High glucose concentrations such as are seen in diabetes mellitus are known to have deleterious effects on cells, but the pathways by which glucose induces these effects are unknown. One hypothesis is that metabolism of glucose to glucosamine might be involved. For example, it has been shown that glucosamine is more potent than glucose in inducing insulin resistance in cultured adipocytes and in regulating the transcription of the growth factor transforming growth factor alpha in smooth muscle cells. The rate-limiting step in glucosamine synthesis is the conversion of fructose-6-phosphate to glucosamine-6-phosphate by the enzyme glutamine:fructose-6-phosphate amidotransferase. To test the hypothesis that this hexosamine biosynthesis pathway is involved in the induction of insulin resistance, we have overexpressed the enzyme glutamine:fructose-6-phosphate amidotransferase in Rat-1 fibroblasts and investigated its effects on insulin action in those cells. We electroporated Rat-1 fibroblasts with expression plasmids that did and did not contain the gene for glutamine:fructose-6-phosphate amidotransferase and measured glycogen synthase activity at varying insulin concentrations. Insulin stimulation was blunted in the glutamine:fructose-6-phosphate amidotransferase-transfected cells, resulting in decreased insulin sensitivity reflected by a rightward shift in the dose-response curve for activation of synthase (ED50 = 7.5 nM vs. 3.4 nM insulin, in glutamine:fructose-6-phosphate amidotransferase and control cells, respectively). Rat-1 fibroblasts incubated with 5.0 mM glucosamine for 3 days exhibited a similar shift in the dose-response curve. The rightward shift in the dose-response curve is seen as early as 2 days after poration. Overexpression of glutamine:fructose-6-phosphate amidotransferase also induced an increase in basal activity of glycogen synthase. The increase in basal glycogen synthase activity in glutamine:fructose-6-phosphate amidotransferase cells developed later than the shift in the dose-response curve; by 4 days after transfection, a 61 ± 14.5% increase in basal glycogen synthase activity had been reached. Overexpression of glutaminerfructose-6-phosphate amidotransferase did not affect total glycogen synthase activity or glucose uptake in these cells. Furthermore, no change occurred in the number or affinity of insulin receptors in the glutamine:fructose-6-phosphate amidotransferase–transfected cells, indicating that the insulin resistance induced by glutamine:fructose-6-phosphate amidotransferase occurs distal to insulin binding.

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