Regulation of insulin receptor substrate 1 (IRS-1)/AKT kinase-mediated insulin signaling by O-Linked beta-N-acetylglucosamine in 3T3-L1 adipocytes

Increased O-linked beta-N-acetylglucosamine (O-GlcNAc) is associated with Insulin resistance in muscle and adipocytes. Upon Insulin treatment of insulin-responsive adipocytes, O-GlcNAcylation of several proteins is increased. Key Insulin signaling proteins, including IRS-1, IRS-2, and PDK1, are substrates for OGT, suggesting potential O-GlcNAc control points within the pathway. To elucidate the roles of O-GlcNAc in dampening Insulin signaling (Vosseller, K., Wells, L., Lane, M. D., and Hart, G. W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5313-5318), we focused on the pathway upstream of Akt. Increasing O-GlcNAc in 3T3-L1 adipocytes decreases phosphoinositide 3-kinase (PI3K) interactions with both IRS-1 and IRS-2. Elevated O-GlcNAc also reduces phosphorylation of the PI3K p85 binding motifs (YXXM) of IRS-1 and results in a concomitant reduction in tyrosine phosphorylation of Y(608)XXM in IRS-1, one of the two main PI3K p85 binding motifs. Additionally, Insulin signaling stimulates the interaction of OGT with PDK1. We conclude that one of the steps at which O-GlcNAc contributes to Insulin resistance is by inhibiting phosphorylation at the Y(608)XXM PI3K p85 binding motif in IRS-1 and possibly at PDK1 as well.