title: Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors.
abstract: Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells requires an increase in intracellular free Ca2+ concentration ([Ca2+]). Glucose uptake into β-cells promotes Ca2+ influx and reactive oxygen species (ROS) generation. In other cell types, Ca2+ and ROS jointly induce Ca2+ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca2+ 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 H2O2 in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H2O2-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca2+] produced by incubation of dissociated β-cells with H2O2. Addition of stimulatory glucose or H2O2 (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 Ca2+ release, induced by the concomitant increases in [Ca2+] and ROS produced by stimulatory glucose, is an essential step in GSIS.
authors.affiliation: Institute for Research in Dental Sciences, Facultad de Odontología, Universidad de Chile, Santiago, Chile; Center of Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Paola
authors.initials: P
authors.lastname: Llanos
authors.affiliation: Institute for Research in Dental Sciences, Facultad de Odontología, Universidad de Chile, Santiago, Chile; Center of Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Ariel
authors.initials: A
authors.lastname: Contreras-Ferrat
authors.affiliation: Physiology and Biophysics Program, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Genaro
authors.initials: G
authors.lastname: Barrientos
authors.affiliation: Center of Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Marco
authors.initials: M
authors.lastname: Valencia
authors.affiliation: Center of Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Santiago, Chile; Human Genetics Program, Institute of Biomedical Sciences, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: David
authors.initials: D
authors.lastname: Mears
authors.affiliation: Center of Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Santiago, Chile; Physiology and Biophysics Program, Facultad de Medicina, Universidad de Chile, Santiago, Chile; Biomedical Neuroscience Institute, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Hidalgo
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D016166:Free Radical Scavengers / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007150:Immunohistochemistry; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects* / Q000378:metabolism; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D018613:Microscopy, Confocal; D016877:Oxidants / Q000494:pharmacology; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism*
chemical_list: D016166:Free Radical Scavengers; D007328:Insulin; D016877:Oxidants; D017382:Reactive Oxygen Species; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002110:Caffeine; D002217:Carbachol; D006861:Hydrogen Peroxide; D005947:Glucose; D002118:Calcium; D000111:Acetylcysteine