Insulin resistance can be induced in vivo by intravenous infusion of glucosamine or in cells by incubation with glucosamine. However, a publication (Hresko, R. C,, ef al, (1998) J, Biol. Chem. 273, 20658-20668) suggests a trivial explanation of glucosamine-induced insulin resistance whereby intracellular ATP pools are depleted presumably due to the phosphorylation of glucosamine to glucosamine 6-phosphate, a hexosamine pathway intermediate. The reduced ATP level impaired insulin receptor (In) autophosphorylation and tyrosine kinase activity toward substrates. The present work describes the development and comparison of two methods for inducing insulin resistance, by treating 3T3-L1 adipocytes overnight using either 25 mM glucose/5 nM insulin or 2 mM glucosamine. Under these conditions basal glucose transport rates were comparable with controls. Insulin-stimulated a-deoxyglucose uptake, however, was reduced by similar to 45% in response to both high glucose/ insulin and glucosamine treatment, relative to control cells. The total relative amounts of the insulin-responsive glucose transporter, Glut4, remained constant under both treatment conditions. The relative phosphotyrosine (Tyr(P)) contents of the insulin receptor and its substrate 1 (IRS-1) were assessed in whole cell homogenates. With both methods to induce insulin resistance, IR/IRS-1 Tyr(P) levels were virtually indistinguishable from those in control cells. insulin-stimulated phosphorylation of Akt on Ser(473) was not impaired in insulin-resistant cells, Furthermore, the relative Tyr(P) content of the PDGF receptor was comparable in high glucose/insulin- or glucosamine-treated 3T3-L1 adipocytes upon subsequent challenge with PDGF. Finally, the relative amounts of glutamine:fructose-6-phosphate amido-transferase and O-linked N-acetylglucosamine transferase, two important hexosamine pathway enzymes, were similar in both treatments when compared with controls. Thus, 3T3-L1 adipocytes can be used as a model system for studying insulin resistance induced by increased influx of glucose. Under appropriate experimental conditions, glucosamine treatment can mimic the effects of increased glucose flux without impairment of tyrosine phosphorylation-based signaling.