Compelling data from many laboratories have documented an essential role for glucosamine metabolism in the induction of insulin-resistance, the underlying cause of type II diabetes. Signaling via the insulin receptor is mediated by a complex, but fairly well studied signal transduction cascade. We and others have described the dynamic O-GlcNAc modification of a myriad of nucleocytoplasmic proteins and its interplay with phosphate on a number of signaling and regulatory proteins. Hyperglycemia induced insulin-resistance is accompanied by a dramatic increase in O-GlcNAc on many proteins. Strikingly, a specific inhibitor of O-GlcNAcase, the enzyme that removes O-GlcNAc from proteins, causes insulin-resistance in the absence of hyperglycemia, providing direct evidence that hyper-O-GlcNAcylation itself is sufficient to disrupt insulin signaling. Given the wealth of knowledge of the insulin signaling pathway, our aims are straight-forward. Aim 1 will systematically determine the site(s) in the insulin signaling pathway which are blocked by hyper-O-GlcNAcylation. Points of involvement of O-GlcNAc in insulin signaling will be mapped by functional analysis at each known step in the pathway, and by a non-biased proteomic analysis of insulin-responsive O-GlcNAc modified proteins. Aim 2 will biochemically characterize the structure/function, site occupancy, and relationship to 0-phosphate of key insulin-signaling proteins that are already known to be O-GlcNAc modified, including GSK3 beta, IRS-1 & 2, Akt/PKB, and PPAR-gamma-RXR. Aim 3 will systematically study the role of insulin-signaling and hyperglycemia in the regulation of the adipocyte enzymes controlling O-GlcNAc cycling, O-GlcNAc Transferase and O-GlcNAcase. The 3T3-L1 adipocyte insulin-signaling system not only provides a great model to elucidate the functions of O-GlcNAc in insulin signaling, but also these studies will likely provide a new understanding of the molecular mechanisms causing diabetes, and may lead to unexpected avenues for its treatment.