Development and comparison of two 3T3-L1 adipocyte models of insulin resistance: Increased glucose flux vs glucosamine treatment

Insulin resistance can be induced in vivo by intravenous infusion of glucos amine or in cells by incubation with glucosamine. However, a publication (H resko, R. C,, ef al, (1998) J, Biol. Chem. 273, 20658-20668) suggests a tri vial explanation of glucosamine-induced insulin resistance whereby intracel lular ATP pools are depleted presumably due to the phosphorylation of gluco samine to glucosamine 6-phosphate, a hexosamine pathway intermediate. The r educed ATP level impaired insulin receptor (In) autophosphorylation and tyr osine kinase activity toward substrates. The present work describes the dev elopment and comparison of two methods for inducing insulin resistance, by treating 3T3-L1 adipocytes overnight using either 25 mM glucose/5 nM insuli n or 2 mM glucosamine. Under these conditions basal glucose transport rates were comparable with controls. Insulin-stimulated a-deoxyglucose uptake, h owever, was reduced by similar to 45% in response to both high glucose/ ins ulin and glucosamine treatment, relative to control cells. The total relati ve amounts of the insulin-responsive glucose transporter, Glut4, remained c onstant under both treatment conditions. The relative phosphotyrosine (Tyr( P)) contents of the insulin receptor and its substrate 1 (IRS-1) were asses sed in whole cell homogenates. With both methods to induce insulin resistan ce, IR/IRS-1 Tyr(P) levels were virtually indistinguishable from those in c ontrol cells. insulin-stimulated phosphorylation of Akt on Ser(473) was not impaired in insulin-resistant cells, Furthermore, the relative Tyr(P) cont ent of the PDGF receptor was comparable in high glucose/insulin- or glucosa mine-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 path way enzymes, were similar in both treatments when compared with controls. T hus, 3T3-L1 adipocytes can be used as a model system for studying insulin r esistance induced by increased influx of glucose. Under appropriate experim ental conditions, glucosamine treatment can mimic the effects of increased glucose flux without impairment of tyrosine phosphorylation-based signaling . (C) 2000 Academic Press.