Effect of norepinephrine on insulin, glucagon, and somatostatin secretion in isolated perifused rat islets.

The rate of insulin, glucagon, and somatostatin secretion was measured from isolated rat islets maintained in a perifusion system. The effect of norepinephrine (NE) was simultaneously determined on the release rate of all three hormones. Norepinephrine was employed at an acute dose of 10 micrometers and in graded doses from 1 nM to 10 micrometers in the presence of high (22 mM) and low (1.4 mM) glucose conditions, insulin secretion was maximally inhibited at 10 micrometers NE concentration and was significantly depressed at 100 mM NE concentration. Under both high and low glucose conditions, glucagon release was maximally stimulated at 10 micrometers NE concentration and was significantly elevated at 10 nM NE concentration. Under high and low glucose conditions, somatostatin release was inhibited by 10 micrometers NE concentration and was significantly depressed at 100 nM NE concentration. During the initial maximal stimulation of glucagon, NE inhibition of somatostatin and insulin was prevented, possibly by the high level of glucagon released. A paracrine effect of glucagon on beta and delta cells is proposed.     

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)     

Alloxan stimulation and inhibition of insulin release from isolated rat islets of Langerhans.

The rate of alloxan-induced insulin release was measured from rat islets maintained in a simple perifusion system. Insulin release during the five-minute exposure to alloxan reached its maximum rate after two to three minutes of the exposure and then rapidly declined. This insulin release was dependent upon extracellular calcium and was associated with an increased 45Ca uptake by isolated islets. Once exposed to alloxan, however, the islets did not release insulin when stimulated again with D-glucose or alloxan. These effects of alloxan on insulin release (stimulation and subsequent inhibition) and the increased 45Ca uptake were prevented by the presence of 3-0-methyl-D-glucose during the alloxan exposure. These findings indicate a close correlation between alloxan-induced insulin release and the subsequent inhibition of further insulin release. D-glucose, when present during the entire five-minute exposure to alloxan, protected competitively against alloxan inhibition of insulin release. In addition, D-glucose, when present immediately after brief (one to three minutes) alloxan exposures, reversed some of the subsequent inhibition of insulin release. These findings suggest that alloxan and D-glucose were competing for a common site on the beta-cell. The possibility of this site being a receptor responsible for the initiation of insulin release is discussed.     

Effect of exogenous phospholipase A2 on insulin secretion from perifused rat islets

Treatment of isolated, perifused rat islets with exogenous PLA2 in amounts ranging from 1 to 1000 mU/ml caused a dose-dependent increase in the rate of insulin secretion. This effect of PLA2 was rapid and seen in the absence of added exogenous fuel. It differed from glucose-induced insulin release in temporal pattern: high concentrations of PLA2 caused a single phase of secretion, and high levels of glucose caused a biphasic pattern of secretion. Like glucose-induced release, PLA2-induced release was partially dependent on extracellular calcium because D600 caused a significant inhibition of release induced by PLA2 at 5 mU/ml. Concentrations of BW755c and NDGA, inhibitors of both the cyclooxygenase and lipoxygenase or only the lipoxygenase pathways of arachidonic acid metabolism, which completely blocked the insulin secretory response to 10 mM glucose, had no effect on the secretory response to 5 mU/ml of PLA2. These inhibitors also inhibited glucose usage by the islets. Finally, although repeated brief exposure of islets to stimulatory concentrations of glucose lead to a progressive increase in the magnitude of both the first and second phases of insulin secretion, repeated brief exposures to PLA2 lead to a progressive decrease in response to each new exposure. Nonetheless, those islets that had been exposed several times to exogenous PLA2, and no longer displayed a response to a further PLA2 exposure, responded normally to the addition of 10 mM glucose. These results indicate that PLA2 is a potent insulin secretagogue, that it shares some of the characteristics of glucose as a secretagogue, but that in many significant ways differs markedly from glucose in its effects on insulin release from isolated islets.     

Effect of 2-bromostearate on glucose-phosphorylating activities and the dynamics of insulin secretion in islets of Langerhans during fasting

Glucose-phosphorylating activity and insulin secretion were measured in homogenates of isolated rat islets and of perfused rat pancreas, respectively. Fasting for 96 h produced a significant decrease of both low-and high-Km glucose-phosphorylating activities and blocked the insulin secretory response to glucose. In the presence of glucose, 0.25 mM 2-bromostearate, a known inhibitor of fatty acid oxidation, partially restored the insulin response to glucose that was lost during fasting. This effect paralleled the restoration of glucose-phosphorylation activities (primarily the high-Km component) seen when islets isolated from 96-h-fasted rats were preincubated with 0.25 mM 2-bromostearate. It is concluded that fasting-induced adaptations of glucose-phosphorylating enzymes could account, at least in part, for the reduced insulin secretory response to glucose. 2-Bromostearate, an inhibitor of fatty acid oxidation, is able to restore both insulin secretory response and glucose-phosphorylating activities, suggesting possible interrelations among the correlated impairment in insulin secretion, islet glucose-phosphorylating activity, islet glucose metabolism, and the oxidation of fatty acids in the B-cell during fasting.     

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.     

Physiologic role of somatostatin. Insulin release from rat islets treated by somatostatin antiserum.

In order to clarify the physiologic role of somatostatin in insulin release, rat pancreatic islets treated by somatostatin antiserum were incubated in media containing various concentrations of glucose. Insulin release from antiserum-treated islets was significantly elevated above that from nontreated ones at 3.3 and 8.3 mM glucose, while the former was not different from the latter at 16.7 mM glucose. It is suggested that somatostatin plays an important role in the regulation of insulin release in the physiologic range of glucose concentration.     

Effect of dynorphin on insulin and somatostatin secretion, calcium uptake, and c-AMP levels in isolated rat islets of Langerhans

Dynorphin-[1-13], at concentrations of 5.8 X 10(-12) to 5.8 X 10(-9) M, stimulated insulin secretion from isolated islets of Langerhans of the rat, in medium containing 6 mM glucose. Higher concentrations of dynorphin had no significant effect on secretion. Dynorphin (5.8 X 10(-9) M) was unable to initiate insulin release from islets in the presence of 2 mM glucose, or to increase insulin secretion further in the presence of 20 mM glucose or 6 and 12 mM glyceraldehyde. Dynorphin-induced insulin secretion from islets was blocked by verapamil (5 microM) or by chlorpropamide (72 microM), but not by a mu opiate receptor antagonist, naloxone (0.11 microM), or by ICI 154129, a specific antagonist for the delta receptor (0.25 microM). Dynorphin had no effect on islet somatostatin secretion, under conditions in which insulin secretion was greatly stimulated. Glucose (20 mM) and glyceraldehyde (6 and 12 mM) significantly increased both insulin and somatostatin secretion. Dynorphin (5.8 X 10(-9) M) increased 45Ca2+ uptake into islets, and also increased intracellular islet c-AMP levels. These changes persisted when higher concentrations of dynorphin were used. These results suggest that (1) dynorphin is a very potent stimulus for insulin secretion; (2) dynorphin does not affect somatostatin secretion in static incubations of islets, in the same way as does glucose and glyceraldehyde; (3) dynorphin's effects may involve increased calcium ion movement and can be blocked by verapamil; (4) dynorphin can also increase islet c-AMP, and could thereby modulate the responsiveness of other secretagogues; (5) the actions of dynorphin on insulin secretion are not mediated by delta or mu opiate receptors in islets.     

Effect of alginate-polylysine-alginate microencapsulation on in vitro insulin release from rat pancreatic islets

We investigated the effect of alginate-polylysine-alginate microencapsulation on glucose-induced insulin secretion by rat islets. Applying the encapsulation method originally described by Lim, we found severely reduced in vitro insulin release (expressed as picomoles of insulin.10 islets-1.45 min-1 when incubated in 16.5 mM glucose), because the insulin release with encapsulated islets was 1.42 +/- 0.49 compared to 13.58 +/- 0.80 with free control islets. This could not be explained by inadequate permeability of the capsule, because insulin release was also severely reduced (2.12 +/- 0.61) when islets were subjected to the procedure but without the membrane-forming polylysine step. Therefore, islets were tested after having been subjected separately to each of the steps of the procedure. Insulin release was not affected by either alginate or CaCl2 but was severely reduced after prolonged suspension in saline or treatment with citrate. When saline and citrate were replaced by Ca2(+)-free Krebs-Ringer bicarbonate buffer (KRBB) and 1 mM EGTA, respectively, insulin release improved significantly both with complete and with incomplete (no polylysine step) encapsulation. This outcome was verified in a set of experiments run in parallel with islets derived from the same isolation procedure. Insulin release was 1.20 +/- 0.23 from islets encapsulated with the method of Lim and 10.73 +/- 1.04 from free control islets. With the modified procedure, insulin release was 9.17 +/- 0.52 vs. 9.61 +/- 1.27 for complete versus incomplete encapsulation, respectively. We conclude that Ca2(+)-free KRBB instead of saline and EGTA instead of citrate should be used to obtain an adequate insulin response from encapsulated islets and that the capsule membrane as such has no influence on glucose and insulin diffusion.     

Effect of muscimol on glucose-stimulated somatostatin and insulin release from the isolated, perfused rat pancreas

This study examines the effect of muscimol, a high affinity, specific gamma-aminobutyric acid (GABA) agonist, on glucose-stimulated somatostatin and insulin release from the isolated, perfused rat pancreas. Perfusion with low glucose (50 mg/dl) conditions resulted in basal somatostatin release of 46 +/- 4 pg/ml. Basal insulin release was less than 20 microU/ml. High glucose (300 mg/dl) conditions stimulated somatostatin and insulin release. Steady-state levels of somatostatin and insulin release under high glucose conditions were 425 +/- 12 pg/ml and 419 +/- 18 microU/ml, respectively. Perfusion with medium containing 1 microM muscimol inhibited glucose-stimulated somatostatin release by 38%, whereas the course of glucose-stimulated insulin release was unaffected. Tentative conclusions from this study are (1) that GABA is potentially a modulator of islet somatostatin but not insulin release, and (2) the fact that somatostatin, an inhibitor of insulin, can be suppressed 38% without coincidental increase in insulin release seems to indicate that, under high glucose conditions, somatostatin is without a significant paracrine effect on the beta-cells.     

Glucose metabolism and pulsatile insulin release from isolated islets

The effects of metabolic inhibition on insulin release and the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) were studied in individually perifused pancreatic islets from ob/ob mice. The modest basal secretion in the presence of 3 mmol/l glucose was pulsatile with a frequency of approximately 0.2/min, although [Ca(2+)](i) was stable at approximately 100 nmol/l. Introduction of 11 mmol/l glucose resulted in large amplitude oscillations of [Ca(2+)](i) and almost 20-fold stimulation of average secretion manifested as increased amplitude of the insulin pulses without change in frequency. Inhibition of glycolysis with iodoacetamide or mitochondrial metabolism with dinitrophenol or antimycin A reduced glucose-stimulated secretion back to basal levels with maintained pulsatility. The [Ca(2+)](i) responses to the metabolic inhibitors were more complex, but in general there was an initial peak and eventually sustained elevation without oscillations. When introduced in the presence of 3 mmol/l glucose, the metabolic inhibitors tended to increase the amplitude of the insulin pulses, although the simultaneous elevation in [Ca(2+)](i) occurred without oscillations. The data indicate that pulsatile secretion is regulated by factors other than [Ca(2+)](i) under basal conditions and after metabolic inhibition. Although pulsatile secretion can be driven by oscillations in metabolism when [Ca(2+)](i) is stable, it was not possible from the present data to determine whether insulin pulses have a glycolytic or mitochondrial origin.