Overexpression of Glutamine:Fructose-6-Phosphate Amidotransferase in Rat-1 Fibroblasts Enhances Glucose-Mediated Glycogen Accumulation via Suppression of Glycogen Phosphorylase Activity*

Abstract

The hexosamine biosynthesis pathway (HBP) mediates many of the adverse effects of excess glucose. We have shown previously that glucose down-regulates basal and insulin-stimulated glycogen synthase (GS) activity. Overexpression of the rate-limiting enzyme in the HBP, glutamine:fructose-6-phosphate amidotransferase (GFA), mimics these effects of high glucose and renders the cells more sensitive to glucose. Here we examine the role of the HBP in regulating cellular glycogen content. Glycogen content and glycogen phosphorylase (GP) activity were determined in Rat-1 fibroblasts that overexpress GFA. In both GFA and controls there was a dose-dependent increase in glycogen content (approximately 8-fold) in cells cultured in increasing glucose concentrations (1-20 mM). There was a shift to the left in the glucose dose-response curve for glycogen content in GFA cells (ED50 for glycogen content = 5.80+/-1.05 vs. 8.84+/-0.87 mM glucose, GFA vs. control). Inhibition of GFA 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 in 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 glycogen 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 eliminate the glucose-mediated down-regulation of GP activity. We conclude that increased flux through the HBP results in enhanced glycogen accumulation due to 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.

Hexosamines Are Unlikely to Function as a Nutrient-Sensor in 3T3-L1 Adipocytes: A Comparison of UDP-Hexosamine Levels after Increased Glucose Flux and Glucosamine Treatment

“…The amount of UDP-glucose in 3T3-L1 adipocytes decreased in a dose-dependent manner when the cells were treated with glucosamine. This might indicate that activation of the hexosamine biosynthesis pathway by incubation of the cells with glucosamine either stimulates processing of UDP-glucose to generate glycogen (20) or that the UDP from UDP-glucose is released for the synthesis of UDPhexosamines (21).…”

Overexpression of the Complementary DNA for Human Glutamine: Fructose-6-Phosphate Amidotransferase in Mesangial Cells Enhances Glucose-Induced Fibronectin Synthesis and Transcription Factor Cyclic Adenosine Monophosphate-Responsive Element Binding Phosphorylation

“…25,54 In addition, we have shown that stable GFA overexpression is a unique model even when compared with culturing in low-dose glucosamine. 11…”

“…9 The HBP pathway and tissue concentrations of UDP-GlcNAc can be increased by various methods such as exposure to prolonged hyperglycemia, glucosamine or uridine infusion, hyperlipidemia, and GFA overexpression. [10][11][12][13] The HBP has been shown to have protean effects on glucose metabolism, insulin action, and other glucose-mediated cellular events. For example, chronic exposure of rat-1 fibroblasts to high glucose decreases both basal and insulin-stimulated glycogen synthase activity, and glucosamine or overexpression of GFA mimics the effects of high glucose.…”

“…For example, chronic exposure of rat-1 fibroblasts to high glucose decreases both basal and insulin-stimulated glycogen synthase activity, and glucosamine or overexpression of GFA mimics the effects of high glucose. [10][11][12][13][14] The activities of several proteins involved in the regulation of glycogen metabolism are regulated by the HBP. 11,15,16 Moreover, transgenic mice overexpressing GFA in skeletal muscle, fat, and β-cells are insulin resistant.…”

“…[10][11][12][13][14] The activities of several proteins involved in the regulation of glycogen metabolism are regulated by the HBP. 11,15,16 Moreover, transgenic mice overexpressing GFA in skeletal muscle, fat, and β-cells are insulin resistant. 13,17,18 Glucosamine has also been shown to be more potent than glucose in the transcriptional regulation of TGF-α and TGF-β.…”