Regulation and function of O-linked N-acetylglucosamine modification of proteins with glucose deprivation.

摘要

O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of proteins that functions as a nutrient sensing mechanism. We have discovered a 7.8-fold induction of O-GlcNAc modification under conditions of glucose deprivation in human hepatocellular carcinoma (HepG2) cells. Some of the targets of glucose deprivation-induced O-GlcNAcylation are distinct from those modified in response to high glucose (HG, 20mM) or glucosamine (GlcN, 10mM) treatment, suggesting differential targeting with glucose deprivation and glucose excess. O-GlcNAcylation of glycogen synthase is significantly increased with glucose deprivation and this O-GlcNAc increase contributes to a 60% decrease in glycogen synthase (GS) activity.;We have investigated the mechanism(s) mediating OGT induction with glucose deprivation. The signal does not appear to be general energy depletion because no differences in AMP-dependent kinase (AMPK) activation are observed between glucose-deprived and normal glucose-treated cells. However, treatment of glucose-deprived cells with a small dose of glucosamine (1mM) dampens the increase in OGT mRNA and O-GlcNAc protein modification seen with glucose deprivation, suggesting that decreased hexosamine flux is the signal for the OGT up regulation. Consistent with this, treatment of glucose-deprived cells with an inhibitor of O-GlcNAcase (PUGNAc) also prevents the subsequent up regulation of OGT. 1mM glucosamine treatment also results in a 40% rescue of the GS activity inhibition normally seen after glucose deprivation. We conclude from these observations that deglycosylation of as yet unidentified proteins within the first few hours of low glucose treatment promotes OGT transcriptional induction. These findings suggest a novel negative feedback regulatory loop for OGT and O-GlcNAc regulation.;Increased O-GlcNAc modification is not mediated by increased UDP-GlcNAc, the rate limiting substrate for O-GlcNAcylation. Rather, the mRNA for nucleocytoplasmic O-linked N-acetylglucosaminyltransferase (ncOGT) increases 3.4-fold within 6 h of glucose deprivation. Within 12 h, OGT protein increases 1.7-fold compared to normal glucose-treated cells. In addition, 12-h glucose deprivation leads to a 49% decrease in O-GlcNAcase protein levels. We conclude that increased O-GlcNAc modification stimulated by glucose deprivation results from increased OGT and decreased O-GlcNAcase levels and these changes affect cell metabolism, inactivating glycogen synthase.