Induction of memory in rat pancreatic islets by tolbutamide. Dependence on ambient glucose level, calcium, and phosphoinositide hydrolysis

The ability of the sulfonylurea tolbutamide to induce insulin output, increase phosphoinositide (PI) hydrolysis, and modulate the insulin response to other agonists was assessed. At 200 microM, tolbutamide increased both insulin release and the efflux of 3H from [3H]inositol-prelabeled islets only in the presence of 5.5 or 7 mM glucose. When the glucose level was maintained at 2.75 mM, tolbutamide (200 microM) had no positive impact on either parameter. The calcium-influx inhibitor nitrendipine (200 nM) blocked the effects of 200 microM tolbutamide (with 7 mM glucose) on 3H efflux and insulin output. Prior exposure of islets to tolbutamide (200 microM) in the presence of 7 mM glucose amplified their subsequent insulin response to 10 mM glucose and 5 mM glyceraldehyde. The effect of 200 microM tolbutamide (with 7 mM glucose) was blocked by nitrendipine. Furthermore, the effect of 200 microM tolbutamide was not observed with 2.75 mM glucose; however, if the level of tolbutamide was increased to 1 mM, both PI hydrolysis and potentiated release to subsequent stimulation with 10 mM glucose were observed. Tolbutamide (200 microM with 7 mM glucose) stimulation for 20 min resulted in an increase in 3H efflux from [3H]inositol-prelabeled islets. Despite the rapid fall in insulin secretion, elevated rates of 3H efflux persisted long after the removal of the sulfonylurea from the medium. The duration of the 3H-efflux response paralleled the duration of potentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of cholecystokinin and glucose in rat pancreatic islets

The effects of sulfated cholecystokinin (CCK-8S) and glucose on insulin secretion and polyphosphoinositide (PPI) metabolism were studied in isolated rat islets. Both agonists stimulate PPI hydrolysis, inositol phosphate accumulation, 3H efflux from [3H]inositol-prelabeled tissue, and 45Ca efflux from prelabeled cells. However, the effects of CCK-8S on PPI metabolism are considerably greater than those of glucose. Furthermore, the effects of CCK-8S on PPI and Ca2+ metabolism are observed whether islets are incubated in either 2.75 or 7 mM glucose, but CCK-8S only stimulates insulin secretion (a biphasic response) when the higher glucose concentration is present. Addition of 1 microM forskolin to islets incubated in media containing 2.75 mM glucose does not influence basal insulin secretion but sensitizes the islets to the action of CCK-8S. In the presence of forskolin, CCK-8S induces a very marked first phase but no second phase of insulin secretion. We postulate that CCK-8S acts in this tissue via receptor-linked PPI hydrolysis, leading to an inositol trisphosphate-induced Ca2+ efflux. These receptor-mediated effects of CCK-8S are not altered either by the ambient glucose concentration or the cAMP content of the islets, but these two factors determine the responsiveness of the islets (in terms of insulin secretion) to a given CCK-8S signal.     

Influence of cAMP and calcium on [3H]inositol efflux, inositol phosphate accumulation, and insulin release from isolated rat islets

The influence of cyclic AMP (cAMP) and extracellular calcium on phosphoinositide (PI) hydrolysis in isolated islets was assessed and related to insulin output. Three stimulants were chosen to activate the beta-cell: sulfated cholecystokinin (CCK-8S, 200 nM), high-level glucose (20 mM), and the sulfonylurea tolbutamide (200 microM). The insulin secretory response to all three agonists was amplified by forskolin (which increases cAMP levels) and reduced by nitrendipine (which decreases calcium influx). All three stimulants increased the hydrolysis of inositol-containing phospholipids, an event monitored by an increase in [3H]inositol efflux from [3H]inositol-prelabeled islets and by the accumulation of labeled inositol phosphates. Forskolin, despite its positive impact on insulin secretion, reduced [3H]inositol efflux and inositol phosphate accumulation in response to all agonists. A similar inhibitory effect on these parameters was noted with nitrendipine; however, nitrendipine abolished secretion in response to all agonists. These findings support the following conclusions: 1) an increase in cellular cAMP levels reduces the quantitative impact of various agonists on these indices of PI hydrolysis; 2) despite this inhibitory effect, cAMP amplifies the insulin secretory response to these agonists; and 3) extracellular calcium is a crucial determinant of both PI hydrolysis and the ensuing insulin secretory response.     

Prior cholecystokinin exposure sensitizes islets of Langerhans to glucose stimulation

Prior, short-term exposure of isolated perifused islets to cholecystokinin (CCK8S) sensitizes them to subsequent glucose stimulation. This sensitization effect develops quickly and persists long after the removal of CCK8S from the perifusion medium. Continued binding of CCK8S to its receptor on the beta-cell and the increase in glucose metabolism noted with glucose stimulation are essential for the full expression of this response. This sensitization process may play an integral role in the postulated incretin effect of the peptide.     

Insulin secretory response to secretagogues by perifused islets from chronically glucose-infused rats

Perifused islets from rats infused for 7 days with 40% glucose exhibited an altered secretory response to selected stimuli. Both phases of insulin release were blunted when 20 mM L-leucine was tested; the secretory response to a subsequent leucine stimulation was also blunted compared with the control group. The ability of 20 mM alpha-ketoisocaproate to stimulate the release of insulin was also greatly diminished in islets from glucose-infused rats. The secretory response to 50 microM tolbutamide plus 7 mM glucose by perifused islets from glucose-infused rats was 45% lower than in the control group. In addition, the response to a subsequent 10 mM glucose stimulation was lost. On the other hand, islets from glucose-infused rats responded to 20 microM forskolin plus 16.7 mM glucose with on significant change in the amount of insulin released during both phases of stimulation compared with the control group. The response to 100 nM phorbol 12-myristate 13-acetate was 3.1-fold higher in islets from glucose-infused compared with saline-infused rats. The finding that chronic infusions of glucose lead to selective impairment of the secretory response to fuel stimuli and agents such as tolbutamide that act on metabolically regulated K+ channels gives support to the notion that alterations in the generation of metabolic coupling signals might be involved in the phenomenon described here.     

Stimulatory effects of cholecystokinin on isolated perifused islets inhibited by potent and specific antagonist L 364718 [corrected

The influence of L 364718 on islet responsiveness to sulfated cholecystokinin (CCK-8S) was investigated. In islets whose inositol-containing phospholipids were prelabeled during a 2-h incubation period, subsequent exposure to L 364718 (1 nM) significantly impaired the secretion of insulin usually noted in response to 200 nM CCK-8S in the simultaneous presence of 7 mM glucose. A higher level of the antagonist (10 nM) completely abolished insulin secretion. L 364718 (1-10 nM) reduced the efflux of 3H from myo-[2-3H]-inositol prelabeled islets in parallel with the reduction in secretion. L 364718 (10 nM) significantly reduced the accumulation of 3H-containing inositol phosphates usually noted with CCK-8S addition. L 364718, at levels 10- to 100-fold greater than those necessary to attenuate CCK-8S-induced insulin secretion, had no adverse effect on the insulin secretory response of freshly isolated islets to 10 mM glucose alone, 5 mM D-glyceraldehyde, 15 mM alpha-ketoisocaproate, or 50 ng/ml gastric inhibitory polypeptide. L 364718 (1000 nM) had no adverse influence on carbamylcholine (1 mM)-induced phosphoinositide hydrolysis. These results establish L 364718 as a potent and highly selective antagonist of cholecystokinin's stimulatory actions on beta-cells. Because of its potency, selectivity, and oral effectiveness, in vivo studies with L 364718, aimed at unraveling the pleiotropic effects of CCK-8S on glucose and insulin homeostasis, seem feasible.     

Morphometric studies of secretory granule distribution and association with microtubules in beta-cells of rat islets during glucose stimulation

Morphometric studies of beta-cell granule positions and interactions with microtubules in areas of exocytosis were performed on islets of rats injected with tolbutamide for 72 h. This treatment depleted granules 85% below normal control values and rendered mapping of their positions and visualization of microtubules more feasible during electron microscopy. Islets were perifused with 2.8 or 16.7 mM glucose and were fixed at 4 min (acute phase) or 20 min (second phase). Photomicrographs of beta-cell sections were digitized, and computer replicas were compared to computer-simulated cells of similar size and shape into which an identical number of granules was inserted randomly. Margination of granules into a zone within 1.5 micron of the plasma membrane was examined. At both 2.8 and 16.7 mM glucose, real cells marginated significantly more secretory granules than did simulated cell pairs. Within the zones of margination during 16.7 mM glucose perifusion, the number of secretory granules associated with microtubules was less than 6% in the acute phase and less than 1% in the second phase. These data suggest that glucose stimulation promotes movement of secretory granules into the beta-cell periphery to a greater extent than can be attributed to chance alone. The role of microtubules in peripheral granule movement into regions of exocytosis appears to be minimal.     

Chloride modulation of insulin release, 86Rb+ efflux, and 45Ca2+ fluxes in rat islets stimulated by various secretagogues

Substitution of extracellular Cl- by impermeant isethionate (5 mM residual Cl-) caused a monophasic inhibition of glucose-stimulated insulin release, accompanied by an initial transient increase and a secondary lasting decrease in 86Rb+ efflux from perifused islets. Cl- reintroduction restored insulin release with an overshoot above control values and successively produced a small decrease and a large increase in efflux. Theophylline potentiated the insulinotropic effect of glucose more markedly at low Cl- than at normal Cl-, but did not restore a normal rate of 86Rb+ efflux. Lowering the concentration of Cl- did not alter the effect of glucose, tolbutamide, or arginine on 86Rb+ efflux, but simply shifted the efflux rates to lower values. The first phase of glucose-stimulated insulin release was not modified, but the second phase was inhibited. The insulinotropic effect of tolbutamide was augmented at low Cl- and that of arginine (at 7 mM glucose) was not affected. In incubated islets, the stimulation of insulin release by glyceraldehyde was barely inhibited when Cl- was substituted by isethionate and the marked decrease of the effect of glucose could be prevented by glutamine. In a glucose-free, low Cl- medium, the insulinotropic effect of leucine, arginine, and lysine was inhibited; this inhibition was reversed by glutamine, but not by theophylline. Lowering the concentration of Cl- had no effect on 45Ca2+ influx or efflux in the absence of glucose, did not alter the increase in influx and efflux during the first 5 min of glucose stimulation, but impaired both influx and efflux during the second phase. Leucine-induced 45Ca2+ uptake was inhibited at low Cl- and this inhibition was prevented by glutamine. In conclusion, islet cells possess a Cl- -activated modality of K efflux, which does not seem to play a role in the stimulus-secretion coupling. Since Cl- substitution by an impermeant anion does not inhibit the stimulation of insulin release by all agents, the role of Cl- ions does not appear to be restricted to a chemiosmotic mechanism of exocytosis. No single mechanism explains the multiple changes in B-cell function resulting from the decrease in Cl- concentration, but it is proposed that some of them could result from modifications of intracellular pH.     

Phosphoinositide hydrolysis and insulin release from isolated perifused rat islets. Studies with glucose

The ability of glucose to promote the hydrolysis of prelabeled [2-3H]inositol-containing phosphoinositides (PI) was assessed by measuring the efflux of 3H in response to glucose and the accumulation of labeled inositol phosphates. The inclusion of nonradioactive inositol (1 mM) in the perifusion medium dramatically improved our ability to monitor glucose-induced increases in 3H efflux. Efflux studies with this method revealed the following. 1) 3H efflux is significantly greater at 7 than at 2.75 mM glucose, and this parallels a small but significant increase in insulin secretion. 2) D-manno-Heptulose reduces 3H efflux with 7 mM glucose to a level approximating that seen in the presence of 2.75 mM glucose and has no effect on 3H efflux with 2.75 mM glucose. 3) In the presence of 20 mM glucose plus 1 mM inositol, 3H efflux is rapid and biphasic, a response that parallels the timing and amplitude of the biphasic pattern of insulin secretion. Direct measurements of labeled inositol and inositol phosphate levels in islets revealed the following. 4) After 50 min of perifusion with 2.75 or 7 mM glucose, labeled inositol phosphates were significantly greater with 7 mM glucose. 5) In response to 20 mM glucose alone, islet levels of free inositol, inositol monophosphate (IP1), and inositol bisphosphate (IP2) increased. 6) In response to 20 mM glucose plus 1 mM cold inositol, islet levels of free inositol increased, whereas islet levels of IP1, IP2, and inositol trisphosphate (IP3) were reduced compared with values obtained with 20 mM glucose alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulatory role of fructose-2,6-bisphosphate in pancreatic islet glucose metabolism remains unsettled

Fructose-2,6-P2 was measured in perifused, isolated rat pancreatic islets. Fructose-2,6-P2 was present in pancreatic islets at low levels approximately equal to fructose-2,6-P2 content of liver from fasted rats. In islets perifused with glucose at physiologic concentrations, fructose-2,6-P2 was increased from 0.8 microM in the presence of 5.5 mM glucose to 1.0 microM at 10 mM glucose and 1.3 microM at 16.7 mM glucose, but did not increase further at higher glucose concentration. Therefore, only modest increases in the phosphofructokinase-1 activator, fructose-2,6-P2, occur at glucose concentrations stimulating insulin secretion.     

Effects of cryopreservation on in vitro and in vivo long-term function of human islets.

BACKGROUND: The possibility of performing transplantation several days after explant seems to be a peculiarity of islet grafts, and the opportunity to cryopreserve human islets may permit an indefinite period for modulating the recipient immune system. The aim of the present study was the evaluation of in vitro and in vivo functional properties of cryopreserved human islets. METHODS: We used six consecutive human islet preparations not suitable for an immediate transplantation in diabetic patients because the limited islet mass separated. The in vitro function of cryo and fresh islets was studied by determination of insulin and glucagon secretion in response to such classical stimuli as glucose (16.7 mM), glucose (16.7 mM) + 3-isobutyl-1-methylxanthine (0.1 mM), arginine (10 mM), and tolbutamide (100 microM). In vivo islet function was assessed through intravenous glucose tolerance tests performed at 15, 30, 60, and 90 days after transplantation of 1000 hand-picked fresh or cryopreserved islets in nude mice. RESULTS: Basal secretion of true insulin was significantly higher in cryopreserved islets than in fresh ones. The response of cryopreserved islets to arginine and glucose + isobutyl-1-methylxanthine seemed partially impaired. Proinsulin-like molecule secretion seemed higher in cryopreserved than in fresh islets in response to all secretagogues used, and the difference was statistically significant for arginine. The capacity of human cryopreserved islets to maintain a correct metabolic control in diabetic nude mice was progressively lost in 3 months. CONCLUSIONS: These findings showed that cryopreservation affects the function of isolated human islets, maintaining in vivo function for a limited period of time.     

Age-dependent stimulation of neonatal insulin release and inositol phosphate accumulation by CCK-8 and carbachol

Development of a robust insulin secretory response to glucose occurs during the early neonatal period. To determine if neuroendocrine agents play a role during this time, we studied the effects of selected peptides and neurotransmitters on insulin release and polyphosphoinositide metabolism in islets isolated from 1- and 3-day neonatal rats. Vasoactive intestinal peptide had no effect on glucose-stimulated release in either islet population. In contrast, sulfated cholecystokinin octapeptide (CCK-8) significantly enhanced glucose-induced insulin release in both islet groups. One-day islets were stimulated only by a concentration of 300 nM, whereas 3-day islets were responsive at 3 nM. Similar to CCK-8, there were clear differences in responses to carbachol between 1- and 3-day islets. One-day islets required a concentration of 200 microM for insulin release to be significantly greater than with glucose alone; 3-day islet insulin release was significant at 2 microM carbachol. Both agonists stimulated inositol phosphate accumulation in 3-day islets, but only CCK-8 caused a significant increase over glucose-induced levels in 1-day islets. These results indicate that islet responsiveness to CCK-8 and carbachol develops in parallel during the early neonatal period. This development may be linked to the maturation of a critical step of stimulus-secretion coupling through which these agents act.     

Nutrient control of insulin secretion in isolated normal human islets

Pancreatic islets were isolated from 16 nondiabetic organ donors and, after culture for approximately 2 days in 5 mmol/l glucose, were perifused to characterize nutrient-induced insulin secretion in human islets. Stepwise increases from 0 to 30 mmol/l glucose (eight 30-min steps) evoked concentration-dependent insulin secretion with a threshold at 3-4 mmol/l glucose, K(m) at 6.5 mmol/l glucose, and V(max) at 15 mmol/l glucose. An increase from 1 to 15 mmol/l glucose induced biphasic insulin secretion with a prominent first phase (peak increase of approximately 18-fold) and a sustained, flat second phase ( approximately 10-fold increase), which were both potentiated by forskolin. The central role of ATP-sensitive K(+) channels in the response to glucose was established by abrogation of insulin secretion by diazoxide and reversible restoration by tolbutamide. Depolarization with tolbutamide or KCl (plus diazoxide) triggered rapid insulin secretion in 1 mmol/l glucose. Subsequent application of 15 mmol/l glucose further increased insulin secretion, showing that the amplifying pathway is operative. In control medium, glutamine alone was ineffective, but its combination with leucine or nonmetabolized 2-amino-bicyclo [2,2,1]-heptane-2-carboxylic acid (BCH) evoked rapid insulin secretion. The effect of BCH was larger in low glucose than in high glucose. In contrast, the insulin secretion response to arginine or a mixture of four amino acids was potentiated by glucose or tolbutamide. Palmitate slightly augmented insulin secretion only at the supraphysiological palmitate-to-albumin ratio of 5. Inosine and membrane-permeant analogs of pyruvate, glutamate, or succinate increased insulin secretion in 3 and 10 mmol/l glucose, whereas lactate and pyruvate had no effect. In conclusion, nutrient-induced insulin secretion in normal human islets is larger than often reported. Its characteristics are globally similar to those of insulin secretion by rodent islets, with both triggering and amplifying pathways. The pattern of the biphasic response to glucose is superimposable on that in mouse islets, but the concentration-response curve is shifted to the left, and various nutrients, in particular amino acids, influence insulin secretion within the physiological range of glucose concentrations.     

Interleukin 1 inhibits insulin secretion from isolated perifused rat islets

Preincubation of collagenase-isolated rat islets for 150 min with 100 U/ml purified human interleukin 1 (IL-1) altered their ability to secrete insulin. Whereas basal release rates with 4 mM glucose were comparable in control and IL-1-treated islets, both the first and second phases of release in response to 20 mM glucose were significantly reduced from IL-1-treated tissue. IL-1 pretreatment also impaired the secretory response to the combination of 100 nM cholecystokinin plus 7 mM glucose. However, the secretory response to 10 mM alpha-ketoisocaproate was comparable in control and IL-1-pretreated islets. Reducing the IL-1 exposure time to 60 min was accompanied by an augmented first phase of release to 20 mM glucose. Second phase secretion was diminished. The use of glucose measured after the perifusion was similar in control and IL-1-treated islets. Similar to other compounds that adversely impact on beta-cell viability, the inhibitory effect of IL-1 on release may presage a cytotoxic action of monokine.     

Islet insulin release and net calcium retention in vitro in vitamin D-deficient rats

In our previous studies, perifused islets from vitamin D-deficient (D-def) rats showed marked impairment of glucose-induced biphasic release, accounted for at least in part by a decrease in food intake. In studies reported here, we test whether D-def rat islets have an impaired response to 5.6 mM glucose or tolbutamide, (T), and if so, whether this impairment is related to a decrease in food intake or a defect in islet calcium metabolism. We isolated islets of normal rats, D-def rats, and rats pair fed (PF) to D-def rats. Biphasic insulin release from perifused islets and net 45Ca retention in lot-incubated islets were measured in response to 5.6 mM glucose, 0.37 mM T, or both. Compared with secretion from normal islets, biphasic insulin release from islets of both D-def rats and PF rats was diminished by greater than 50% in response to 5.6 mM glucose alone or 5.6 mM glucose plus T. Insulin secretion was not significantly different between islets of D-def rats and islets of PF rats. In contrast, net calcium retention in islets of D-def rats was decreased to 68% of retention in islets of PF rats. However, net calcium retention in islets of both PF and D-def rats increased in response to T. The pair-feeding experiments suggest that the decrease in insulin release from islets of D-def rats is due to the decrease in food intake associated with the D-def state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of alpha- and beta-cell secretory responses in islets isolated with collagenase and in the isolated perfused pancreas of rats.

The inhibitory actions of somatostatin (100 ng./ml.) on insulin release, stimulated by high glucose (20 mM), and on glucagon release, stimulated by arginine (15 mM), were studied with two in vitro systems: the isolated perifused rat islets prepared by the collagenase procedure and the isolated perfused rat pancreas. Suppression of arginine-induced glucagon release by glucose (20 mM) and glyceraldehyde (5 mM) was also assessed in both systems. With the perfused pancreas, somatostatin caused 32 per cent inhibition of glucose-mediated insulin release and inhibited arginine-induced glucagon release by 72 per cent. In the same system, glucose and glyceraldehyde were similarly potent inhibitors of arginine-induced glucagon secretion. In contrast to the isolated perfused pancreas, there was no significant somatostation suppression of glucose-induced insulin release or arginine-induced glucagon release whether the inhibitor was present prior to or was added during stimulation by glucose or arginine. Furthermore, glucose was only minimally active and glyceraldehyde ineffective in inhibiting glucagon secretion due to arginine in the perifusion system. The most plausible explanation for the difference in the endocrine response of islet cells in the two types of widely used in vitro systems is that the alpha and beta cells have lost inhibitory receptors in the plasma membrane as a result of the collagenase isolation technic.     

Differential effects of age versus glycemic stimulation on the maturation of insulin stimulus-secretion coupling during culture of fetal rat islets

We have cultured islets from 21.5-day-old fetal rats for 1-7 days in RMPI 1640/10% fetal calf serum containing 2.8 or 11.1 mM glucose to evaluate the differential effects of age vis-à-vis glycemic stimulation on the maturation of selected components of stimulus-secretion coupling. After 1 day of culture in either media, acute stimulation with 3.0 mg/ml glucose during basal perifusion with 0.5 mg/ml glucose elicited only a small first phase of stimulated insulin secretion and no second phase. The acute exposure to 3.0 mg/ml glucose produced no change in the prevailing high rates of oxygen consumption (QO2) and caused only minor increments in phosphate efflux (i.e., peak values for phosphate flush of 126 +/- 16% of baseline for islets that had been cultured in 11.1 mM glucose and 162 +/- 30% for islets cultured in 2.8 mM glucose). After 7 days of culture in 11.1 mM glucose, acute stimulation with 3.0 mg/ml glucose increased Qo2 (as in adult islets) and effected acute increases in the AT32P and GT32P content of prelabeled islets. The 3.0 mg/ml glucose also triggered phosphate flush to 599 +/- 45% of baseline and elicited first as well as early second phases of stimulated insulin secretion that replicated the performance of adult islets. By contrast, after 7 days of culture in 2.8 mM glucose, similar acute exposures of fetal islets to 3.0 mg/ml glucose effected only a small first phase and a negligible second phase of stimulated insulin secretion despite the occurrence of the same increments in Qo2 as after culture in 11.1 mM glucose, highly significant increases in AT32P and GT32P, and phosphate flushes that peaked at 306 +/- 16% of basal values. Thus, the ontogeny of individual components of stimulus-secretion coupling may occur in asynchronous fashion and varying require glycemic stimulation in addition to aging per se. The capacities to augment efflux of orthophosphate, enhance respiration, and heighten nucleotide turnover in response to acute stimulation with glucose seem to mature in large measure in time-dependent fashion, whereas some chronic exposure to ambient glucose in excess of basal levels may be required to establish and/or maintain the other coupled components that underlie biphasic stimulated insulin release. However, we did not achieve full exocytotic maturation even after 7 days of culture with 11.1 mM glucose.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of insulin secretion from isolated rat islets by SaRI 59-801

Oral administration of SaRI 59-801 (DL-alpha-[dimethylaminomethyl]-2-[3-ethyl-5-methyl-4-isoxazolyl]-1H- indole-3-methanol) has been reported to decrease blood glucose in several species and to elevate plasma insulin in rats and mice. In studies with isolated rat pancreatic islets incubated 1 h with 3 mM glucose, 0.05 mM 59-801 produced a significant increase in insulin secretion, and 0.3 mM produced maximum release. 59-801 (0.3 mM) stimulated insulin release 4-5-fold from islets incubated with 0, 3, or 5 mM glucose but had little effect on the high rates of release obtained at 10 or 20 mM glucose. Ten millimolar mannoheptulose, which inhibits phosphorylation of glucose and blocks glucose-stimulated insulin release, had little effect on the stimulation of insulin release by 0.3 mM 59-801 from islets incubated with 3 mM glucose. Stimulation of insulin release in the absence of glucose or in the presence of 3 mM glucose plus 10 mM mannoheptulose suggests that glucose metabolism is not required for the action of 59-801. The rate of conversion of 5 mM [5(-3)H]-glucose to 3H2O by islets, a measure of the rate of glycolysis, was not affected by 59-801. The potency, dependency on glucose concentration, lack of inhibition by mannoheptulose, and lack of effect on glycolysis of 59-801 were similar to that of tolbutamide. However, proinsulin synthesis by islets incubated with 5.55 mM glucose was not affected by 0.5 mM 59-801, but was inhibited 72% and 67% by 0.5 mM tolbutamide and 0.1 mM glibenclamide, respectively.     

Evolution of insulin secretory response to glucose by perifused islets from lean (FA/FA) rats chronically infused with glucose

Chronic infusion of nondiabetic rats with glucose for up to 7 days modified the insulin secretory response by subsequently perifused islets. Thus, on the 1st day of infusion with 40% glucose, the islets responded to 16.7 mM glucose with a 3.9-fold increase in insulin release during the first 10 min with no significant change during the second-phase insulin output compared with the control group. On the 2nd day, there was a 2-fold enhancement of insulin release during the initial 10 min of stimulation, the second phase being a similar to control islets. On the 5th day of infusion, the pattern of insulin release was not significantly different from the control group. After 7 day of infusion, there was a 46% decrease in first-phase and a 33% decrease in second-phase insulin response to glucose. The response to 10 mM arginine plus 5 mM glucose was not modified by chronic glucose infusion. Priming experiments indicate that islets from rats infused for 7 days were not able to recover the normal pattern of secretion in vitro. Islets from 7-day glucose-infused rats contained 75% more protein and had a significantly higher insulin content than control islet, suggesting that insulin synthesis is not involved in the loss of the response observed. Glucose metabolism by the islets was modified by glucose infusion with a significant increase in glucose utilization and no changes in glucose oxidation, suggesting that alterations in mitochondrial oxidative events are involved in the phenomenon of desensitization.     

Effect of fasting on the release of insulin and somatostatin from perifused islets of Langerhans.

Release of somatostatin and insulin from perifused islets of fasted and control rats was compared. After a fasting period of 48 h glucose-induced insulin release but not somatostatin release was diminished. Islets from fasted rats released significantly more somatostatin in the presence of 3.3 mM glucose than islets from controls. Simultaneously, the somatostatin content of isolated islets from fasting rats was significantly decreased. The results indicate that the low secretory activity of islet B cells in the fasting state is associated with a high secretory activity of islet D cells.