The hexosamine biosynthesis pathway has been hypothesized to mediate some of the regulatory as well as the deleterious effects of glucose. We have stably overexpressed the cDNA for human glutamine:fructose-6-phosphate amidotransferase (GFA), the rate-limiting enzyme in the hexosamine biosynthesis pathway, in rat-1 fibroblasts. Two cell lines expressing the human RNA were selected by Northern analysis, and they exhibited 51-95% increases in GFA activity. Insulin-stimulated glycogen synthase (GS) activity and net glycogen synthesis were assayed, and GFA cells revealed decreased insulin sensitivity for both GS and net glycogen synthesis. The ED50 for insulin stimulation of GS was 2.45 ± 0.4 nmol/l insulin in controls and 5.29 ± 1.01 nmol/l in GFA cells (P < 0.005). For insulin-stimulated glycogen synthesis, the ED50 was 3.43 ± 0.88 nmol/l in controls and 5.54 ± 0.98 nmol/l in GFA cells (P < 0.005). There were no significant differences in maximally insulin-stimulated or total GS activities, insulin binding or receptor number, or glucose uptake between GFA and control cells. We also examined the effects of glucose on GS activity. GFA cells had a twofold increase in GS activity at low glucose (0.5 mmol/l) when compared with controls (P < 0.025). Both GFA and control cells had an ∼75–80% decrease in GS activity as glucose concentration was increased from 0.5 to 20 mmol/l. This change in GS activity was not observed until after 12 h in culture. GFA cells were more sensitive to the effects of glucose. The ID50 values for glucose were 1.72 ± 0.08 mmol/1 in GFA cells and 5.60 ± 2.05 mmol/l in control cells (P ? 0.05). Glucosamine mimicked the effects of glucose but at a much higher potency with an ED50 of 0.667 ± 0.15 mmol/1 in control cells cultured in low glucose. The changes in activity seen with glucose are not due to the osmotic effects of glucose or the carryover of intracellular effector molecules into the in vitro GS assay. In sum, the stable overexpression of the cDNA for human GFA leads to decreased insulin sensitivity of GS and glycogen synthesis and is accompanied by an increased sensitivity to inhibition of GS by high glucose. We conclude that glucose regulates glycogen synthesis through the metabolism of glucose to hexosamines.

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