High glucose/low dose insulin-mediated insulin resistance of glucose transport was studied in 3T3-L1 adipocytes. In this model proximal insulin signalling, including IRS-1-bound PI3-kinase activation is preserved, but insulin-stimulated Akt activation is markedly impaired. To assess a difference in acute insulin stimulated production of PtdIns(3,4,5)P₃ cells were labelled with ³²P-orthophosphate and glycerophosphoinositides were quantified by HPLC. While basal PtdIns(3,4,5)P₃ was similar, insulin stimulated its production 33.6% more in controls (p<0.03) than in insulin-resistant cells. PTEN protein, a lipid phosphatase, which dephosphorylates PtdIns(3,4,5)P₃ in the 3-position, was significantly and specifically increased in insulin resistant cells. Treatment with rapamycin, (a specific inhibitor of mTORC1), inhibited increased PTEN expression and partially restored insulin-stimulated glucose transport and Akt activation to insulin resistant cells. Acute insulin markedly stimulated Ser 636/639-phosphorylation of IRS-1, this was rapamycin inhibited, but was significantly decreased in cells that had been preexposed to insulin, while total IRS-1 was unaffected. These findings were essentially paralleled by changes in the activation of p70-S6kinase and S6-ribosomal protein. Overexpression of uncoupling protein-1 or manganese superoxide dismutase did not prevent the development of insulin resistant glucose transport and impaired Akt activation in high glucose/low insulin pretreated cells. The insulin resistance associated with glucotoxicity in our model reflects in part decreased availability of PtdIns(3,4,5)P₃ which correlates with increased PTEN protein expression. Chronic activation of mTORC1 plays a role in stimulating PTEN expression and possibly in activation or induction of a phosphoprotein phosphatase. No evidence was found for a role for increased mitochondrial superoxide production in this model.