Alginate/aminopropyl-silicate/alginate membrane immunoisolatability and insulin secretion of encapsulated islets

We utilized the sol-gel reaction to prepare an immunoisolatable membrane for a microcapsule-shaped bioartificial pancreas. The membrane, derived from two precursors, 3-(aminopropyl)trimethoxysilane (APTrMOS) and tetramethoxysilane (TMOS), was formed onto calcium-alginate gel beads via electrostatic interaction. The molecular weight cutoff point of less than 150 000 required for immunoisolation was achieved at molar ratios ([APTrMOS]/[TMOS]) ranging from 0.60 to 2.40 with the amount of APTrMOS fixed at 3.40 mmol/(10 mL of calcium-alginate). When encapsulated in a membrane prepared at the molar ratio of 0.60, the islets contracted in volume and showed no response to stimulation by a high glucose concentration. However, islets in a membrane prepared at the molar ratio of 2.40 showed no contraction and responded to the glucose stimulation at almost the same level as free islets. These results demonstrated that the molar ratio of the precursors was a dominant factor affecting membrane permeability and the insulin secretion activity of the encapsulated islets.     

Leptin suppresses basal insulin secretion from rat pancreatic islets

The effects of leptin on insulin secretion from pancreatic islets of Sprague–Dawley rats were examined in vitro. In a basal glucose medium (5.5 mM), insulin secretion from isolated islets was significantly decreased after addition of a recombinant leptin (80 nM) (3.20±0.14 nmol/10 islets/h) compared with that before the addition (4.41±0.30 nmol/10 islets/h). Although significant leptin suppression of insulin secretion was not observed under a glucose-stimulated (11.1 mM) condition, these results suggest that a negative feedback system may exist between leptin and insulin, which increases the production of leptin from adipose tissues.     

Propionate inhibits glucose-induced insulin secretion in isolated rat pancreatic islets

Dietary fibers, probably by generating short chain fatty acids (SCFA) through enterobacterial fermentation, have a beneficial effect on the control of glycemia in patients with peripheral insulin resistance. We studied the effect of propionate on glucose-induced insulin secretion in isolated rat pancreatic islets. Evidence is presented that propionate, one of the major SCFA produced in the gut, inhibits insulin secretion induced by high glucose concentrations (11.1 and 16.7 mM) in incubated and perfused pancreatic islets. This short chain fatty acid reduces [U-(14)C]-glucose decarboxylation and raises the conversion of glucose to lactate. Propionate causes a significant decrease of both [1-(14)C]- (84%) and [2-(14)C]-pyruvate (49%) decarboxylation. These findings indicate pyruvate dehydrogenase as the major site for the propionate effect. These observations led us to postulate that the reduction in glucose oxidation and the consequent decrease in the ATP/ADP ratio may be the major mechanism for the lower insulin secretion to glucose stimulus induced by propionate.     

Regulation of leucine-stimulated insulin secretion and glutamine metabolism in isolated rat islets

Glutamate dehydrogenase (GDH) is regulated by both positive (leucine and ADP) and negative (GTP and ATP) allosteric factors. We hypothesized that the phosphate potential of beta-cells regulates the sensitivity of leucine stimulation. These predictions were tested by measuring leucine-stimulated insulin secretion in perifused rat islets following glucose depletion and by tracing the nitrogen flux of [2-(15)N]glutamine using stable isotope techniques. The sensitivity of leucine stimulation was enhanced by long time (120-min) energy depletion and inhibited by glucose pretreatment. After limited 50-min glucose depletion, leucine, not alpha-ketoisocaproate, failed to stimulate insulin release. beta-Cells sensitivity to leucine is therefore proposed to be a function of GDH activation. Leucine increased the flux through GDH 3-fold compared with controls while causing insulin release. High glucose inhibited flux through both glutaminase and GDH, and leucine was unable to override this inhibition. These results clearly show that leucine induced the secretion of insulin by augmenting glutaminolysis through activating glutaminase and GDH. Glucose regulates beta-cell sensitivity to leucine by elevating the ratio of ATP and GTP to ADP and P(i) and thereby decreasing the flux through GDH and glutaminase. These mechanisms provide an explanation for hypoglycemia caused by mutations of GDH in children.     

Astragalin augments basal calcium influx and insulin secretion in rat pancreatic islets

Astragalin is a flavonol glycoside with several biological activities, including antidiabetic properties. The objective of this study was to investigate the effects of astragalin on glycaemia and insulin secretion, in vivo, and on calcium influx and insulin secretion in isolated rat pancreatic islets, ex vivo. Astragalin (1 and 10 mg / kg) was administered by oral gavage to fasted Wistar rats and serum glucose and plasma insulin were measured. Isolated pancreatic islets were used to measure basal insulin secretion and calcium influx. Astragalin (10 mg/ kg) decreased glycaemia and increased insulin secretion significantly at 15–180 min, respectively, in the glucose tolerance test. In isolated pancreatic cells, astragalin (100 μM) stimulated calcium influx through a mechanism involving ATP-dependent potassium channels, L-type voltage-dependent calcium channels, the sarcoendoplasmic reticulum calcium transport ATPase (SERCA), PKC and PKA. These findings highlight the dietary coadjuvant, astragalin, as a potential insulin secretagogue that may contribute to glucose homeostasis.     

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.     

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.     

Human and rat amylin have no effects on insulin secretion in isolated rat pancreatic islets

Amylin, an islet amyloid peptide secreted by the pancreatic beta cell, has been proposed as a humoral regulator of islet insulin secretion. Four separate preparations of amylin were tested for effects on hormone secretion in both freshly isolated and cultured rat islets and in HIT-T15, hamster insulinoma cells. With all three experimental models, exposure to human amylin acid and human and rat amylin at concentrations as high as 100 nM had no significant effect on rates of insulin or glucagon secretion. These observations suggest that amylin, even at concentrations appreciably higher than those measured in peripheral plasma, is not a significant humoral regulator of islet hormone secretion.     

Islet amyloid polypeptide inhibits glucose-stimulated insulin secretion from isolated rat pancreatic islets

Islet amyloid polypeptide has 37 amino acids and is a major component of amyloid deposition in pancreatic islets of patients with type 2 diabetes mellitus. To determine whether the peptide is involved in the impaired insulin secretion in this type of diabetes mellitus, we synthesized islet amyloid polypeptide and its fragments and examined its effect on insulin secretion. Islet amyloid polypeptide inhibited the glucose-stimulated insulin secretion from isolated rat pancreatic islets, as calcitonin gene-related peptide did, but the fragments failed to inhibit the secretion. Thus, we propose that amyloid deposition may be an important factor in the impairment of insulin secretion in type 2 diabetes mellitus.     

Oxygen and temperature dependence of stimulated insulin secretion in isolated rat islets of Langerhans

The effects of lowered O2 tension on insulin secretion and changes in cellular energy parameters were investigated in isolated rat pancreatic islets perifused with buffers equilibrated with 21, 9, 5, and 1% oxygen and containing 5 mM glucose. Decreasing the external [O2] reduced the amount of insulin released in response to 16 mM glucose, 20 mM alpha-ketoisocaproic acid, and 40 mM KCl. Secretion elicited by high glucose or KCl had declined significantly at 9% oxygen, whereas that caused by alpha-ketoisocaproic acid became inhibited below 5% O2. Lowering the oxygen tension also decreased the ability of islets to respond with a rise in [ATP]/[ADP] upon stimulation with metabolic secretagogues. This reduction in the evoked increase in the nucleotide ratios paralleled the inhibition of stimulated insulin secretion. Addition of 2 mM amytal markedly decreased the islet energy level and eliminated the secretory response to 16 mM glucose. The results suggest that enhancement of B-cell energy production and a consequent rise in [ATP] (or [ATP]/[ADP]) are a necessary event for the hormone release elicited by high glucose and alpha-ketoisocaproic acid. A decrease in temperature inhibited insulin secretion with all three secretagogues tested. The energies of activation were similar for high glucose and KCl-induced secretion, about 20 kcal/mol, but were higher for alpha-ketoisocaproic acid, about 35 kcal/mol. At 28 degrees C, the [ATP]/[ADP] was larger than that at 38 degrees C (8 versus 5) and was not increased further upon addition of 16 mM glucose. It is suggested that a decrease in the rate of energy production at lowered temperatures may contribute to the inhibition of insulin release caused by metabolic secretagogues.     

Adenosine and the regulation of insulin secretion by isolated rat islets of Langerhans.

The effect of adenosine in insulin secretion and adenylate cyclase activity of rat islets of Langerhans was investigated. Adenosine inhibited insulin secretion stimulated by glucose, glucagon, prostaglandin E2, tolbutamine and theophylline. Adenosine decreased basal adenylate cyclase activity of the islets as well as that stimulated by glucagon prostaglandin E2 and GTP, although fluoride-stimulated activity was not affected. Neither insulin secretion nor adenylate cyclase activity of the islets was affected by adenine, AMP or ADP. The inhibitory effect of adenosine on adenylate cyclase activity was not altered by either phenoxybenzamine (alpha-adrenergic blocker) or propranolol (beta-adrenergic blocker), suggesting that the effect is not mediated through the adrenergic receptors of the islet cells. These results suggest that the intracellular concentration of adenosine in the beta-cell may play a role in regulating insulin secretion and that this effect may be mediated via alterations in the activity of adenylate cyclase in the beta-cell.     

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.     

Dissociation between intracellular calcium mobilization and insulin secretion in isolated rat islets of Langerhans

The neuropeptide bombesin provoked a dose-dependent stimulation of 45Ca2+ efflux from pre-loaded islets of Langerhans. This response occurred rapidly, was not sustained and did not depend on the presence of extracellular calcium, suggesting that it resulted from the mobilization of intracellular calcium stores. Under conditions when large increases in 45Ca2+ efflux were observed, bombesin completely failed to stimulate the rate of insulin secretion. Similar results were also obtained with the muscarinic cholinergic agonist, carbachol. The data suggest that the release of calcium from intracellular pools is not sufficient to induce an increase in insulin secretion in normal islet cells.     

Transglutaminase involvement in the secretion of insulin from electropermeabilised rat islets of langerhans

Ca²⁺-Induced insulin release from electropermeabilised islets is inhibited by the transglutaminase inhibitors monodansylcadaverine, glycine methylester, methylamine and cystamine but not by the control compounds dimethyl monodansylcadaverine and sarcosine methylester which lack the primary amine group. Neither monodansylcadaverine nor glycine methylester inhibited insulin secretion induced by either cAMP or the phorbol ester PMA at basal levels (10 nM) of Ca²⁺. These data provide further evidence for the involvement of transglutaminase in Ca²⁺ induced insulin secretion, they also suggest that insulin secretion induced by either cAMP or PMA may act in part by a mechanism independent of that induced by Ca²⁺.     

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.     

Phosphoinositides turnover and insulin secretion in pancreatic islets

The relationship between glucose-induced insulin secretion and metabolism of inositol phospholipid was investigated by means of an islet perifusion method and direct measuring of inositol phosphates after sonicating the islets. The results showed that the time course of inositol phospholipid breakdown is coincident with the first phase of glucose-induced insulin secretion. Analysis of the effluent perifusate as well as the water soluble inositol-containing substance after sonication of stimulated islets revealed that most of the metabolite of inositol phospholipid is inositol-triphosphate, the hydrolysis product of phosphatidylinositol-4,5-bisphosphate. On the other hand, perifusion of islets with exogenous inositol-triphosphate showed a monophasic and dose-dependent response of insulin secretion. Thus, the initial process of glucose stimulation is accompanied with the formation of inositol-triphosphate, which is a possible candidate for the triggering of first phase insulin secretion.     

Blockade of IRS1 in isolated rat pancreatic islets improves glucose-induced insulin secretion

Several neural, hormonal and biochemical inputs actively participate in the balance of insulin secretion induced by blood glucose fluctuations. The exact role of insulin as an autocrine and paracrine participant in the control of its own secretion remains to be determined, mostly due to insufficient knowledge about the molecular phenomena that govern insulin signaling in pancreatic islets. In the present experiments we demonstrate that higher insulin receptor and insulin receptor substrates-1 and -2 (IRS1 and IRS2) concentrations are predominantly encountered in cells of the periphery of rat pancreatic islets, as compared to centrally located cells, and that partial blockade of IRS1 protein expression by antisense oligonucleotide treatment leads to improved insulin secretion induced by glucose overload, which is accompanied by lower steady-state glucagon secretion and blunted glucose-induced glucagon fall. These data reinforce the inhibitory role of insulin upon its own secretion in isolated, undisrupted pancreatic islets.     

Difference in mechanism between glyceraldehyde- and glucose-induced insulin secretion from isolated rat pancreatic islets

The effects of D-glyceraldehyde and glucose on islet function were compared in order to investigate the difference between them in the mechanism by which they induce insulin secretion. The stimulation of insulin secretion from isolated rat islets by 10 mM glyceraldehyde was not completely inhibited by either 150 microM diazoxide (an opener of ATP-sensitive K(+) channels) or 5 microM nitrendipine (an L-type Ca(2+)-channel blocker), whereas the stimulation of insulin secretion by 20 mM glucose was completely inhibited by either drug. The insulin secretion induced by glyceraldehyde was less augmented by 100 microM carbachol (a cholinergic agonist) than that induced by glucose. The stimulation of myo-inositol phosphate production by 100 microM carbachol was more marked in islets incubated with the hexose than with the triose. The content of glyceraldehyde 3-phosphate, a glycolytic intermediate, in islets incubated with glyceraldehyde was far higher than that in islets incubated with glucose, whereas the ATP content in islets incubated with the triose was significantly lower than that in islets incubated with the hexose. These results suggest that glyceraldehyde not only mimics the effect of glucose on insulin secretion but also has the ability to cause the secretion of insulin without the influx of Ca(2+ )through voltage-dependent Ca(2+) channels. The reason for the lower potency of the triose than the hexose in stimulating insulin secretion is also discussed.     

Effects of dehydrouramil on protein phosphorylation and insulin secretion in rat islets of Langerhans.

Dehydrouramil hydrate hydrochloride (DHU), a stable analogue of alloxan, inhibited the phosphorylation of an endogenous protein of Mr 53,000 catalysed by a Ca2+-calmodulin-dependent protein kinase in extracts of islets of Langerhans. The concentration of DHU required for 50% inhibition was 0.09 mM. DHU did not inhibit islet cyclic AMP-dependent protein kinase and caused only slight inhibition of Ca2+-phospholipid-dependent protein kinase. Inhibition of Ca2+-calmodulin-dependent protein kinase was neither prevented nor reversed by dithiothreitol. DHU did not affect the ability of calmodulin to activate cyclic AMP phosphodiesterase. In intact islets, pre-exposure to DHU impaired the insulin-secretory response to glucose and blocked the potentiatory effect on insulin secretion of forskolin, an activator of adenylate cyclase, and of tetradecanoylphorbol acetate (TPA), an activator of Ca2+-phospholipid-dependent protein kinase. The increase in islet cyclic AMP elicited by forskolin was not affected by DHU. The data are consistent with the hypothesis that protein phosphorylation catalysed by a Ca2+-calmodulin-dependent protein kinase may play a central role in the regulation of insulin secretion.