Overexpression of glutamine : fructose-6-phosphate amidotransferase in rat-1 fibroblasts enhances glucose-mediated glycogen accumulation via suppression of glycogen phosphorylase activity

The hexosamine biosynthesis pathway (HBP) mediates many of the adverse effe cts of excess glucose. We have shown previously that glucose down-regulates basal and insulin-stimulated glycogen synthase (GS) activity. Overexpressi on of the rate-limiting enzyme in the HBP, glutamine:fructose-6-phosphate a midotransferase (GFA), mimics these effects of high glucose and renders the cells more sensitive to glucose. Here we examine the role of the HBP in re gulating cellular glycogen content. Glycogen content and glycogen phosphory lase (GP) activity were determined in Rat-1 fibroblasts that overexpress GF A. In both GFA and controls there was a dose-dependent increase in glycogen content (similar to 8-fold) in cells cultured in increasing glucose concen trations (1-20 mM). There was a shift to the left in the glucose dose-respo nse curve for glycogen content in GFA cells (ED50 for glycogen content = 5. 80 +/- 1.05 vs. 8.84 +/- 0.87 mM glucose, GFA us. control). Inhibition of G FA reduced glycogen content by 28.4% in controls cultured in 20 mM glucose. In a dose-dependent manner, glucose resulted in a more than 35% decrease i n GP activity in controls. GP activity in GFA cells was suppressed compared with that in controls, and there was no glucose-induced down-regulation of GP activity. Glucosamine and uridine mimicked the effects of glucose on gl ycogen content and GP activity. However, chronic overexpression of GFA is a unique model of hexosamine excess, as culturing control cells in low dose glucosamine (0.1-0.25 mM) did not suppress GP activity and did not eliminat e the glucose-mediated down-regulation of GP activity. We conclude that inc reased flux through the HBP results in enhanced glycogen accumulation due t o suppression of GP activity. These results demonstrate that the HBP is an important regulator of cellular glucose metabolism and supports its role as a cellular glucose/satiety sensor.