The early phase of glucose-stimulated insulin secretion requires nitric oxide.

Nitric oxide (nitrogen monoxide, NO) acts as a signal transducer in a variety of cells. In the present study rat pancreatic islets were perifused with physiologically relevant glucose concentrations in the presence or absence of various NO-modulating agents. Perifusion in the presence of 0.1-1 mmol/l of the NO synthase inhibitor, NG-monomethyl-L-arginine or of 10 micromol/l of the NO-scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), resulted in an inhibition of the early phase of glucose-stimulated insulin secretion by 60-65% and 46%, respectively. Light- and electron-microscopic studies revealed that pancreatic islets constitutively express NO-synthase in alpha and delta cells, where it is confined to the secretory granules. Therefore, these data indicate that NO may be important in the signal transduction pathway of the early phase of glucose-stimulated insulin secretion.

Tetrahydrobiopterin synthesis precedes nitric oxide-dependent inhibition of insulin secretion in INS-1 rat pancreatic beta-cells.

Interleukin 1 (IL-1) induces pancreatic beta-cell dysfunction mainly due to overproduction of nitric oxide (NO). Since tetrahydrobiopterin (BH4) is a obligatory cofactor of NO synthases, we examined the temporal relationship of BH4 synthesis, NO production and insulin secretion in a pancreatic beta-cell line (INS-1) which was exposed to IL-1. IL-1 affected BH4 synthesis in a time- and concentration-dependent manner. At a concentration of 10 ng/ml IL-1 caused an increase in intracellular BH4 with peak levels being observed at 6 hours followed by a steady decline in the cellular BH4 content. The increase in BH4 synthesis was followed by enhanced NO production and, consecutively, inhibition of insulin secretion. The concentration-dependent regulation of BH4 synthesis, NO production and suppression of insulin secretion indicate a functional link between these parameters in pancreatic beta-cells.

Interferon-gamma inhibits insulin release and induces cell death in the pancreatic beta-cell line INS-1 independently of nitric oxide production.

Interferon-gamma is among the cytokines which have been implicated as effector molecules of beta-cell destruction in autoimmune diabetes. Its mechanism of action is, however, largely unknown. In the present study rat pancreatic beta-cells, INS-1, were incubated with rat interferon-gamma (rIRN-gamma) for 24 h. rIFN-gamma at 1-1000 U/ml caused a dose-dependent inhibition of insulin release and cell metabolism with maximal inhibition being observed at 100 U/ml (insulin release: 51.2%, cell metabolism: 43.3% of control, respectively). In addition, 100 U/ml rIFN-gamma induced a 4- and 8.3-fold increase in apoptotic cell death after 24 and 48 h of incubation, respectively. These effects were not mediated by nitric oxide (NO), since IFN-gamma failed to induce nitric oxide synthase and NO production. Similarly, beta-cell dysfunction and death were not prevented by coincubation of the INS-1 cells with the poly(ADP-ribose) polymerase inhibitors benzamide, 3-aminobenzamide, and 4-aminobenzamide, the oxygen free radical scavenger Trolox, and the antioxidant N-acetylcysteine, indicating that NO, poly(ADP-ribose) polymerase, and oxygen free radicals are not involved in IFN-gamma induced beta-cell dysfunction and death.

Interleukin 10 inhibits insulin release from and nitric oxide production in rat pancreatic islets.

Interleukin 10 was found to prevent cytokine-induced nitric oxide production in murine macrophages. Because, in rat islets, interleukin 1 beta induces beta-cell dysfunction, mainly due to overproduction of nitric oxide, we studied if this effect could be counteracted by interleukin 10. Rat pancreatic islets were cultured for 24 h in the presence or absence of 0.02-20 ng/ml recombinant human interleukin 10. Interleukin 10 dose-dependently inhibited insulin secretion with maximal inhibition (27 +/- 4%, p < 0.05) at 2 ng/ml without impairment of islet cell viability. However, incubation of pancreatic islets with interleukin 10 resulted in a 61.5% decrease of nitric oxide production. Co-incubation of islets with interleukin 10 (2 ng/ml) and recombinant human interleukin 1 beta (0.15 ng/ml) resulted in a more pronounced suppression of basal insulin release than with interleukin 1 beta alone (55 +/- 3.6% vs 44 +/- 3.6% with interleukin 1 beta alone, p < 0.05) but did not reduce interleukin 1 beta-stimulated NO production or reverse the effect of interleukin 1 beta on cell viability. Thus, in pancreatic islets interleukin 10 is not capable of counteracting the interleukin 1 beta induced beta-cell dysfunction, but rather enhances the inhibitory effect of interleukin 1 beta by a different mechanism.

Nitric oxide (nitrogen monoxide, NO) stimulates insulin secretion by inducing calcium release from mitochondria.

Nitric oxide (nitrogen monoxide, NO) acts as messenger molecule in a variety of cells and may also be involved in the insulin secretory pathway of islet beta-cells. We report here that NO at a low micromolar concentration stimulates epinephrine-sensitive insulin secretion from cells of the beta-cell line, INS-1. Insulin secretion is paralleled by a reversible decrease of the mitochondrial membrane potential and by an increase of the cytosolic calcium. Chelation of intracellular, but not of extracellular calcium prevents the NO-induced insulin secretion. These data indicate that NO can stimulate insulin secretion by deenergizing mitochondria and thereby triggering mitochondrial calcium release.

Oxidants in mitochondria: from physiology to diseases.

Reactive oxygen species (ROS: superoxide radical, O2.-; hydrogen peroxide, H2O2; hydroxyl radical, OH.), which arise from the univalent reduction of dioxygen are formed in mitochondria. We summarize here results which indicate that ROS, and also the radical nitrogen monoxide ('nitric oxide', NO), act as physiological modulators of some mitochondrial functions, but may also damage mitochondria. Hydrogen peroxide, which originates in mitochondria predominantly from the dismutation of superoxide, causes oxidation of mitochondrial pyridine nucleotides and thereby stimulates a specific Ca2+ release from intact mitochondria. This release is prevented by cyclosporin A (CSA). Hydrogen peroxide thus contributes to the maintenance of cellular Ca2+ homeostasis. A stimulation of mitochondrial ROS production followed by an enhanced Ca2+ release and re uptake (Ca2+ 'cycling') by mitochondria causes apoptosis and necrosis, and contributes to hypoxia/reperfusion injury. These kinds of cell injury can be attenuated at the mitochondrial level by CSA. When ROS are produced in excessive amounts in mitochondria nucleic acids, proteins, and lipids are extensively modified by oxidation. Physiological (sub-micromolar) concentrations of NO potently and reversibly deenergize mitochondria at oxygen tensions that prevail in cells by transiently binding to cytochrome oxidase. This is paralleled by mitochondrial Ca2+ release and uptake. Higher NO concentrations or prolonged exposure of cells to NO causes their death. It is concluded that ROS and NO are important physiological reactants in mitochondria and become toxic only when present in excessive amounts.