Insulin promotes cell growth and has been shown to increase radical generation. The most prominent cellular sources of reactive oxygen species (ROS) are NADPH oxidases. We therefore hypothesized that insulin promotes formation of adipocytes via increasing ROS formation and determined the underlying mechanism using 3T3 fibroblasts.

Insulin promotes differentiation of 3T3 cells to adipocytes and acutely activates MAP kinases. Stimulation with H2O2 also induced differentiation and increased MAP kinase phosphorylation. In contrast, reduction of ROS 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 the expression of their mRNA is increased in the cause of differentiation. Overexpression of Nox4 promoted differentiation of 3T3 cells into adipocytes. SiRNA directed against Nox4 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 during the process. Blockade of individual Nox subunit could be utilized to prevent the negative effects of hyperinsulinemia.