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813 DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS INTO FAT INVOLVES REACTIVE OXYGEN SPECIES AND UPREGULATION OF ANTIOXIDANT ENZYMES

Objectives: Both reactive oxygen species (ROS) and the transcription factor forkhead box protein (FOXOs) family are involved in regulating adipogenic differentiation of pre-adipocytes and stem cells to produce fat. While FOXOs has a pivotal role in maintaining cellular redox homeostasis, the interactions between ROS and FOXOs during adipogenesis are not clear. Here we examined how ROS and FOXOs regulated adipogenesis in human adipose-derived stem cells (hASC). Methods: hASC from abdominal fat were isolated and characterized by mesenchymal stem cell markers (CD29, CD44, CD73, CD90 and CD105, but not CD45 or CD31). The standard adipogenic cocktail medium (IDII; insulin, dexamethasone, indomethacin and IBMX) was used to induce adipogenic differentiation of hASC to adipocytes. Results: Using IDII cocktail, adipogenesis was induced in hASC, which was accompanied by enhanced H2O2 generation and upregulation of FOXO1 and FOXO3. Treatment with the antioxidant N-acetylcysteine (NAC) or silencing FOXO1 expression partially inhibited IDII-induced adipogenesis. Overexpressing a H2O2-generating NADPH oxidase Nox4 mimicked IDII-induced H2O2 production, but only weakly stimulated adipogenesis as compared to IDII. Nox4 overexpression had no effects on FOXO1 levels. Antioxidant enzymes including SOD2, catalase and GPx were upregulated by IDII during adipogenesis, and these effects were blunted by FOXO1 silencing. Conclusion: Our data therefore suggest that ROS and FOXO1 have independent effects in promoting adipogenic differentiation of hASC. FOXO1 may act as a self-regulatory mechanism which protects cells from excessive oxidative stress by maintaining a balance of ROS generation and antioxidant enzyme expression in hASC undergoing adipogenesis, to avoid oxidative stress and cell damage.