Tumor Necrosis Factor (TNF)-α-induced Repression of GKAP42 Protein Levels through cGMP-dependent Kinase (cGK)-Iα Causes Insulin Resistance in 3T3-L1 Adipocytes

Insulin receptor substrates (IRSs) have been shown to be major mediators of insulin signaling. Recently, we found that IRSs form high-molecular weight complexes, and here, we identify by yeast two-hybrid screening a novel IRS-1-associated protein: a 42-kDa cGMP-dependent protein kinase-anchoring protein (GKAP42). GKAP42 knockdown in 3T3-L1 adipocytes suppressed insulin-dependent IRS-1 tyrosine phosphorylation and downstream signaling, resulting in suppression of GLUT4 translocation to plasma membrane induced by insulin. In addition, GLUT4 translocation was also suppressed in cells overexpressing GKAP42-N (the IRS-1 binding region of GKAP42), which competed with GKAP42 for IRS-1, indicating that GKAP42 binding to IRS-1 is required for insulin-induced GLUT4 translocation. Long term treatment of 3T3-L1 adipocytes with TNF-alpha, which induced insulin resistance, significantly decreased the GKAP42 protein level. We then investigated the roles of cGMP-dependent kinase (cGK)-I alpha, which bound to GKAP42, in these changes. cGK-I alpha knockdown partially rescued TNF-alpha-induced decrease in GKAP42 and impairment of insulin signals. These data indicated that TNF-alpha-induced repression of GKAP42 via cGK-I alpha caused reduction of insulin-induced IRS-1 tyrosine phosphorylation at least in part. The present study describes analysis of the novel TNF-alpha-induced pathway, cGK-I alpha-GKAP42, which regulates insulin-dependent signals and GLUT4 translocation.