We examined insulin signaling in rat epididymal adipocytes which developed insulin resistance by the in vivo infusion of glucosamine. Insulin-stimulated 2-deoxyglucose uptake into the adipocytes isolated from rats which were infused glucosamine for 4 hours was diminished by 26%. To analyze insulin signaling in adipocytes, the epididymal fat tissues were harvested 5 minutes after insulin administration (10U/kg), which was administered immediately after glucosamine infusion. Glucosamine had no effect on the insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate (IRS)-1. Glucosamine infusion decreased insulin-stimulated phosphatidylinositol (PI) 3-kinase activity by 66%. Glucosamine infusion also inhibited insulin-stimulated PI 3-kinase activity associated with IRS-1, 2, 3 by 30%, 43%, and 44%, respectively. There was no difference in the association of the 85kDa subunit of PI 3-kinase with the IRS-1 and IRS-2 protein. PI 3-kinase activity in adipocytes from rats treated with glucosamine that were administered platelet derived growth factor (3microg/kg) for 5 minutes was also reduced by 39%. When we measured the kinase activity of protein kinase C (PKC) lamda, which is the downstream effector of PI 3-kinase in isolated adipocytes, we found that glucosamine inhibited insulin stimulated PKClamda kinase activity by 33%. These results suggest that glucosamine infusion contributes to the development of insulin resistance by mainly modulating the PI 3-kinase molecules.