Insulin-dependent metabolic and inotropic responses in the heart are modulated by hydrogen peroxide from NADPH-oxidase isoforms NOX2 and NOX4

Abstract Rationale Hydrogen peroxide (H 2 O 2 ) is a stable reactive oxygen species (ROS) that has long been implicated in insulin signal transduction in adipocytes. However, H 2 O 2 's role in mediating insulin's effects on the heart are unknown. Objective We investigated the role of H 2 O 2 in activating insulin-dependent changes in cardiac myocyte metabolic and inotropic pathways. The sources of insulin-dependent H 2 O 2 generation were also studied. Methods and results In addition to the canonical role of insulin in modulating cardiac metabolic pathways, we found that insulin also inhibited beta adrenergic-induced increases in cardiac contractility. Catalase and NADPH oxidase (NOX) inhibitors blunted activation of insulin-responsive kinases Akt and mTOR and attenuated beta adrenergic receptor-mediated responses. These insulin responses were lost in a mouse model of type 2 diabetes, suggesting a role for these H 2 O 2 -dependent pathways in the diabetic heart. The H 2 O 2 -sensitive fluorescent biosensor HyPer revealed rapid increases in cytosolic and caveolar H 2 O 2 concentrations in response to insulin treatment, which were blocked by NOX inhibitors and attenuated in NOX2 KO and NOX4 KO mice. In NOX2 KO cardiac myocytes, insulin-mediated phosphorylation of Akt and mTOR was blocked, while these responses were unaffected in cardiac myocytes from NOX4 KO mice. In contrast, insulin's effects on contractility were lost in cardiac myocytes from NOX4 KO animals but were retained in NOX2 KO mice. Conclusions These studies identify a proximal point of bifurcation in cardiac insulin signaling through the simultaneous activation of both NOX2 and NOX4. Each NOX isoform generates H 2 O 2 in cardiac myocytes with distinct time courses, with H 2 O 2 derived from NOX2 augmenting Akt-dependent metabolic effects of insulin, while H 2 O 2 from NOX4 blocks beta adrenergic increases in inotropy. These findings suggest that insulin resistance in the diabetic heart may lead to potentially deleterious potentiation of beta adrenergic responses.