REGULATION OF INSULIN-STIMULATED GLYCOGEN-SYNTHASE ACTIVITY BY OVEREXPRESSION OF GLUTAMINE - FRUCTOSE-6-PHOSPHATE AMIDOTRANSFERASE IN RAT-1FIBROBLASTS

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 meta bolism of glucose to glucosamine might be involved. For example, it ha s been shown that glucosamine is more potent than glucose in inducing insulin resistance in cultured adipocytes and in regulating the transc ription of the growth factor transforming growth factor alpha in smoot h 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 hy pothesis that this hexosamine biosynthesis pathway is involved in the induction of insulin resistance, we have overexpressed the enzyme glut amine:fructose-6-phosphate amidotransferase in Rat-1 fibroblasts and i nvestigated its effects on insulin action in those cells. We electropo rated Rat-1 fibroblasts with expression plasmids that did and did not contain the gene for glutamine:fructose-6-phosphate amidotransferase a nd measured glycogen synthase activity at varying insulin concentratio ns. Insulin stimulation was blunted in the glutamine:fructose-6-phosph ate 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 glut amine:fructose-6-phosphate amidotransferase and control cells, respect ively). Rat-1 fibroblasts incubated with 5.0 mM glucosamine for 3 days exhibited a similar shift in the dose-response curve. The rightward s hift in the dose-response curve is seen as early as 2 days after porat ion. 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-p hosphate amidotransferase cells developed later than the shift in the dose-response curve; by 4 days after transfection, a 61 +/- 14.5% incr ease in basal glycogen synthase activity had been reached. Overexpress ion of glutamine:fructose-6-phosphate amidotransferase did not affect total glycogen synthase activity or glucose uptake in these cells. Fur thermore, no change occurred in the number or affinity of insulin rece ptors in the glutamine:fructose-6-phosphate amidotransferase-transfect ed cells, indicating that the insulin resistance induced by glutamine: fructose-6-phosphate amidotransferase occurs distal to insulin binding .