Inhibition Of Glucose-stimulated Insulin Release Research Articles

Islet amyloid polypeptide acts on glucose‐ stimulated beta cells to reduce voltage‐gated calcium channel activation, intracellular Ca2+ concentration, and insulin secretion

the mechanism by which islet amyloid polypeptide (IAPP) inhibits insulin release is unclear. We hypothesized that reduced voltage-gated calcium channel activity and intracellular Ca(2+) concentration might contribute to IAPP-mediated inhibition of glucose-stimulated insulin release. rat islet beta cells were cultured and treated with various extracellular concentrations of IAPP, and insulin release was stimulated via addition of glucose. Activation voltage, high voltage-gated calcium channel currents, intracellular Ca(2+) concentration, and insulin secretion were detected by patch clamp electrophysiology, fluorescent digital imaging microscopy using calcium-sensitive fluorescent dye, and radioimmunoassay, respectively. high voltage-gated calcium channel currents, intracellular Ca(2+) concentration, and insulin secretion increased in a dose-dependent manner when rat beta cells were exposed to glucose. After short-term IAPP treatment (5 or 10 µM), these parameters decreased significantly in glucose-stimulated beta cells. However, no significant changes were observed with lower doses of IAPP. glucose-stimulated islet beta-cell high voltage-gated calcium channels were activated in conjunction with insulin secretion, while high extracellular concentrations of IAPP inhibited beta-cell high voltage-gated calcium channel activation and insulin secretion in a dose-dependent manner.

Expression of islet inducible nitric oxide synthase and inhibition of glucose-stimulated insulin release after long-term lipid infusion in the rat is counteracted by PACAP27

Chronic exposure of pancreatic islets to elevated plasma lipids (lipotoxicity) can lead to beta-cell dysfunction, with overtime becoming irreversible. We examined, by confocal microscopy and biochemistry, whether the expression of islet inducible nitric oxide synthase (iNOS) and the concomitant inhibition of glucose-stimulated insulin release seen after lipid infusion in rats was modulated by the islet neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP)27. Lipid infusion for 8 days induced a strong expression of islet iNOS, which was mainly confined to beta-cells and was still evident after incubating islets at 8.3 mmol/l glucose. This was accompanied by a high iNOS-derived NO generation, a decreased insulin release, and increased cyclic GMP accumulation. No iNOS expression was found in control islets. Addition of PACAP27 to incubated islets from lipid-infused rats resulted in loss of iNOS protein expression, increased cyclic AMP, decreased cyclic GMP, and suppression of the activities of neuronal constitutive (nc)NOS and iNOS and increased glucose-stimulated insulin response. These effects were reversed by the PKA inhibitor H-89. The suppression of islet iNOS expression induced by PACAP27 was not affected by the proteasome inhibitor MG-132, which by itself induced the loss of iNOS protein, making a direct proteasomal involvement less likely. Our results suggest that PACAP27 through its cyclic AMP- and PKA-stimulating capacity strongly suppresses not only ncNOS but, importantly, also the lipid-induced stimulation of iNOS expression, possibly by a nonproteasomal mechanism. Thus PACAP27 restores the impairment of glucose-stimulated insulin release and additionally might induce cytoprotection against deleterious actions of iNOS-derived NO in beta-cells.

Effects of Antioxidants Coenzyme Q10 and Lipoic Acid on Interleukin-1β-Mediated Inhibition of Glucose-Stimulated Insulin Release from Cultured Mouse Pancreatic Islets

Effects of Antioxidants Coenzyme Q10 and Lipoic Acid on Interleukin-1β-Mediated Inhibition of Glucose-Stimulated Insulin Release from Cultured Mouse Pancreatic Islets

Effects of Antioxidants Coenzyme Q10 and Lipoic Acid on Interleukin-1β-Mediated Inhibition of Glucose-Stimulated Insulin Release from Cultured Mouse Pancreatic Islets

During the development of the autoimmune disease, insulin-dependent diabetes mellitus (IDDM) islet cell death is thought to be mediated in part by oxygen and nitrogen free radicals and interleukin 1β (IL-1β), secreted by activated macrophages. Free radicals disrupt the homeostasis of biological systems by damaging major constituent molecules such as lipids, proteins, and DNA. Islet cells are quite susceptible to oxidative damage due to low levels of antioxidant enzymes involved in free radical consumption. If IDDM is associated with an imbalance of oxidative stresses and antioxidant responses in islet cells, then it may be possible to ameliorate disease by supplementating antioxidant defenses. In this study, the antioxidants coenzyme Q10 and lipoic acid were able to block IL-1β-mediated inhibition of glucose-stimulated insulin secretion from islet cells at 10− 12 M and 10− 9 M, respectively.

Inhibition of glucose-induced electrical activity in rat pancreatic β-cells by DCPIB, a selective inhibitor of volume-sensitive anion currents

We have investigated the effects of the ethacrynic acid derivative 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB), an inhibitor of the volume-sensitive anion channel (VSAC), on electrical activity and insulin secretion in rat pancreatic β-cells. DCPIB inhibited whole-cell VSAC currents in β-cells with IC 50 values of 2.2 and 1.7 μM for inhibition of outward and inward currents, respectively. DCPIB also inhibited the VSAC at the single channel level in cells activated by glucose. In intact cells, DCPIB caused a net increase in β-cell input conductance and evoked an outward current that was sensitive to inhibition by tolbutamide, suggesting K ATP channel activation. However, no K ATP channel activation was evident under conventional whole-cell conditions, suggesting that the drug might activate the channel in intact cells via an indirect mechanism, possibly involving nutrient metabolism. DCPIB suppressed glucose-induced electrical activity in β-cells, hyperpolarised the cell membrane potential at a substimulatory glucose concentration and prevented depolarisation when the glucose concentration was raised to stimulatory levels. The suppression of electrical activity by DCPIB was associated with a marked inhibition of glucose-stimulated insulin release from intact islets. It is concluded that DCPIB inhibits electrical and secretory activity in the β-cell as a combined result of a reciprocal inhibition of VSAC and activation of K ATP channel activities, thus producing a marked hyperpolarisation of the β-cell membrane potential.

Evidence for a local angiotensin-generating system and dose-dependent inhibition of glucose-stimulated insulin release by angiotensin II in isolated pancreatic islets

A local angiotensin-generating system has been found in the exocrine pancreas. This study aimed, primarily, to investigate the existence of a local angiotensin-generating system in the pancreatic islets and, secondly, to elucidate its role in regulating insulin secretion. Real-time RT-PCR and western blot were used to investigate if angiotensin-generating components are present in the mouse pancreatic islets, which are subject to regulation by islet transplantation. The localisation of AT1-receptors in islets was investigated by immunohistochemistry. Batch-type incubations of isolated islets were applied for studying the influence of angiotensin II on the glucose-stimulated insulin release, glucose oxidation and (pro)insulin, and total protein biosynthesis. Major components, namely angiotensinogen, ACE, AT1- and AT2-receptors, were expressed in endogenous islets. AT1-receptors were localised to pancreatic beta cells. Exposure of the isolated islets to angiotensin II induced a dose-dependent inhibition of glucose-stimulated insulin release and inhibited (pro)insulin biosynthesis. This inhibitory action was fully preventable by pretreatment of the islets with losartan, an AT1-receptor antagonist. We also investigated if the expression of these components was changed after islet transplantation. Notably, a markedly increased expression of mRNA for the AT1-receptor was observed in islets retrieved from 4-week-old syngeneic islet transplants, a finding that was confirmed at the protein level. These data indicate the existence of an islet angiotensin-generating system of potential importance in the physiological regulation of glucose-induced insulin secretion, thus diabetes mellitus. The increased expression of the AT1-receptor in islet transplants could have relevance to islet-graft function.

Interferon regulatory factor-1 down-regulates cytokine-induced IP-10 expression in pancreatic islets

Background. Interferon (IFN)-γ acts synergistically with interleukin (IL)-1β and tumor necrosis factor (TNF)-α to activate isoform of nitric oxide synthetase (iNOS) gene expression, induce apoptosis, and impair glucose-stimulated insulin release (GSIR) in pancreatic islets. This effect is an important mechanism of islet dysfunction in models of pancreatitis, type I diabetes, and islet allograft rejection. We tested the hypothesis that transcription factor interferon regulatory factor (IRF)-1 plays a regulatory role in both this cytokine-induced islet injury and cytokine-induced gene expression for chemotactic chemokines. Methods. Isolated islets from wild-type (WT) or IRF-1−/− C57BL/6 mice were cultured in a mixture of IL-1β, TNF-α, and IFN-γ ± the iNOS inhibitor L-NMMA. The following end points were assessed: i) GSIR; ii) rates of apoptosis; and iii) gene expression for iNOS, IRF-1 and inducible protein (IP)-10, monocyte chemoattractive protein (MCP)-1, macrophage inflammatory protein-1α, and macrophage inflammatory protein-1β. Results. Cytokine-treated WT islets demonstrated an increase in IRF-1 and iNOS gene expression, inhibition of GSIR, increased rates of apoptosis, and increased gene transcription and protein release for IP-10 and MCP-1. Cytokine-treated IRF-1−/− islets demonstrated relatively less iNOS gene expression, preserved GSIR, reduced rates of apoptosis, and a more marked increase in transcription for IP-10 and MCP-1 and in IP-10 protein release. L-NMMA-cotreated WT islets were completely resistant to cytokine-induced dysfunction and apoptosis but demonstrated the same degree of cytokine-induced chemokine gene expression as cytokine-treated WT without L-NMMA. Conclusion. IFN-γ, IL-1β, and TNF-α in combination induce chemokine gene expression in pancreatic islets. Transcription factor IRF-1 mediates cytokine-induced islet dysfunction, apoptosis, and iNOS gene expression but down-regulates IP-10 gene expression. (Surgery 2003;134:134-41.)

Effects of interleukin-15 on suppression of rat pancreatic islets in vitro induced by proinflammatory cytokines

The cytokine IL-15 might contribute to inflammatory processes, but also act as an inhibitor of apoptosis in different cell lines. Furthermore, it has been reported that islet cells express IL-15 after exposure to proinflammatory cytokines, which could indicate a defence reaction. We aimed in this study to investigate if IL-15 could influence cell death and/or functional impairment of rat pancreatic islets induced by in vitro exposure to a combination of cytokines (25 U/ml IL-1β+1000 U/ml IFN-γ+1000 U/ml TNF-α). The effect of IL-15 itself on the function of rat pancreatic islets was also studied. Isolated rat islets were exposed for 24 h to IL-15 at different concentrations in the presence or absence of the cytokine mixture. The cytokines caused a strong inhibition of glucose-stimulated insulin release and the glucose oxidation rates. IL-15 (0.1–10 ng/ml) could not prevent the functional suppression caused by these effects. The cytokine combination caused a decline in whole islet DNA content and a marked increase in non-viable cells analysed by propidium iodide (PI) and annexin V staining. However, there was no significant decrease in whole islet DNA content when IL-15 (0.1 or 1.0 ng/ml) was present together with the cytokine mixture. On the other hand, IL-15 failed to influence the increase in cell death after PI and annexin V staining. If anything, IL-15 alone had a slight stimulatory effect (glucose oxidation rate) on islet cells. In conclusion, we can not exclude that IL-15 might antagonize some cytokine mediated cell death in islet cells, however, IL-15 fails to counteract functional suppression induced by cytokines.

Mastoparan Stimulates GABA Release from MIN6 Cells: Relationship between SNARE Proteins and Mastoparan Action

We examined the action of mastoparan on β cell exocytosis. Mastoparan stimulated GABA and insulin release from MIN6 β cells. On the other hand, mastopraran-induced GABA release was decreased by expressing the tetanus toxin C1 light chain in MIN6 cells. We have then investigated the relationship between SNARE proteins and mastoparan action using adenovirus-mediated gene transfer system. Overexpression of t-SNAREs, syntaxin 1A, and SNAP-25 inhibited the mastoparan-induced insulin release by approximately half-fold of control levels, however, the mastoparan-induced GABA release was not affected by these t-SNAREs overexpression. The overexpression of mutant α-SNAP (1–285), which inhibits the wild-type α-SNAP function in a dominant negative manner, did not influence either mastoparan-induced GABA or insulin release in spite of its marked inhibition of glucose-stimulated insulin release. Our data indicate that mastoparan stimulates GABA exocytosis via vesicular transport; however, SNARE proteins are differently involved in the exocytosis of insulin and GABA.

Progesterone inhibits insulin secretion by a membrane delimited, non-genomic action.

In rat islets, progesterone caused a prompt concentration-dependent inhibition of glucose-stimulated insulin release with an IC50 of 10 microM at 8.4mM glucose. The inhibition was specific since both testosterone and 17beta-estradiol had no such effect. The degree of inhibition was similar in islets from male and female rats. The inhibition was not blocked in PTX-treated islets thus ruling out the Gi/Go proteins as mediators of the inhibition. Progesterone inhibited both glucose- and BayK-8644-stimulated insulin secretion in HIT-T15 cells and the IC50 vs. 10 mM glucose was also 10 microM. There was no effect on intracellular cyclic AMP concentration in the presence 0.2 and 10 mM glucose. Progesterone decreased [Ca2+]i under all conditions tested. The decrease in [Ca2+]i was due to blockade of the L-type voltage-dependent Ca2+ channels. Under Ca(2+)-free conditions, progesterone did not inhibit the stimulation of insulin release due to the combination of glucose, phorbol ester and forskolin. Thus blockade of Ca2+ entry appears to be the sole mechanism by which progesterone inhibits insulin release. As progesterone covalently linked to albumin had a similar inhibitory effect as progesterone itself, it is concluded that the steroid acts at the outer surface of the beta-cell plasma membrane. These effects would be classified as either AI or AIIb in the Mannheim classification of nongenomically initiated steroid actions.

Apoptosis and mitochondrial damage in INS-1 cells treated with alloxan.

To evaluate the participation of mitochondrial damage, oxygen radicals and cell death in diabetes mellitus, we designed a way to investigate INS-1 cells, rat pancreatic beta-cell line, to die by treatment with alloxan which generate reactive oxygen species (ROS). Incubation of INS-1 cells with alloxan for 24 h resulted in a decrease in viability of cells as well as inhibition of glucose-stimulated insulin release; this could be prevented by antioxidants, vitamin E and butylated hydroxyanisol (BHA). The formation of a DNA ladder and the distribution of phosphatidylserine at the external surface of plasma membrane were observed as indicators of apoptosis in the cells treated with alloxan at concentrations below 0.5 mM. The formation of DNA ladder was prevented by vitamin E, BHA and catalase, suggesting that the ROS is involved in the process of apoptosis in INS-1 cells treated with alloxan. Lower levels of intracellular ATP, collapse of mitochondrial membrane potential and release of cytochrome c from mitochondria were also observed in INS-1 cells treated with alloxan, suggesting that alloxan caused the damage of mitochondria in cells and was related to the process of apoptosis. In contrast, rat liver RLC-18 cells treated with alloxan were not observed in the decrease of viability. It follows from the present study that mitochondrial damages by ROS generated from alloxan is linked to apoptosis in INS-1 cells.

Nitric oxide, islet acid glucan-1,4-alpha-glucosidase activity and nutrient-stimulated insulin secretion.

The mechanism of nutrient-evoked insulin release is clearly complex. One part of that mechanism is postulated to be the activation of the glycogenolytic enzyme acid glucan-1,4-alpha-glucosidase. As nitric oxide (NO) has been found to be a potent inhibitor of glucose-stimulated insulin secretion, we have now investigated a possible influence of exogenous NO and inhibition of endogenous NO production on islet acid glucan-1,4-alpha-glucosidase activity in relation to insulin release stimulated by glucose and l-arginine. In isolated islets, NO derived from the intracellular NO donor hydroxylamine inhibited the activation of acid glucan-1, 4-alpha-glucosidase and its isoform acid alpha-glucosidase in parallel with inhibition of glucose-stimulated insulin release. In comparison, other lysosomal enzymes were largely unaffected. Similarly, the spontaneous NO donor sodium nitroprusside, as well as NO gas, when added to islet homogenates, suppressed the activities of these acid alpha-glucosidehydrolases and, to a lesser extent, the activities of other lysosomal enzymes. Finally, in the presence of the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester, insulin release from isolated islets stimulated by glucose or l-arginine was markedly potentiated in parallel with an accompanying increase in the activities of acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase. Other lysosomal enzymes and neutral alpha-glucosidase were not influenced. We propose that an important inhibitory effect of NO on the insulin secretory processes stimulated by glucose and l-arginine is exerted via inactivation of islet acid glucan-1,4-alpha-glucosidase, a putative key enzyme in nutrient-stimulated insulin release.

Protective mechanism of glucose against alloxan-induced β-cell damage: pivotal role of ATP

Glucose prevents the development of diabetes induced by alloxan. In the present study, the protective mechanism of glucose against alloxan-induced beta-cell damage was investigated using HIT-T 15 cell, a Syrian hamster transformed beta-cell line. Alloxan caused beta-cell damages with DNA fragmentation, inhibition of glucose-stimulated insulin release, and decrease of cellular ATP level, but all of these beta-cell damages by alloxan were prevented by the presence of 20 mM glucose. Oligomycin, a specific inhibitor of ATP synthase, completely abolished the protective effects of glucose against alloxan-induced cell damage. Furthermore, treatment of nuclei isolated from HIT-T15 cells with ATP significantly prevented the DNA fragmentation induced by Ca2+. The results indicate that ATP produced during glucose metabolism plays a pivotal role in the protection of glucose against alloxan-induced beta-cell damage.

Interleukin-1β-induced Rat Pancreatic Islet Nitric Oxide Synthesis Requires Both the p38 and Extracellular Signal-regulated Kinase 1/2 Mitogen-activated Protein Kinases

Interleukin-1beta (IL-1beta) is cytotoxic to rat pancreatic beta-cells by inhibiting glucose oxidation, causing DNA damage and inducing apoptosis. Nitric oxide (NO) is a necessary but not sufficient mediator of these effects. IL-1beta induced kinase activity toward Elk-1, activation transcription factor 2, c-Jun, and heat shock protein 25 in rat islets. By Western blotting with phosphospecific antibodies and by immunocomplex kinase assay, IL-1beta was shown to activate extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (p38) in islets and rat insulinoma cells. Specific ERK1/2 and p38 inhibitors individually reduced but in combination blocked IL-1beta-mediated islet NO synthesis, and reverse transcription-polymerase chain reaction of inducible NO synthase mRNA showed that ERK1/2 and p38 controlled IL-1beta-induced islet inducible NO synthase expression at the transcriptional level. Hyperosmolarity caused phosphorylation of Elk-1, activation transcription factor 2, and heat shock protein 25 and activation of ERK1/2 and p38 in islets comparable to that induced by IL-1beta but did not lead to NO synthesis. Inhibition of p38 but not of ERK1/2 attenuated IL-1beta-mediated inhibition of glucose-stimulated insulin release. We conclude that ERK1/2 and p38 activation is necessary but not sufficient for IL-1beta-mediated beta-cell NO synthesis and that p38 is involved in signaling of NO-independent effects of IL-1beta in beta-cells.

Acute and Chronic Effects of Leptin on Glucose Utilization in Lean Mice

Experiments described here show that in vivo glucose uptake is impaired in mice given 30 μg leptin by intraperitoneal injection 2 hours before an oral glucose tolerance test (GTT). When mice were infused for 7 days with 10 μg/day leptin, the 4-fold increase in circulating leptin caused a transient hypophagia, a sustained weight loss and significantly inhibited insulin release in response to an oral GTT. Adipocytes from these mice were not insulin responsive whereas insulin-stimulated muscle and liver glycogen synthesis were increased. In contrast, leptin added to 2 hour in vitro incubations had an insulin-like effect on muscle glucose utilization and augmented insulin stimulation of adipocyte lipid synthesis. Thus, normal mice treated chronically with leptin develop tissue specific changes in insulin sensitivity and compensate for inhibition of glucose-stimulated insulin release. The contrasting response to acute leptin exposure suggests these changes are not a direct effect of the protein.

Insulin-releasing action of the novel antidiabetic agent BTS 67 582.

1. BTS 67582 (1,1-dimethyl-2-(2-morpholinophenyl)guanidine fumarate) is a novel antidiabetic agent with a short-acting insulin-releasing effect. This study examined its mode of action in the clonal B-cell line BRIN-BD11. 2. BTS 67582 increased insulin release from BRIN-BD11 cells in a concentration-dependent manner (10[-8] to 10[-4] M) at both non-stimulating (1.1 mM) and stimulating (16.7 mM) concentrations of glucose. 3. BTS 67582 (10[-4] M) potentiated the insulin-releasing effect of a depolarizing concentration of K+ (30 mM), whereas the K+ channel openers pinacidil (400 microM) and diazoxide (300 microM) inhibited BTS 67582-induced release. 4. Suppression of Ca+ channel activity with verapamil (20 microM) reduced the insulin-releasing effect of BTS 67582 (10[-4] M). 5. BTS 67582 (10[-4] M) potentiated insulin release induced by amino acids (10 mM), and enhanced the combined stimulant effects of glucose plus either the fatty acid palmitate (10 mM), or agents which raise intracellular cyclic AMP concentrations (25 microM forskolin and 1 mM isobutylmethylxanthine), or the cholinoceptor agonist carbachol (100 microM). 6. Inhibition of glucose-stimulated insulin release by adrenaline or noradrenaline (10 microM) was partially reversed by BTS 67582 (10[-4] M). 7. These data suggest that the insulin-releasing effect of BTS 67582 involves regulation of ATP-sensitive K+ channel activity and Ca2+ influx, and that the drug augments the stimulant effects of nutrient insulin secretagogues and agents which enhance adenylate cyclase and phospholipase C. BTS 67582 may also exert insulin-releasing effects independently of ATP-sensitive K+ channel activity.

Nitric oxide donor SIN-1 inhibits insulin release.

Preceding the onset of insulin-dependent diabetes mellitus, pancreatic islets are infiltrated by macrophages secreting interleukin-1 beta, which exerts cytotoxic and inhibitory actions on islet beta-cell insulin secretion through induction of nitric oxide (NO) synthesis. The influence of the NO donor 3-morpholinosydnonimine (SIN-1) on insulin secretion from isolated pancreatic islets in response to various secretagogues was investigated. Stimulation of insulin release evoked by glucose, phospholipase C activation with carbachol, and protein kinase C activation with phorbol ester were obtained by SIN-1, whereas the response to adenylyl cyclase activation or K(+)-induced depolarization was not affected. It is concluded that enzymes involved in glucose catabolism, phospholipase C or protein kinase C, may be targeted by NO. Reversal of SIN-1 inhibition of glucose-stimulated insulin release by dithiothreitol suggests that NO may inhibit insulin secretion partly by S-nitrosylation of thiol residues in key proteins in the stimulus-secretion coupling. These adverse effects of NO on the beta-cell stimulus-secretion coupling may be of importance for the development of the impaired insulin secretion characterizing diabetes mellitus.

Effects of aminoguanidine on rat pancreatic islets in culture and on the pancreatic islet blood flow of anaesthetized rats

Aminoguanidine ( AG; ⩽0.5 mM) is a potent inhibitor of the inducible form of nitric oxide synthase (iNOS) and, at higher concentrations, is also able to prevent advanced glycosylation of proteins. Due to these properties, AG might be an interesting therapeutic compound for prevention of the development of diabetes and for prevention of diabetes complications. In the present study, we examined the effect of AG (0.1, 0.5, 1.0, 5.0, or 10 mM) on prolonged in vitro culture of isolated rat pancreatic islets. Furthermore, the acute effect of AG on pancreatic and islet blood flow in anaesthetized rats was studied with a microsphere technique. Culture for 6 days of pancreatic islets at either 11.1 mM or 28 mM glucose, in the presence of 0.1–1.0 mM AG, was not toxic to the islet cells or impaired insulin secretion. However, when islets were cultured for 8 days with the addition of 5 mM AG at 11.1 mM or 28 mM glucose, a 50% inhibition of glucose-stimulated insulin release was observed. Rats injected intravenously with AG (1, 10, or 50 mg/kg body weight) had a decreased pancreatic blood flow 30 min later. Glucose injection (1 g/kg body weight) increased the islet blood flow, and this effect was not attenuated by AG. The present data suggest that AG, when used in concentrations that inhibit iNOS, can affect pancreatic blood flow, but appears not to be directly harmful to β-cell function.

Interleukin-10 Stimulates Rat Pancreatic Islets in Vitro, but Fails to Protect against Interleukin-1

Rat pancreatic islets were cultured for 42 h in medium supplemented without or with 0.1 - 10 ng/ml of human interleukin-10 (IL-10). Medium insulin accumulation was increased by 50% after culture with 0.1 ng/ml IL-10. All concentrations of IL-10 increased the islet insulin content, but did not affect the islet DNA content. IL-10 did neither affect basal and glucose-stimulated insulin secretion nor (pro)insulin and total protein biosynthesis rates. Addition of 25 U/ml of interleukin-1β during the last 24 h of culture, led to a marked inhibition of glucose-stimulated insulin release and a 5-6 fold increase in nitrite production by rat insulinoma cells. These events could not be counteracted by any concentration of IL-10. The present data suggest that IL-10 might in some aspects stimulate pancreatic β-cell function, but it fails to block the inhbitory action of IL-1β.

Ca2+ deficiency, selective alpha-glucosidehydrolase inhibition, and insulin secretion

We investigated the relation between activities of islet glycogenolytic alpha-glucosidehydrolases and insulin secretion induced by glucose and 3-isobutyl-1-methylxanthine (IBMX) by means of suppressing 1) insulin release (Ca2+ deficiency) and 2) islet alpha-glucosidehydrolase activity (selective inhibition by the deoxynojirimycin derivative miglitol). Additionally, the in vivo insulin response to both secretagogues was examined. We observed that, similar to glucose-induced insulin release, islet glycogenolytic hydrolases (acid amyloglucosidase, acid alpha-glucosidase) were highly Ca2+ dependent. Acid phosphatase, N-acetyl-beta-D-glucosaminidase, or neutral alpha-glucosidase (endoplasmic reticulum) was not influenced by Ca2+ deficiency. In Ca2+ deficiency IBMX-induced insulin release was unaffected and was accompanied by reduced activities of islet alpha-glucosidehydrolases. Miglitol strongly inhibited glucose-induced insulin release concomitant with a marked suppression of islet alpha-glucosidehydrolase activities. Direct addition of miglitol to islet homogenates suppressed acid amyloglucosidase [half-maximal effective concentration (EC50) approximately 10(-6) M] and acid alpha-glucosidase. Acid phosphatase and N-acetyl-beta-D-glucosaminidase were unaffected. The miglitol-induced inhibition of glucose-stimulated insulin release was dose dependent (EC50 approximately 10(-6) M) and displayed a remarkable parallelism with the inhibition curve for acid amyloglucosidase. The in vivo insulin secretory response to glucose was markedly reduced in dystrophic mice (low amyloglucosidase), whereas the response to IBMX was unaffected. In summary, islet glycogenolytic hydrolases are Ca2+ dependent, and acid amyloglucosidase is directly involved in the multifactorial process of glucose-induced insulin release. In contrast the mechanisms of IBMX-stimulated insulin secretion operate independently of these enzymes. The effects of miglitol, a drug currently used in diabetes therapy, deserves further investigation.