||Attachment of O-linked (3-N-acetylglucosamine (O-GlcNAc) to the hydroxyl group of serine and/or threonine residues of nucleocytoplasmic proteins is one of the more common post-translational modifications of proteins. Although O-GlcNAc has been shown to be essential for cell viability, aberrant increases in O-GlcNAcylation of proteins in different tissues have been shown to mediate pathological effects of hyperglycemia as observed in the type 2 diabetes. The physiological function of O-GlcNAc in normal physiology, however, has not been well studied. We have therefore overexpressed the enzyme p-iV-acetylglucosaminidase (O-GlcNAcase), which catalyzes the removal of the O-GlcNAc modification from proteins, in the liver or pancreatic P-cells of normal C57BL/6J mice. We demonstrate that overexpressing O-GlcNAcase in euglycemic mouse liver through tail vein injection of adenovirus resulted in increased Akt signaling, an important arm of the insulin signal transduction cascade. We also observed that the increased in Akt phosphorylation was not due to increased activity of signaling pathway upstream of Akt. Despite a significant decrease in the transcript level of the gluconeogenic enzyme glucose-6-phosphatase, consistent with increased Akt activity, there were no significant differences in the glucose or insulin levels after 3 days of O-GlcNAcase overexpression, although fasting blood glucose levels were moderately lowered after 5 days of overexpression. These findings demonstrate the role of hexosamine biosynthesis pathway (HBP) as a nutrient sensing pathway not only in pathologic states of diabetes or caloric excess but also in normal physiology. In P-cells, O-GlcNAcase was overexpressed in transgenic mice under control of the rat insulin promoter. O-GlcNAcase overexpression in younger (3 months old) mice results in decreased circulating fasting insulin levels. This decrease was paralleled by decreased insulin mRNA and islet insulin content. Further studies demonstrated an increase in pancreatic insulin content with increased p-cell mass, suggesting a possible defect in insulin exocytosis associated with O-GlcNAcase overexpression. However, when the mice were challenged with a high fat diet, O-GlcNAcase seems to protect the pcell function as determined by fasting insulin and glucose levels. Together, the present studies demonstrate that O-GlcNAc function to regulate metabolic pathway under physiologic and normoglycemic condition. In addition, decreased O-GlcNAc protects from the adverse effects of hyperglycemia as seen in type 2 diabetes, both through increased Akt signaling and protection of the P-cell function under conditions of nutrient stress.