It has been hypothesized that glucose-induced insulin resistance is mediated by accumulation of UDP-N-acetylhexosamines (UDP-HexNAcs). In a previous study on rat epitrochlearis muscles incubated with high concentrations of glucose and insulin, we found that insulin resistance developed even when the increase in UDP-HexNAcs was prevented (Kawanaka K, D-H Han, J Gao, LA Nolte, and JO Holloszy: Development of glucose-induced insulin resistance in muscle requires protein synthesis. J.Biol.Chem. 276:20101-20107, 2001.). Furthermore, actinomycin D completely prevented glucose-induced insulin resistance despite a greater accumulation of UDP-HexNAcs. In the present study, we used the same epitrochlearis muscle preparation as well as the rat hemidiaphragm to determine whether, like glucose, glucosamine causes insulin resistance by an actinomycin D inhibitable process. Incubation of diaphragm muscles with 10 mM glucosamine for 3h resulted in a ~5-fold increase in UDP-HexNAcs, a ~50% reduction in insulin responsiveness of glucose transport, and a 58% reduction in ATP concentration. These effects of glucosamine were not prevented by actinomycin D. Incubation of epitrochlearis muscles with 20 mM glucosamine for 3h or with 10 mM glucosamine for 5h also caused large decreases in insulin responsiveness of glucose transport, but with no reduction in ATP concentration. Actinomycin D did not prevent the glucosamine- induced insulin resistance. The insulin-induced increases in tyrosine phosphorylation of IRS-1 and binding of PI 3-kinase to IRS-1 were decreased ~60% in epitrochlearis muscles exposed to glucosamine. This is in contrast to glucose-induced insulin resistance, which was not associated with impaired insulin signaling. These results provide evidence that glucosamine and glucose induce insulin resistance by different mechanisms.