Hypoxic activation of AMPK is dependent on mitochondrial ROS but independent of an increase in AMP/ATP ratio

AMP-activated protein kinase (AMPK) is a sensor of cellular energy status found in metazoans that is known to be activated by stimuli that increase the cellular AMP/ATP ratio. Full activation of AMPK requires specific phosphorylation within the activation loop of the catalytic domain of the α-subunit by upstream kinases such as the serine/threonine protein kinase LKB1. Here we show that hypoxia activates AMPK through LKB1 without an increase in the AMP/ATP ratio. Hypoxia increased reactive oxygen species (ROS) levels and the antioxidant EUK-134 abolished the hypoxic activation of AMPK. Cells deficient in mitochondrial DNA (ρ⁰ cells) failed to activate AMPK during hypoxia but are able to in the presence of exogenous H₂O₂. Furthermore, we provide genetic evidence that ROS generated within the mitochondrial electron transport chain and not oxidative phosphorylation is required for hypoxic activation of AMPK. Collectively, these data indicate that oxidative stress and not an increase in the AMP/ATP ratio is required for hypoxic activation of AMPK.