This proposal is directed at a better understanding of how organisms adapt to nutrient excess and how that may contribute to type 2 diabetes. The hexosamine biosynthesis pathway (HBP) serves a nutrient sensing function. In the short term HBP signaling promotes the storage of fat. Chronically excess HBP flux, however, mimics many aspects of the type 2 diabetes syndrome. Overexpression of the rate limiting enzyme of the HBP (GFA) in fat leads to altered regulation of adiponectin, insulin resistance in skeletal muscle, hyperleptinemia, and activation of carbohydrate uptake and fat synthesis in adipocytes. We have shown that the effects of the HBP are mediated by the enzymatic addition of N-acetylglucosamine (GlcNAc) to serine and threonine residues of cytosolic and nuclear proteins by the enzyme O-GlcNAc transferase (OGT). Using glycogen synthase (GS) as a model, we have demonstrated that 0-linked GlcNAc inactivates GS in a manner analogous to phosphate, leaving GS unresponsive to kinase/phosphatase signaling. This has led to the hypothesis that the HBP acts as a nutrient sensor that modulates kinase/phosphatase signaling. In the setting of high nutrient flux, the net result is to augment fat storage and limit carbohydrate storage. In the fat cell, the HBP somewhat paradoxically activates both fuel storage and fuel utilization pathways, overriding the normal control mechanisms of AMP-activated kinase {AMPK). Because of the central role of adipocytes in regulating metabolism and energy/nutrient balance, we propose to focus our studies on the mechanisms for the effects of excess HBP flux in fat.
Our Aims are: 1: Investigate the crosstalk between the AMPK and HBP/OGT pathways. Evidence has accumulated that AMPK is a key regulator of metabolism, and we present preliminary data that AMPK is activated in adipose tissue with high HBP flux. This allows fatty acid oxidation to proceed even in the face of chronic nutrient and fuel excess that would normally shut down AMPK and prevent fatty acid oxidation. We will define the effect of the HBP on AMPK, and vice versa. We will determine the mechanism for these effects and the degree to which the results of HBP activation are dependent on AMPK. 2: Determine the precise mechanism by which O-GlcNAc on glycogen synthase (GS) alters its function. We have described the effects of global changes in O-GlcNAc modification on the enzyme kinetics and hormone responsiveness of GS. We will map the sites of glycosylation on glycogen synthase and determine which sites are responsible for its inactivation. 3: Determine if over expression of the O-GlcNAc-ase in fat and muscle will prevent nutrient-induced insulin resistance. An extension of our earlier studies that showed sufficiency of the HBP in inducing insulin resistance, this transgenic model will determine the necessity of the pathway.
