Abstract

Insulin signaling includes generation of low levels of H2O2; however, its origin and contribution to insulin-stimulated glucose transport are unknown. We tested the impact of H2O2 on insulin-dependent glucose transport and GLUT4 translocation in skeletal muscle cells. H2O2 increased the translocation of GLUT4 with an exofacial Mycepitope tag between the first and second transmembrane domains (GLUT4myc), an effect additive to that of insulin. The anti-oxidants N-acetyl L-cysteine and Trolox, the p47(phox)-NOX2 NADPH oxidase inhibitory peptide gp91-ds-tat or p47(phox) knockdown each reduced insulin-dependent GLUT4myc translocation. Importantly, gp91-dstat suppressed insulin-dependent H2O2 production. A ryanodine receptor (RyR) channel agonist stimulated GLUT4myc translocation and insulin stimulated RyR1-mediated Ca2+ release by promoting RyR1 S-glutathionylation. This pathway acts in parallel to insulinmediated stimulation of inositol-1,4,5-trisphosphate (IP3)-activated Ca2+ channels, in response to activation of phosphatidylinositol 3kinase and its downstream target phospholipase C, resulting in Ca2+ transfer to the mitochondria. An inhibitor of IP3 receptors, Xestospongin B, reduced both insulin-dependent IP3 production and GLUT4myc translocation. We propose that, in addition to the canonical a, b phosphatidylinositol 3-kinase to Akt pathway, insulin engages both RyR-mediated Ca2+ release and IP3-receptormediated mitochondrial Ca2+ uptake, and that these signals jointly stimulate glucose uptake.