Objective— Insulin promotes differentiation of preadipocytes into adipocytes. Insulin also stimulates reactive oxygen species (ROS) production, and the NADPH oxidases Nox1 and Nox4 are important sources of ROS. We determined in human and mouse preadipocytes whether Nox proteins contribute to ROS formation and differentiation in response to insulin.

Methods and Results— The expression of Nox1 and Nox4 was increased during insulin-induced differentiation, and insulin increased ROS production. SiRNA against Nox4 but not Nox1 inhibited insulin-induced differentiation and ROS production but promoted proliferation. Nox4 overexpression yielded the opposite effect. As observed by siRNA and overexpression, Nox4 controlled the expression of MAP kinase phosphatase-1 (MKP-1), which reduces insulin-induced ERK1/2 activation. Consequently, downregulation of Nox4 promoted ERK1/2 signaling: Proliferation was increased and through phosphorylation of the inhibitory site serine612, ERK1/2 inhibited the activation of the insulin-receptor substrate-1 (IRS-1) and thereby prevented differentiation in response to insulin. Inhibition of ERK1/2 or overexpression of MPK-1 promoted insulin-induced differentiation. Accordingly, insulin-induced proliferation was enhanced by siRNA against MKP-1, whereas inhibition of ERK1/2 or overexpression of MKP-1 attenuated proliferation.

Conclusions— Nox4 acts as a switch from insulin-induced proliferation to differentiation by controlling MKP-1 expression, which limits ERK1/2 signaling.