Analysis of intracellular mechanisms contributing to insulin secretion from pancreatic β-cells

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METHODS : Pancreatic islets and β-cells were isolated from male Wistar rats (9-12 weeks old) by collagenase digestion technique. (1) Double immunostaining with antiserum to NOS1 or NOS3 and that to insulin or glucagon was performed in the isolated islets, and the fluorescent images were analyzed in a confocal laser scanning microscope. (2) The production of NO in islet cells was measured with a fluorescent NO indicator, DAF-2 DA in a confocal laser scanning microscope. (3) [Ca^<2+>]_i was measured in fura 2-loaded β-cells by Argus-50/CA system. (4) The secretion of insulin from isolated islets was measured by the batch incubation method and EIA.RESULTS AND DISCUSSION : (1) Double immunostaining showed that both NOS1- and NOS3-immunoreactivity could be detected in rat β-cells. (2) The amount of NO in β-cells was elevated by glucose in a concentration-dependent manner. (3) In the presence of L-NNA, an NO donor, NOC 7, at 0.5 μM increased the amplitude of [Ca^<2+>]_i oscillations induced by 11.1 mM glucose, and at 10 μM terminated them. A soluble guanylyl cyclase inhibitor suppressed the stimulatory action of NOC7 at low concentrations, but did not affect the inhibitory one at high concentrations, suggesting that the former is mediated by cGMP while the latter is not. (4) Insulin secretion induced by 11.1 mM glucose was facilitated by 0.5 μM NOC7, while it was suppressed by 10 μM NOC7.CONCLUSION : NO is an endogenous regulator of insulin secretion, which facilitates and inhibits glucose-induced insulin secretion at low and high concentrations, respectively.