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 (H(2)O(2)) 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 H(2)O(2) levels and reversible cysteine modifications across mouse and human skeletal muscle models. Utilizing advanced redox tools-including genetically encoded H(2)O(2) sensors and non-reducing immunoblotting-we consistently observed no increase in subcellular H(2)O(2) 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 H(2)O(2) generation in skeletal muscle and suggest that an insulin-stimulated pro-reductive shift modulates certain aspects of insulin signal transduction.