Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca(2+) Release via ROS-Stimulated Ryanodine Receptors

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells requires an increase in intracellular free Ca(2+) concentration ([Ca(2+)]). Glucose uptake into β-cells promotes Ca(2+) influx and reactive oxygen species (ROS) generation. In other cell types, Ca(2+) and ROS jointly induce Ca(2+) release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca(2+) release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H(2)O(2) in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H(2)O(2)-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca(2+)] produced by incubation of dissociated β-cells with H(2)O(2). Addition of stimulatory glucose or H(2)O(2) (in basal glucose) to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca(2+) release, induced by the concomitant increases in [Ca(2+)] and ROS produced by stimulatory glucose, is an essential step in GSIS.