Oxidative stress activates AMPK in cultured cells primarily by increasing cellular AMP and/or ADP

•AMPK is activated by oxidative stress generated using glucose oxidase.•Activation is largely, but not solely, mediated by increases in AMP and/or ADP.•Increases in Thr172 phosphorylation are mediated by inhibition of dephosphorylation. AMPK is known to be activated by oxidative stress. Addition of glucose oxidase to cells generates H2O2 at a constant rate that is opposed by enzymic degradation, providing a good model for physiological oxidative stress. AMPK activation by glucose oxidase correlated with increases in cellular AMP:ATP and was greatly reduced in cells expressing an AMP-insensitive AMPK mutant, although a small degree of activation remained. The effects of increased AMP were partly due to inhibition of Thr172 dephosphorylation. These results suggest that changes in adenine nucleotides, rather than direct oxidative modification, are the major drivers of AMPK activation during oxidative stress.