Pulsatile insulin secretion in isolated rat islets

The pancreas secretes insulin in an oscillatory fashion, but the precise site of the pacemaker for pulsatile insulin secretion has not been identified. These studies were designed to determine whether islets also secrete insulin in a pulsatile fashion if they are isolated from their pancreatic milieu. Isolated rat islets (80-100) were perifused 8 h in culture medium after overnight incubation, and samples were collected at 3.3-min intervals. Insulin secretion was evaluated for pulsatility with the Clifton Cycle Detection Program. Perifusion of islets was associated with a spontaneous, persistent, and regular pulsatility of insulin secretion, which was observed in all conditions tested. Perifusion with medium containing 5.5 mM glucose (n = 11) demonstrated oscillations with a mean periodicity of 17.6 +/- 1.1 min and a mean amplitude of 4.8 +/- 0.4 microU/ml when overall mean insulin concentration was 16.7 +/- 2.4 microU/ml. When the glucose concentration was 16.7 mM (n = 9), overall mean insulin concentration was 54.4 +/- 2.6 microU/ml, with increases in periodicity (22.0 +/- 1.3 min) and amplitude (10.7 +/- 0.5 microU/ml). All measurements were significantly different from those observed during perifusion with 5.5 mM glucose (P less than 0.02-0.001). Theophylline (1 mM) also enhanced the overall mean insulin concentration and amplitude (69.4 +/- 10.4 and 14.2 +/- 1.2 microU/ml, respectively) compared with control studies without theophylline (16.7 +/- 5.3 and 4.3 +/- 0.5 microU/ml) (P less than 0.01). The period of the cycle was also increased from 17.5 +/- 1.1 to 26.4 +/- 6.3 min, but this was not significantly different from the control group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arachidonic acid metabolism and insulin secretion by isolated human pancreatic islets

Isolated human pancreatic islets converted [3H8]arachidonate to compounds with the high-performance liquid-chromatographic mobility of cyclooxygenase products, including prostaglandin E2 (PGE2), PGF2 alpha, and the lipoxygenase product 12-HETE. Human islet synthesis of PGE2, PGF2 alpha, and 12-HETE from endogenous arachidonate was demonstrated with stable isotope dilution-gas chromatographic-negative ion-chemical ionization-mass spectrometric analysis. Pharmacologic inhibition of arachidonate metabolism by both lipoxygenase and cyclooxygenase pathways with BW 755C strongly suppressed glucose-induced insulin secretion from perifused human islets, and the selective cyclooxygenase inhibitor indomethacin enhanced insulin secretion. These findings are similar to those reported for islets isolated from rats and suggest that arachidonate metabolites may modulate glucose-induced insulin secretion in humans.     

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.     

Evaluation of insulin secretion of isolated rat islets cultured in extracellular matrix

Islet isolation involves enzymatic digestion of the interstitial matrix and mechanical disruption of the tissue. It is possible that a fundamental change of islet biology resulting from the loss of critical factors required for islet function or survival will occur. Extracellular matrix (ECM) is one of the most important components of the islet microenvironment. Reconstruction of the cell-matrix relationship seems to be effective for improving the loss of differentiated islet structure and function. The purpose of this study was to characterize and compare the effects of collagen gel mixture or Matrigel on beta-cell function and islet cell survival. After isolation by the collagenase digestion technique, rat islets were divided and cultured with various types of collagen gel mixture. They were assessed for their glucose-stimulated insulin secretion and cell viability. Glucose-induced insulin secretion of islets cultured with collagen type I gel or a mixture of collagen type I and IV was improved after 11 days in culture. In conclusion, a type of gel composed of collagen type I and/or type IV as an islet microenvironment is sufficient to maintain glucose responsiveness and may be useful for islet transplantation.     

Cyclosporin-induced inhibition of insulin secretion in isolated rat islets and HIT cells

The effects of cyclosporin, an immunosuppressive agent used in diabetic and nondiabetic patients, on in vitro glucose-induced insulin secretion were evaluated in isolated islets and in a glucose-responsive clonal beta-cell line (HIT cells). Cyclosporin inhibited glucose-induced insulin secretion in a dose-response manner at concentrations commonly found in human blood. With isolated islets, four time periods (0-5, 5-15, 15-30, and 30-60 min) were examined after stimulation with 300 mg/dl glucose. Inhibition of insulin secretion by cyclosporin was evident by 5-15 min as well as during later times with progressively smaller drug concentrations. With HIT cells, longer-term effects were examined after 16 h of incubation with various drug concentrations. Inhibition of insulin secretion was again observed, and these inhibitory effects were not reversed by drug washout. A cyclosporin concentration of 0.1 microgram/ml, which is a therapeutic blood level in humans, was sufficient to inhibit insulin secretion in both in vitro models. Patients using this drug should be carefully monitored for signs of deficient insulin secretion.     

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.     

Lack of islet amyloid polypeptide regulation of insulin biosynthesis or secretion in normal rat islets

We examined the effects of rat islet amyloid polypeptide (IAPP) on insulin biosynthesis and secretion by isolated rat islets of Langerhans. Culture of islets for 24 h in the presence of 10(-6) M IAPP and 5.5 mM glucose had no effect on insulin mRNA levels. Similarly, the rates of proinsulin biosynthesis were not altered in islets incubated in 10(-4)-10(-9) M IAPP and 5.5 mM glucose, nor was the rate of conversion of proinsulin to insulin changed at 10(-6) M IAPP. Addition of 10(-5) M IAPP to islets incubated in 11 mM glucose decreased the fractional insulin secretion rate; however, the secretion of newly synthesized proinsulin and insulin was not affected. These data indicate that it is unlikely that IAPP is a physiologically relevant modulator of insulin biosynthesis or secretion.     

Interleukin 1 is potent modulator of insulin secretion from isolated rat islets of Langerhans

The effects of interleukin 1 (IL-1) on glucose-induced insulin secretion from isolated rat islets of Langerhans have been examined. IL-1 both inhibits and stimulates glucose-induced insulin secretion depending on the experimental design. Inhibition of glucose-induced insulin secretion was observed after a 15-h treatment of islets with either purified IL-1, murine recombinant IL-1 (rIL-1), or human rIL-1, rIL-1 inhibition of glucose-induced insulin secretion was dose dependent with half-maximal inhibition observed at 25 pM human rIL-1. Basal insulin secretion was not affected by rIL-1 treatment. Mannose- and leucine-induced insulin secretion was also inhibited by a 15-h treatment with human rIL-1. Islets treated 15 h with inhibitory concentrations of murine IL-1 were morphologically intact, well granulated, and retained normal concentrations of insulin compared with control islets. Furthermore, human rIL-1 treatment did not affect the islet plasma membrane permeability as assessed by the measurement of the islet intracellular volume. Finally, the viability of islets treated 15 h with murine rIL-1 was demonstrated by the observation that the inhibitory effects of murine rIL-1 on glucose-induced insulin secretion were reversible. In addition to the inhibitory effects of IL-1 on glucose-induced insulin secretion, purified IL-1 and human rIL-1 had stimulatory effects on glucose-induced insulin secretion under the following conditions: a 90-min incubation with purified IL-1 (10% vol/vol) or in the presence of human rIL-1 (1400 pM) or a 15-h incubation with relatively low concentrations of human rIL-1 (0.5 or 5 pM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of monensin on conversion of proinsulin to insulin and secretion of newly synthesized insulin in isolated rat islets

When isolated rat islets were incubated with 10(-10) - 10(-6) M monensin, a sodium and proton ionophore, glucose-stimulated insulin release was inhibited in a concentration- and time-dependent manner. After removal of monensin, inhibition of insulin secretion persisted during stimulation with a variety of secretagogues, including 5 mM glucose plus 15 mM arginine, 20 mM glucose, and 20 mM glucose plus 1 mM 3-isobutyl-1-methylxanthine. Within the same low range of monensin concentrations, proteolytic conversion of newly synthesized proinsulin to insulin was also blocked. At each concentration, prohormone-to-hormone conversion was inhibited to almost the same extent as inhibition of insulin secretion. Therefore, both processes may have equal or common dependency on a subcellular ionic gradient. Although monensin decreased total insulin secretion, the glucose-regulated marking process was unaffected. Regardless of the monensin concentration or the overall rate of insulin secretion, the percentage of secreted newly synthesized versus older insulin remained the same, and the threefold differences in the fractional secretory rates of newly synthesized versus total insulin also remained the same. Thus, rather than specifically blocking protein traffic through the Golgi apparatus of the beta cell, monensin probably first inhibited insulin secretion by disrupting proton gradients in secretory vesicles and, thereby, also inhibited other processes occurring within this organelle.     

Factors influencing insulin secretion from encapsulated islets

Adequate regulation of glucose levels by a microencapsulated pancreatic islet graft requires a minute-to-minute regulation of blood glucose. To design such a transplant, it is mandatory to have sufficient insight in factors influencing the kinetics of insulin secretion by encapsulated islets. The present study investigates factors influencing the glucose-induced insulin response of encapsulated islets in vitro. We applied static incubations and did the following observations. (i) Small islets (90-120 microm) showed a similar instead of a lower glucose-induced insulin response, suggesting that inclusion of only small islets, which are associated with lower protrusion and failing rates, has no consequences for the functional performance of the graft. (ii) A capsule diameter of 800 microm showed identical rather than lower glucose-induced insulin responses as smaller, 500-microm capsules. (iii) Capsule membranes constructed with a conventional permeability interfered with diffusion of insulin, as illustrated by a lower response of islets in capsules with a 10-min poly-L-lysine (PLL) membrane than islets in capsules with a 5-min PLL membrane. (iv) Irrespective of the tested porosity, the capsules provided sufficient immunoprotection because the 10-min PLL membranes did block diffusion of the cytokines IL-1beta (17 kDa) and TNF-alpha (70 kDa) while the 5-min PLL membranes interfered with the diffusion of the vast majority of the cytokines. We conclude that capsules containing small islets (90-120 microm) and a membrane with a lower permeability than routinely applied is preferred in order to obtain a graft with adequate glucose-induced insulin responses.     

Human islet amyloid polypeptide oligomers disrupt cell coupling, induce apoptosis, and impair insulin secretion in isolated human islets

Insulin secretion from the 2,000-3,000 beta-cells in an islet is a highly synchronized activity with discharge of insulin in coordinate secretory bursts at approximately 4-min intervals. Insulin secretion progressively declines in type 2 diabetes and following islet transplantation. Both are characterized by the presence of islet amyloid derived from islet amyloid polypeptide (IAPP). In the present studies, we examined the action of extracellular human IAPP (h-IAPP) on morphology and function of human islets. Because oligomers of h-IAPP are known to cause membrane disruption, we questioned if application of h-IAPP oligomers to human islets would lead to disruption of islet architecture (specifically cell-to-cell adherence) and a decrease in coordinate function (e.g., increased entropy of insulin secretion and diminished coordinate secretory bursts). Both hypotheses are affirmed, leading to a novel hypothesis for impaired insulin secretion in type 2 diabetes and following islet transplantation, specifically disrupted cell-to-cell adherence in islets through the actions of membrane-disrupting IAPP oligomers.     

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.     

Adenovirus-mediated XIAP gene transfer reverses the negative effects of immunosuppressive drugs on insulin secretion and cell viability of isolated human islets

Immunosuppressive drugs are routinely used to provide tolerance after whole pancreas and islet cell transplantations. While they are essential in inhibiting graft rejection, little is known about their effect on islet function and beta-cell viability. In this study, we report that tacrolimus, sirolimus, and mycophenolic acid, when added to cultures of freshly isolated human islets, induce a downregulation of the synthesis and secretion of insulin. These functional changes are associated with decreased islet cell viability. All three agents induce a decrease of intracellular levels of Bcl-2 and Bcl-xL, with an increased level of Smac, indicating that they are capable of promoting a downregulation of anti-apoptotic factors and an accumulation of pro-apoptotic mediators. Transduction of islet cells with the anti-apoptotic gene XIAP prevents the negative effects of these drugs on the function and viability of islets. XIAP-infected cells show a higher expression of phospho-CREB (cAMP-responsive element binding protein) and a reduced level of Smac, resulting in a significant reduction of apoptotic cells and a preservation of the glucose-dependent secretion of insulin. In conclusion, the present study demonstrates that genetically modified human islets expressing XIAP are resistant to the negative effects of immunosuppressive drugs on insulin secretion and cell viability.     

G-proteins and hormonal inhibition of insulin secretion from HIT-T15 cells and isolated rat islets.

G-proteins are important mediators of hormonal inhibition of insulin secretion. To characterize the pertussis toxin-sensitive substrates present in HIT cell membranes, we performed immunoblots with specific antisera and found evidence for the presence of Gi alpha 1, Gi alpha 2, Gi alpha 3, and three forms of Go alpha. We observed that pertussis toxin-sensitive substrates mediate all of the effects of SRIF, and a major portion of the effects of EPI, on insulin secretion from rat islets during static incubations. These results agree with our previously reported studies examining phasic glucose-induced insulin secretion from HIT cells. To ascertain whether inhibition of adenylate cyclase, presumably involving coupling of the catalytic subunit to Gi, may be a common mechanism for both hormones, we studied the effects of 8-bromo-cyclic AMP and found that this agent partially prevented the inhibitory effects of both hormones. We also observed that the inhibitory effects of SRIF and EPI on insulin were nonadditive, that both hormones were additive to nickel chloride during inhibition of insulin release, and that they noncompetitively inhibited glipizide-induced insulin secretion through pertussis toxin-sensitive mechanisms. Together, these results suggest that both hormones exert their effects on insulin secretion at multiple G-protein-regulated sites including adenylate cyclase and sites distal to the glipizide-binding site on the KATP channel.     

Phosphate depletion impairs insulin secretion by pancreatic islets.

Phosphate depletion (PD) is associated with resistance to the peripheral action of insulin and with glucose intolerance. However, data on the effect of PD on insulin secretion are not consistent, and were derived indirectly by measurements of blood levels of insulin during intravenous glucose tolerance test (IVGTT) or with hyperglycemic clamp technique. Direct evidence for an effect of PD on insulin secretion by pancreatic islets is not available, and the potential mechanisms through which PD may affect insulin secretion are not known. We performed IVGTT, examined in vitro insulin secretion by pancreatic islets, and evaluated various factors involved in insulin secretion in PD and pair weighed (PW) rats. PD animals had fasting hyperglycemia and normal plasma insulin levels, and displayed abnormal IVGTT as compared to PW rats. Both initial and late phases of D-glucose-induced insulin secretion from islets were markedly and significantly (P less than 0.01) lower than from islets of PW rats. In contrast, D-glyceraldehyde-induced insulin release in PD rats was similar to that of PW rats. [H3]2-deoxyglucose uptake by islets and their cyclic AMP content after exposure to D-glucose, D-glyceraldehyde or forskolin were not different among the two groups of animals. Insulin content in PD islets was modestly but significantly (P less than 0.01) higher than PW islets. In PD islets, ATP content and the ATP/ADP ratio at basal state and after incubation with 16.7 mM D-glucose were significantly (P less than 0.01) lower and resting cytosolic calcium was significantly (P less than 0.01) higher than in PW islets.(ABSTRACT TRUNCATED AT 250 WORDS)