Tumor necrosis factor alpha-mediated insulin resistance, but not dedifferentiation, is abrogated by MEK1/2 inhibitors in 3T3-L1 adipocytes

Tumor necrosis factor-alpha (TNF alpha) has been implicated as a contributi ng mediator of insulin resistance observed in pathophysiological conditions such as obesity, cancer-induced cachexia, and bacterial infections. Previo us studies have demonstrated that TNF alpha confers insulin resistance by p romoting phosphorylation of serine residues on insulin receptor substrate 1 (IRS-1), thereby diminishing subsequent insulin-induced tyrosine phosphory lation of IRS-1. However, little is known about which signaling molecules a re involved in this process in adipocytes and about the temporal sequence o f events that ultimately leads to TNF alpha-stimulated IRS-1 serine phospho rylation. In this study, we demonstrate that specific inhibitors of the MAP kinase kinase (MEK)1/2-p42/44 mitogen-activated protein (MAP) kinase pathw ay restore insulin signaling to normal levels despite the presence of TNF a lpha. Additional experiments show that MEK1/2 activity is required for TNF alpha-induced IRS-1 serine phosphorylation, thereby suggesting a mechanism by which these inhibitors restore insulin signaling. We observe that TNF alpha requires 2.5-4 h to markedly reduce insulin-trigg ered tyrosine phosphorylation of IRS-1 in 3T3-L1 adipocytes. Although TNF a lpha activates p42/44 MAP kinase, maximal stimulation is observed within 10 -30 min. To our surprise, p42/44 activity returns to basal levels well befo re IRS-1 serine phosphorylation and insulin resistance are observed. These activation kinetics suggest a mechanism of p42/44 action more complicated t han a direct phosphorylation of IRS-1 triggered by the early spike of TNF a lpha-induced p42/44 activity. Chronic TNF alpha treatment (>> 72 h) causes adipocyte dedifferentiation, a s evidenced by the loss of triglycerides and down-regulation of adipocyte-s pecific markers. We observe that this longer term TNF alpha-mediated dediff erentiation effect utilizes alternative, p42/44 MAP kinase-independent intr acellular pathways. This study suggests that TNF alpha-mediated insulin resistance, but not adi pocyte dedifferentiation, is mediated by the MEK1/2-p42/44 MAP kinase pathw ay.