Enhanced sensitivity of insulin-resistant adipocytes to vanadate is associated with oxidative stress and decreased reduction of vanadate (+5) to vanadyl (+4)

Vanadate (sodium orthovanadate), an inhibitor of phosphotyrosine phosphatas es (PTPs), mimics many of the metabolic actions of insulin in vitro and in vivo. The potential of vanadate to stimulate glucose transport independent of the early steps in insulin signaling prompted us to test its effectivene ss in an in vitro model of insulin resistance. In primary rat adipocytes cu ltured for 18 h in the presence of high glucose (15 mM) and insulin (10(-7) M), sensitivity to insulin-stimulated glucose transport was decreased. In contrast, there was a paradoxical enhanced sensitivity to vanadate of the i nsulin-resistant cells (EC50 for control, 325 +/- 7.5 muM; EC50 for insulin -resistant, 171 +/- 32 muM; p < 0.002). Enhanced sensitivity was also prese nt for vanadate stimulation of insulin receptor kinase activity and autopho sphorylation and Akt/protein kinase B Ser-473 phosphorylation consistent wi th more effective PTP inhibition in the resistant cells. Investigation of t his phenomenon revealed that 1) depletion of GSH with buthionine sulfoximin e reproduced the enhanced sensitivity to vanadate while preincubation of re sistant cells with N-acetyleysteine (NAC) prevented it, 2) intracellular GS H was decreased in resistant cells and normalized by NAC, 3) exposure to hi gh glucose and insulin induced an increase in reactive oxygen species, whic h was prevented by NAC, 4) EPR (electron paramagnetic resonance) spectrosco py showed a decreased amount of vanadyl (+4) in resistant and buthionine su lfoximine-treated cells, which correlated with decreased GSH and increased vanadate sensitivity, while total vanadium uptake was not altered, and 5) i nhibition of recombinant PTP1B in vitro was more sensitive to vanadate (+5) than vanadyl (+4). In conclusion, the parodoxical increased sensitivity to vanadate in hyperglycemia-induced insulin resistant adipocytes is due to o xidative stress and decreased reduction of vanadate (+5) to vanadyl (+4). T hus, sensitivity of PTP inhibition and glucose transport to vanadate is reg ulated by cellular redox state.