Glucocorticoids, such as dexamethasone, enhance protein breakdown via ubiquitin–proteasome system. However, the role of autophagy in organelle and protein turnover in the glucocorticoid-dependent a...

Insulin is a major regulator of glucose metabolism, stimulating its mitochondrial oxidation in skeletal muscle cells. Mitochondria are dynamic organelles that can undergo structural remodeling in order to cope with these ever-changing metabolic demands. However, the process by which mitochondrial morphology impacts insulin signaling in the skeletal muscle cells remains uncertain. To address this question, we silenced the mitochondrial fusion proteins Mfn2 and Opa1 and assessed insulin-dependent responses in L6 rat skeletal muscle cells. We found that mitochondrial fragmentation attenuates insulin-stimulated Akt phosphorylation, glucose uptake and cell respiratory rate. Importantly, we found that insulin induces a transient rise in mitochondrial Ca²⁺ uptake, which was attenuated by silencing Opa1 or Mfn2. Moreover, treatment with Ruthenium red, an inhibitor of mitochondrial Ca²⁺ uptake, impairs Akt signaling without affecting mitochondrial dynamics. All together, these results suggest that control of mitochondrial Ca²⁺ uptake by mitochondrial morphology is a key event for insulin-induced glucose uptake.

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 Myc-epitope 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-ds-tat 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 insulin-mediated stimulation of inositol-1,4,5-trisphosphate (IP₃)-activated Ca²⁺ channels, in response to activation of phosphatidylinositol 3-kinase 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 α,β phosphatidylinositol 3-kinase to Akt pathway, insulin engages both RyR-mediated Ca²⁺ release and IP₃-receptor-mediated mitochondrial Ca²⁺ uptake, and that these signals jointly stimulate glucose uptake.