GLUCOSAMINE-INDUCED INSULIN-RESISTANCE IN 3T3-L1 ADIPOCYTES IS CAUSEDBY DEPLETION OF INTRACELLULAR ATP

Glucosamine, which enters the hexosamine pathway downstream of the rat e-limiting step, has been routinely used to mimic the insulin resistan ce caused by high glucose and insulin. We investigated the effect of g lucosamine on insulin-stimulated glucose transport in 3T3-L1 adipocyte s. The Delta-insulin (insulin-stimulated minus basal) value for S-deox yglucose uptake was dramatically inhibited with increasing concentrati ons of glucosamine with an ED50 of 1.95 mM. Subcellular fractionation experiments demonstrated that reduction in insulin-stimulated 2-deoxyg lucose uptake by glucosamine was due to an inhibition of translocation of both Glut 1 and Glut 4 from the low density microsomes (LDM) to th e plasma membrane. Analysis of the insulin signaling cascade revealed that glucosamine impaired insulin receptor autophosphorylation, insuli n receptor substrate (IRS-1) phosphorylation, IRS-l-associated PI 3-ki nase activity in the LDM, and AKT-1 activation by insulin. Measurement of intracellular ATP demonstrated that the effects of glucosamine wer e highly correlated with its ability to reduce ATP levels. Reduction o f intracellular ATP using azide inhibited Glut 1 and Glut 4 translocat ion from the LDM to the plasma membrane, insulin receptor autophosphor ylation, and IRS-1 tyrosine phosphorylation. Additionally, both the re duction in intracellular ATP and the effects on insulin action caused by glucosamine could be prevented by the addition of inosine, which se rved as an alternative energy source in the medium. We conclude that d irect administration of glucosamine can rapidly lower cellular ATP lev els and affect insulin action in fat cells by mechanisms independent o f increased intracellular UDP-N-acetylhexosamines and that increased m etabolism of glucose via the hexosamine pathway may not represent the mechanism of glucose toxicity in fat cells.