of anti-insulin serum, nifedipine or wortmannin to the medium with 16.7 mM glucose decreased 14CO2 and 3H2O production in control but not in SRD islets. Whereas wortmannin did not decrease insulin release induced by 16.7 mM glucose in SRD hamsters, it did in controls. We can conclude that the autocrine stimulatory effect of insulin upon glucose metabolism observed in normal islets is attenuated or even absent in islets from IR animals. Such decreased islet sensitivity to insulin did not prevent the compensatory secretion of insulin from maintaining glucose homeostasis, suggesting that, at least in this model, the islets can put forward alternative mechanisms to overcome such defect.     

Inositol cycling and phosphoinositide metabolism in rat pancreatic islets.

The effects of extracellular inositol and LiCl on intra-islet inositol cycling were investigated in isolated rat islets. Islets were cultured for 7 days in inositol-free RPMI 1640 containing 11.1 mM glucose and labeled with 3.7 MBq myo-[2-3H] inositol for the final 3 days. The labeled islets were then perifused under various conditions. There was a persistent increase in [3H] efflux from labeled islets stimulated with 16.7 mM glucose for 60 min. Addition of 5 mM inositol resulted in marked release of [3H] from islets and a decrease in radioactive inositol-lipid. When islets were perifused with 5 mM LiCl, the glucose-induced efflux of [3H] was greatly inhibited. The inhibitory effect of LiCl on [3H] efflux was partially corrected by the addition of 5 mM inositol. A prominent effect of LiCl was an increase in inositol monophosphate, indicating increased phospholipase C activity. This was detected within 5 min after glucose stimulation. The present data suggest that there is always very active intra-islet inositol cycling and that glucose can augument inositol-lipid metabolism.     

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.     

Effects of lactate on pancreatic islets. Lactate efflux as a possible determinant of islet-cell depolarization by glucose.

The secretion of insulin from perifused rat pancreatic islets was stimulated by raising the glucose concentration from 5.6 to 20 mM or by exposure to tolbutamide. The addition of sodium lactate (40 mM) to islets perifused in the presence of glucose (5.6 mM) resulted in a small, transient, rise in the rate of secretion. The subsequent removal of lactate, but not glucose or tolbutamide, from the perifusate produced a dramatic potentiation of insulin release. The rate of efflux of 45Ca2+ was also increased when islets were exposed to a high concentration of glucose or lactate or to tolbutamide, and again subsequently upon withdrawal of lactate. Efflux of 86Rb+ was modestly inhibited upon addition of lactate and markedly enhanced by the subsequent withdrawal of lactate from islets. The output of [14C]lactate from islets incubated in the presence of [U-14C]glucose increased linearly with increasing concentrations of glucose (1-25 mM). It is proposed that the activation of islets by the addition or withdrawal of lactate is not due to increased oxidative flux, but occurs as a result of the electrogenic passage of lactate ions across the plasma membrane, resulting in islet-cell depolarization, Ca2+ entry and insulin secretion. The production of lactate via the glycolytic pathway, and the subsequent efflux of lactate from the islet cells with concomitant exchange of H+ for Na+, could be a major determinant of depolarization and hence insulin secretion, in response to glucose.     

Stimulation of insulin secretion by transforming growth factor-beta

Effects of transforming growth factor-beta (TGF-beta) on insulin secretion were studied in rat pancreatic islets. When islets were incubated in a batch incubation system with various concentrations of TGF-beta in the presence of 2.8 mM glucose, TGF- beta increased insulin release in a concentration-dependent manner. Both TGF- beta 1 and TGF- beta 2 were equally effective. The stimulatory action of TGF- beta was greater in the presence of stimulatory concentration of glucose. In perifusion system, TGF- beta induced an immediate monotonic increase in insulin secretion. These results indicate that TGF- beta is a stimulator of insulin secretion.     

Insulin release and metabolism of calcium, adenine and adenosine 3',5'-cyclic monophosphate.

In order to assess further the mechanisms involved in insulin release, we prelabeled rat pancreatic islets of Langerhans by incubating either 45Ca or [2-3H]adenine. When prelabeled islets were perfused with a glucose-free medium (the experiment with 45Ca) and a medium containing 2.8 mM glucose (the experiment with [2-3H]adenine) respectively, a constant rate of efflux of the radioactivity was established by 30 min in each case. D-Glucose at 16.7 mM concentration elicited a rapid efflux of 45Ca and [2-3H]adenine derivatives ([3H]Ad) within 4 to 6 min after commencing the step-wise stimulation by glucose, concomitantly with insulin release. However, L-glucose and D-galactose littel stimulated both 45Ca and [3H]Ad release. Lanthanum chloride caused a burst peak of 45Ca release in the absence of glucose. A rapid efflux of 45Ca was caused by beta-D-glucose and D-glyceraldehyde to much lesser extent than by alpha-D-glucose. The slowly rising concentration of glucose at 0.1 mM/min of gradient level failed to elicit any rapid efflux of 45Ca or [3H]Ad, although insulin release occurred in accordance with an increase in glucose concentration. Even when the gradient of glucose concentration was raised to 0.7 mM/min, glucose failed to stimulate an efflux of [3H]Ad but the subsequent stimulation by 16.7 mM glucose caused a rapid efflux of [3H]Ad concomitantly with the release of insulin. No rapid efflux of 45Ca was observed under a slow-rise glucose stimulation until the gradient level of the glucose concentration was raised to 6.7 mM. Analysis of distribution of the radioactive adenine derivatives after incubation showed that the adenosine fraction had the highest radioactivity in the medium followed by the ATP, adenine and cAMP fraction in that order, and the ATP fraction had the highest radioactivity in the islet. The ratio of radioactivity in the cAMP fraction in the medium to the total count was the highest among all. On the basis of these results, it was suggested that the discharge of [3H]Ad and 45Ca might occur with the alteration of the membrane permeability induced by a rapid change of the glucose concentration, and that their discharge might perhaps link to the glucoreceptor mechanism directly controlling insulin release.     

Successful cultivation of isolated islets of Langerhans without attachment: relationship between Glucose- and theophylline-induced insulin release and insulin content in rats islets after cultivation.

The effect of various inhibitors of insulin secretion such as mannoheptulose (20 mM), atropine (1 mM), diphenylhydantoin (20 microng/ml), high concentration of Mg++ (5.3 mM) in the presence of 20 mM glucose (control) on insulin content and secretion from collagenase-isolated rat pancreatic islets was studied in vitro by cultivation of islets up to 5 or 9 days in glass Petri dishes without attachment. In a following short-term incubation for 60 min the glucose-induced insulin release without and with theophylline (5 mM) was investigated. Islets cultivated at 5 mM glucose and at 20 mM glucose with the inhibitors mannoheptulose or atropine lost the responsiveness to glucose and theophylline whereas such islets cultivated at 20 mM glucose alone or with diphenylhydantoin (DPH) or 5.3 mg Mg++ showed a stimulation of insulin secretion by glucose and theophylline. Compared, however, with freshly isolated islets all cultivated islets were restricted in their maximal glucose response and this defect was not evoked alone by quantitative changes in islet insulin content. Nevertheless, culture conditions which facilitate a net increase of insulin (content and release) during cultivation influenced also positively the glucose-induced insulin release without and with 5 mM theophylline in the following short-term experiments.     

Dual effect of formycin a upon the hydrolysis of phosphoinositides in perifused pancreatic islets

Formycin A (1.0 mM) caused a rapid, sustained and rapidly reversible inhibition of effluent radioactivity in rat pancreatic islets prelabelled with myo-[2-³H]inositol and perifused in the presence of 8.3 mM -glucose. This coincided with a progressive decrease in islet ATP content and transient inhibition of insulin release. Theraafter, however, formycin A increased glucose-induced insulin release. Moreover, in islets that were preincubated with myo-[2-³H]inositol and then exposed during perifusion to a rise in -glucose concentration from 2.8 to 16.7 mM, the release of insulin and ³H fractional outflow rate at both the low and high hexose concentrations were much higher when both the preincubation and perifusion were conducted in the presence, rather than absence, of formycin A. It is concluded that formycin A first inhibits and later enhances both the hydrolysis of phosphoinositides and release of insulin, these effects being possibly related to changes in the islet cell content of adenosine and/or formycin A triphosphates.