We have examined the hypothesis that glucosamine (GlcN) can rapidly induce insulin resistance through an allosteric mechanism. When insulin-treated adipocytes were exposed to 2 mM GlcN, glucose uptake was rapidly reduced by similar to 60% with a T-1/2 Of 2 min. We also observed an increase in intracellular GlcN-6-P (at 5 min) from undetectable levels to similar to 260 nmol/g. Continued GlcN treatment resulted in additional accumulation of GlcN-6-P (> 1200 nmol/g at 2 h), but caused no further decrease in glucose uptake. Although the acute inhibitory action of GlcN Could be completely reversed by removing extracellular GlcN, a slow and progressive decrease in insulin-stimulated glucose transport was observed with longer treatment times (T-1/2 of 45 min, 62% loss by 5 h). From these data, we conclude that: (1) GlcN elevates intracellular GlcN-6-P levels within minutes, resulting in desensitization of the glucose transport system through allosteric inhibition of hexokinase; (2) prolonged treatment elevates GlcN-6-P to levels that cannot be effectively lowered by cell washing; and (3) residual levels of GlcN-6-P continue to allosterically inhibit glucose uptake, resulting in a slower rate of desensitization that is temporally similar to glucose-induced desensitization, but mechanistically different. (c) 2005 Elsevier Inc. All rights reserved.