O-Linked N-acetylglucosamine (O-GlcNAc (1-4). In the cell, the HBP converts imported glucose and glucosamine to UDP-GlcNAc. Glutamine:fructose-6-phosphate amidotransferase is the rate-limiting enzyme in this pathway. OGT catalyzes GlcNAc transfer to serine and threonine residues of target proteins, whereas O-O-GlcNAc is known to affect multiple metabolic pathways and has been implicated specifically as a contributor to insulin resistance and type 2 diabetes (5-7). Chronically elevated HBP flux, a result of hyperglycemia, is known to exacerbate metabolic dysregulation in part by targeting metabolic enzymes. For example, in diabetic mice, glycogen synthase (GS) becomes resistant to insulin stimulation as its level of O-GlcNAc modification increases (8, 9). AMP-activated protein kinase is activated in adipocytes with elevated HBP flux, resulting in O-GlcNAc-mediated elevation of fatty acid oxidation (10). To date, the majority of reports of O-GlcNAc-mediated metabolic changes attribute increased O-GlcNAc modification to increased HBP flux. We report a novel and significant induction of O-GlcNAc modification in glucose-deprived HepG2 cells that is independent of increased HBP flux and appears distinct from previously reported stress-induced O-GlcNAc induction. Rather, increased O-GlcNAc with glucose deprivation is mediated by induction of OGT and down-regulation of O-GlcNAcase. Increased O-GlcNAcylation of GS in these conditions contributes to decreased GS activity.

We have investigated the mechanism by which high concentrations of glucose inhibit insulin stimulation of glycogen synthase. In NIH-3T3-L1 adipocytes cultured in low glucose (LG; 2.5 mM), the half-maximal activation concentration (A 0 Glycogen synthase, which incorporates activated glucose into glycogen, is a major gatekeeper of carbohydrate metabolism. The enzyme is highly regulated, both allosterically and by several kinases and phosphatases (1, 2). Phosphorylation inactivates the enzyme. The process is complex, since there are nine phosphorylation sites, targeted by several kinases, each with different effects on enzyme activity (3-5). The inactivation of glycogen synthase can be overcome by allosteric interaction with glucose 6-phosphate (G6P) 1 (6). Insulin stimulates glycogen synthase primarily through the phosphatidylinositol 3-kinase/Akt pathway, resulting in inhibition of glycogen synthase kinase-3 and dephosphorylation of the enzyme by protein phosphatase 1 (1,7,8). Protein phosphatase 1 is itself regulated by stimulation and by specific targeting to glycogen (9 -11). With activation by insulin, glycogen synthase becomes more sensitive to G6P, and basal, G6P-independent activity is increased. In type 2 diabetes mellitus there is resistance to the stimulation of glycogen synthase by insulin and a reduction of glycogen synthase activity (12)(13)(14). How this insulin resistance is triggered is not known. Work from Marshall's laboratory originally suggested that insulin resistance could be mediated by an increase in carbohydrate flux through the hexosamine biosynthesis pathway (HBP) (15). Consistent with that hypothesis, acute infusions of glucosamine or transgenic overexpression in muscle and fat of the rate-limiting enzyme in the HBP, glutamine:fructose-6-phosphate amidotransferase, result in insulin resistance (16,17)..The terminal metabolites of the hexosamine pathway are UDP-hexosamines. UDP-N-acetylglucosamine is a substrate for the cytosolic UDP-N-acetylglucosamine:peptide glycosyltransferase (OGT), which glycosylates nuclear and cytosolic proteins with a single N-acetylglucosamine moiety on serine and threonine residues (O-GlcNAc) (18,19). This recently described protein modification is in many ways analogous to phosphorylation; it is dynamic and has been shown to occur exclusively on phosphoproteins. Additionally, it has been shown to often have a reciprocal relationship with the degree of phosphorylation of a protein (20,21). Recent studies have suggested possible links between the O-GlcNAc modification on proteins and the pathogenesis of diabetes and insulin resistance. For example, insulin resistance of endothelial nitric-oxide synthase stimulation results when the Akt phosphorylation site of endothelial nitric-oxide synthase is modified by O-GlcNAc (22). Transgenic overexpression of OGT in skeletal muscle and fat results in the development of insulin resistance in mice, mimicking the effects of increased hexosamine flux (23).We have previously demonstrated that treatment of fibroblasts with h...

O-Linked N-acetylglucosamine (O-GlcNAc

The hexosamine biosynthesis pathway (HBP) regulates the posttranslational modification of nuclear and cytoplasmic protein by O-linked N-acetylglucosamine ( O-GlcNAc). Numerous studies have demonstrated that, in hyperglycemic conditions, excessive glucose flux through this pathway contributes to the development of insulin resistance. The role of the HBP in euglycemia, however, remains largely unknown. Here we investigated the effect of O-GlcNAc on hepatic Akt signaling at physiological concentrations of glucose. In HepG2 cells cultured in 5 mM glucose, removal of O-GlcNAc by adenoviral-mediated overexpression of O-GlcNAcase increased Akt activity and phosphorylation. We also observed that Akt was recognized by succinylated wheat germ agglutinin (sWGA), which specifically binds O-GlcNAc. Overexpression of O-GlcNAcase in HepG2 cells reduced the levels of Akt in sWGA precipitates. The increased Akt activity was accompanied by increased phosphorylation of Akt substrates and reduced mRNA for glucose-6-phosphatase and phospho enolpyruvate carboxykinase (PEPCK). The increased Akt activity was not a result of activation of its upstream activator phosphoinositide 3-kinase (PI 3-kinase). Further demonstrating Akt regulation by O-GlcNAc, we found that overexpression of O-GlcNAcase in the livers of euglycemic mice also significantly increased Akt activity, resulting in increased phosphorylation of downstream targets and decreased mRNA for glucose-6-phosphatase. Together, these data suggest that O-GlcNAc regulates Akt signaling in hepatic models under euglycemic conditions.

Glycogen synthase is post-translationally modified by both phosphate and O-linked N-acetylglucosamine (OGlcNAc). In 3T3-L1 adipocytes exposed to high concentrations of glucose, O-GlcNAc contributes to insulin resistance of glycogen synthase. We sought to determine whether O-GlcNAc also regulates glycogen synthase in vivo. Glycogen synthase activity in fat pad extracts was inhibited in streptozotocin (STZ)-treated diabetic mice. The half-maximal activation concentration for glucose 6-phosphate (A 0.5 ) was increased to 830 ؎ 120 M compared with 240 ؎ 20 M in control mice (C, p < 0.01), while the basal glycogen synthase activity (%I-form) was decreased to 2.4 ؎ 1.4% compared with 10.1 ؎ 1.8% in controls (p < 0.01). Glycogen synthase activity remained inhibited after compensatory insulin treatment. The rate-limiting enzyme in glycogen metabolism, glycogen synthase, is a major determinant of overall glucose metabolism (1, 2). Because of its central role in glucose metabolism glycogen synthase is responsive to endocrine factors, including insulin, glucagon, and catecholamines, as well as to metabolic status, such as the concentration of the allosteric activator glucose 6-phosphate (G6P) 1 (3, 4). Glycogen synthase activity is modulated by phosphorylation that directly inhibits the enzyme and renders it less sensitive to allosteric activation by G6P (5). Insulin stimulation leads to removal of phosphate by protein phosphatase 1 (PP1), resulting in increased sensitivity to activation by G6P and a higher level of G6P-independent activity (6, 7). Glycogen levels, glycogen synthase activity, and responsiveness to insulin signaling are all reduced in diabetes (8 -11). Both endogenous and exogenous phosphatases are also less able to fully activate glycogen synthase in streptozotocin (STZ)-diabetic rats (12).Glycogen synthase activity is also affected by the hexosamine biosynthetic pathway, which produces UDP-N-acetylhexosamines (13-17). UDP-N-acetylglucosamine is a substrate for O-linked N-acetylglucosaminyltransferase, which transfers the monosaccharide onto serine and threonine residues of cytosolic and nuclear proteins. Data recently published by our laboratory showed that glycogen synthase from extracts of 3T3-L1 adipocytes was modified by O-GlcNAc in a glucose-dependent manner (18). This modification inhibited the enzyme in a manner analogous to phosphate, and only after enzymatic removal of O-GlcNAc could the enzyme be fully activated by exogenous PP1 (18). This illustrated a direct link between increased glucose uptake, modification by O-GlcNAc, glycogen synthase inhibition, and resistance of the synthase to activation by insulin signaling. We therefore investigated the relative roles of OGlcNAc and phosphate in regulating glycogen synthase in vivo in mice made diabetic by low dose STZ treatment. We show that hyperglycemia results in elevated O-GlcNAc on glycogen synthase and that removal of O-GlcNAc facilitates activation of the enzyme by PP1 especially in diabetes. This confirms that O-GlcNAc has an important regul...