Insulin Induces both H2O2 Production and IP3-Dependent Mitochondria Ca2+ Uptake. H2O2 Oxidizes RyR to Elicit Ca2+ Release and GLUT4 Translocation in Skeletal Muscle Cells

Abstract

We have described that insulin induces both NADPH oxidase-dependent ROS production and Ca2+ increase in skeletal muscle cells and although insulin-regulated GLUT4 traffic has been widely explored, the role of intracellular Ca2+ handling in this process is poorly understood. We studied insulin-induced GLUT4 traffic in GLUT4myc permanently transfected L6 skeletal muscle cells.Insulin-induced exofacial exposure of myc epitope, glucose uptake and Akt activation were independent of extracellular Ca2+ levels. Insulin increased H2O2 production measured with the cytosolic molecular sensor HyPer which was inhibited by NAC, a potent antioxidant. p47 subunit of NADPHox labeled only differentiated myotubes. Antioxidant agents, NAC and trolox partly reduced the externalization of myc epitope as did a specific inhibitor of NADPHox and overexpression of catalase. Superoxide dismutase 1 appears to potentiate the insulin effect. H2O2 induced Ca2+ release, which was inhibited by pre-incubation with ryanodine. Cytosol directed parvalbumin-DsRed reduced insulin-dependent exofacial exposure of myc epitope. Moreover, both ryanodine and xestospongin B partly inhibited myc exposure with an additive effect. Insulin induced an increase in the S-glutathionylation state of RyR1, detected by proximity ligation assay. Insulin induced an increase in mitochondrial ROS and Ca2+ levels measured with ratiometric HyPer and PeriCam respectivelly. The mitochondrial potential and oxygen consumption were also increased by insulin and inhibited by pre-incubation with IP3R blockers. Insulin-dependent GLUT4myc translocation to cell surface was strongly reduced in presence of ruthenium red. These data suggest that insulin induces an increase in cytoplasmic Ca2+ levels by NADPH-dependent RyR1 modifications and IP3R-dependent mitochondrial regulation in skeletal muscle myotubes.This work was supported by FONDECYT 3110170, FONDECYT 3110105, FONDECYT 1080436, ACT1111.