Glucose Deprivation Stimulates O-GlcNAc Modification of Proteins through Up-regulation of O-Linked N-Acetylglucosaminyltransferase

Abstract

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.

Glucose Deprivation-induced Increase in Protein O-GlcNAcylation in Cardiomyocytes Is Calcium-dependent

“…Fig. 1A it can be seen that removal of glucose from NRVMs resulted in a time-dependent increase in O-GlcNAc levels; this increase was apparent, particularly in high molecular mass proteins, within 3 h of glucose deprivation, and overall O-GlcNAc levels were significantly increased by 6 h and reached maximal levels by 12 h. In contrast to studies in other cell lines we did not observe an initial decrease in O-GlcNAc levels in response to glucose deprivation (25,27). Glucose is a primary energy source for cardiomyocytes; therefore, glucose deprivation was repeated for 6 h with and without 1 mM and 10 mM pyruvate, which is readily oxidized by cardiomyocytes for energy production.…”

“…2A). This contrasts with other reports that glucose deprivation resulted in increased levels of OGT protein and decreased OGA protein (25,27). To determine whether the changes in OGA and OGT protein levels were due to altered transcription, we determined the effects of glucose deprivation on OGA and OGT mRNA levels and found that transcript levels for both were increased following removal of glucose (Fig.…”

“…Taylor et al were the first to report the paradoxical result that glucose deprivation leads to increased O-GlcNAcylation in HepG2 cells and found that this was accompanied by increased OGT and decreased OGA protein (25); they subsequently reported that the increase in O-GlcNAc was independent of AMPK activation and appeared to be mediated at least in part by a decrease in HBP flux (27). On the other hand, Cheung and Hart reported that in Neuro-2a neuroblastoma cells, the effects of glucose deprivation on O-GlcNAcylation were mediated in part by AMPK as well as by p38 MAPK (26); however, in A549 lung cancer cells Kang et al, reported that the glucose deprivation-induced increase in O-GlcNAc was not APMK-dependent, but rather appeared to be due to increased glycogen degradation (24).…”

“…Interestingly, however, a number of studies have recently reported that glucose deprivation significantly induced O-GlcNAc modification in Hep2G, Neuro-2a neuroblastoma cells, and other cancer cell lines (24 -27). Studies in Hep2G cells demonstrated that the glucose deprivation-induced increase in O-GlcNAc levels was not mediated by increased HBP flux but rather by up-regulation of OGT and down-regulation of OGA (25,27). In Neuro-2a cells it was shown that glucose deprivation increased O-GlcNAc levels by up-regulation of OGT expression in an AMP-activated protein kinase (AMPK)-dependent manner and also that substrate targeting of OGT was regulated by p38 MAPK (26).…”

Enzymatic analysis of uridine diphosphate N-acetyl-d -glucosamine

“…Inhibitors of bacterial protein synthesis cause an increase in the concentrations of peptidoglycan precursors, including UDP-GlcNAc [3]. These physiological changes in UDP-GlcNAc pools have created a need for rapid assays to analyze the large number of samples produced during time course experiments [4].…”