Abstract 694: Insulin-Induced Adipocyte Differentiation Critically Depends on Nox4
Hyperinsulinemia is a risk factor for cardiovascular diseases (CVD) and insulin promotes cell growth. CVD are associated with oxidative stress and insulin has been shown to modulate radical generation. The most prominent cellular sources of reactive oxygen species (ROS) in the vascular system are NADPH oxidases. We therefore hypothesized that insulin induces differentiation of fibroblasts to adipocytes via increasing ROS formation and determined the underlying mechanisms. Insulin as well as H2O2 promoted the differentiation of 3T3 fibroblasts to adipocytes and activated MAP kinases, as determined by FACS analysis and Western blot analysis. Reduction of ROS concentrations by apocynin, catalase and SOD prevented both effects. qRT-PCR revealed that 3T3-cells as well as differentiated adipocytes express the NADPH oxidase homologues Nox1 and Nox4 and that there was a 5-fold induction of their mRNAs was increased by 5 times in the course of differentiation. Nox4 mRNA was also increased after stimulation of cells with H2O2. SiRNA directed against Nox1 but not Nox4 prevented the insulin-stimulated activation of p38 MAP kinase. Lentiviral-mediated overex-pression of the Nox4 homologue increased the basal phosphorylation of MAP kinases, suggesting that Nox4 might be involved in the long-term effects of insulin. Indeed, overexpression of Nox4 promoted differentiation of 3T3 cells into adipocytes. SiRNA directed against Nox4 as well as siRNA directed against Nox1 completely prevented the differentiation of 3T3-fibroblasts to adipocytes.
In conclusion: Insulin-induced differentiation critically dependents on ROS generated by Nox1- as well as Nox4-containing NADPH oxidases. Nox1 mediates the immediate responses to insulin, whereas the subsequent long-lasting process of differentiation is regulated by Nox4, which is induced in response to insulin. Blockade of individual Nox subunits could be utilized to prevent the negative effects of hyperinsulinemia.