The hexosamine biosynthesis pathway has been hypothesized to be involved in mediating some of the adverse effects of high glucose. We have previously shown that glucose downregulates basal glycogen synthase (GS) activity in Rat-1 cells and that overexpressing the rate-limiting enzyme in the hexosamine biosynthesis pathway (glutamine:fructose-6-phosphate amidotransferase [GFA]) makes the cells more sensitive to these effects of glucose. GFA overexpression also leads to a reduction in insulin sensitivity of GS. Here we examine the effects of glucose and glucosamine on insulin-stimulated GS activity and on protein phosphatase-1 (PP1) activity. These activities were assayed in cytoplasmic extracts from Rat-1 fibroblasts overexpressing human GFA and cultured in varying glucose concentrations. Both maximal insulin-stimulated GS activity and insulin sensitivity decreased with increasing glucose. Overexpression of GFA leads to a further reduction in insulin sensitivity but not in maximal insulin-stimulated GS activity. Because there were no differences in total (glucose-6-phosphate-dependent) GS activity between cell lines or as a function of glucose concentration, these results most likely reflect a change in the phosphorylation state of the synthase. Activity of PP1, a potential mediator of these effects, was responsive to glucose and hexosamines. Control cells showed a 9.3 ± 4.3% decrease in PP1 activity with increasing glucose. GFA cells showed a greater response to glucose, with PP1 activity decreasing 34.2 ± 5.5% with increasing glucose. Glucosamine was more potent than glucose in decreasing PP1 activity in control cells. Cells overexpressing the normal human insulin receptor (HIRc-B) were used to facilitate analysis of insulin-stimulated PP1 activity. Stimulation with 1.7 mmol/l insulin led to a 37.6 ± 9.9% increase in PP1 activity in HIRc-B cells cultured in 1 mmol/l glucose, while cells cultured in 5 mmol/l glucosamine or 20 mmol/l glucose demonstrated only 3.79 ± 0.60 or 1.6 ± 0.75% increases, respectively. We conclude that both basal and insulin-stimulable GS and PP1 activity are downregulated by high glucose in fibroblasts and this regulation is mediated by products of the hexosamine biosynthesis pathway.

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