Superoxide is the major reactive oxygen species regulating autophagy

Autophagy is involved in human diseases and is regulated by reactive oxygen species (ROS) including superoxide (O-2(center dot-)) and hydrogen peroxide (H2O2). However, the relative functions of O-2(center dot-) and H2O2 in regulating autophagy are unknown. In this study, autophagy was induced by starvation, mitochondrial electron transport inhibitors, and exogenous H2O2. We found that O-2(center dot-) was selectively induced by starvation of glucose, L-glutamine, pyruvate, and serum (GP) whereas starvation of amino acids and serum (AA) induced O-2(center dot-) and H2O2. Both types of starvation induced autophagy and autophagy was inhibited by overexpression of SOD2 (manganese superoxide dismutase, Mn-SOD), which reduced O-2(center dot-) levels but increased H2O2 levels. Starvation-induced autophagy was also inhibited by the addition of catalase, which reduced both O-2(center dot-) and H2O2 levels. Starvation of GP or AA also induced cell death that was increased following treatment with autophagy inhibitors 3-methyladenine, and wortamannin. Mitochondrial electron transport chain (mETC) inhibitors in combination with the SOD inhibitor 2-methoxyestradiol (2-ME) increased O-2(center dot-) levels, lowered H2O2 levels, and increased autophagy. In contrast to starvation, cell death induced by mETC inhibitors was increased by 2-ME. Finally, adding exogenous H2O2 induced autophagy and increased intracellular O-2(center dot-) but failed to increase intracellular H2O2. Taken together, these findings indicate that O-2(center dot-) is the major ROS-regulating autophagy. Cell Death and Differentiation (2009) 16, 1040-1052; doi: 10.1038/cdd.2009.49; published online 1 May 2009