Revisiting insulin-stimulated hydrogen peroxide dynamics reveals a cytosolic reductive shift in skeletal muscle

The intracellular redox state is crucial for insulin responses in peripheral tissues. Despite the longstanding belief that insulin signaling increases hydrogen peroxide (H₂O₂) production leading to reversible oxidation of cysteine thiols, evidence is inconsistent and rarely involves human tissues. In this study, we systematically investigated insulin-dependent changes in subcellular H₂O₂ levels and reversible cysteine modifications across mouse and human skeletal muscle models. Utilizing advanced redox tools-including genetically encoded H₂O₂ sensors and non-reducing immunoblotting-we consistently observed no increase in subcellular H₂O₂ levels following insulin stimulation. Instead, stoichiometric cysteine proteome analyses revealed a selective pro-reductive shift in cysteine modifications affecting insulin transduction related proteins, including Cys179 on GSK3β and Cys416 on Ras and Rab Interactor 2 (RIN2). Our findings challenge the prevailing notion that insulin promotes H₂O₂ generation in skeletal muscle and suggest that an insulin-stimulated pro-reductive shift modulates certain aspects of insulin signal transduction.