Many authors suggest that an increased production of ROS in

Many authors suggest that an increased production of ROS in the beginning of differentiation is an essential factor for adipogenesis (Liu et al., 2012; Zhou et al., 2014; Wang and Hai, 2015). ROS could, in small quantities, act in cell signaling (Mc Bride et al., 2006; Zhang and Gutterman, 2007) and influence the function of several proteins involved in adipogenesis (Lowe et al., 2011). One of the reasons for that could be the suppression of pathways involved on osteoblast differentiation (Atashi et al., 2015). Mitochondria are the major source of intracellular ROS as a result of mitochondrial ETC activity (Zhou et al., 2014) and here we observed an increased mitochondrial activity in 7days of adipogenic differentiation. We also analyzed ROS production by A-366 and, surprisingly, an increased production of ROS (hydrogen peroxide) was only detected in 3days of induction, which may represent a stimulus for differentiation and can be related to cell commitment to adipogenesis. For 7days of induction, however, ROS levels were the same as in the control group, showing that the antioxidant defenses were possibly stimulated in the cell. Thus, the presence of ROS is important in the first steps of adipogenesis, but not in excess, since high concentrations may result in oxidative stress and cell damage. Here we only detected ROS in the first days of stimulus, followed by a return to control levels, indicating a cytoprotective activity. To confirm this hypothesis, we evaluated the NPT/GSH concentration and SOD and catalase activities, three essential antioxidants for redox milieu regulation (Zhou et al., 2014). The NPT concentration was lower for the induced cells (3 and 7days), indicating the presence of high levels of ROS and oxidative stress (Spangenberg et al., 2013). SOD activity was significantly higher in induced cells, since the first days of differentiation, but catalase (CAT) activity was increased only after 7days of induction. The SOD-CAT system usually work together, with SOD converting superoxide in hydrogen peroxide and CAT degrading it to water and molecular oxygen. SOD is widely distributed in cell compartments, but catalase is mainly peroxisomal in mammals (Glorieux et al., 2015). Thus, the increase of catalase activity and decrease of hydrogen peroxide after 7days of induction is a preliminary evidence of the involvement of peroxisomes in ROS production and in the process of differentiation into adipocytes. Lipid peroxidation can be initiated by hydroxyl radicals produced through Fenton and Harber-Weiss reactions (Das et al., 2015). Considering that lipid peroxidation decreased after 3 and 7days of induction, efficient cytoprotective activity acted shortly after the differentiation stimulus initiation. In cells, the threshold between physiological/signaling levels of ROS and pathological ones is an increase in lipid oxidation (Singh et al., 2015). Thus, ROS produced in the first steps of differentiation may be at a signaling level. ROS required for the commitment of differentiation may have come from other sources that not mitochondria, such as NADPH oxidase (NOX) (Kanda et al., 2011) and peroxisomes, since we observed an increase in ROS production but not in mitochondrial activity and mitochondrial superoxide generation in early stages of differentiation. ROS, as second messengers, are predominantly generated by the NOX family of proteins in the form of superoxide, a short-lived type of ROS (Pendyala and Natarajan, 2010). Taken together, our data suggest that NOX could be the source of ROS after 3-day induction of differentiation; in addition, SOD activity was also increased at day 3. To test our hypothesis, we used apocynin (Sigma-Aldrich®) as a NADPH oxidase inhibitor (Sun et al., 2015) and we measured ROS generation using DCFH-DA, as previously described. Cells treated with apocynin and induced to differentiation did not show alteration in ROS generation at the same time point when compared to control cells (Fig. 5), supporting our idea. In summary, we believe that superoxide can be produced by NOX and converted to hydrogen peroxide by SOD, and these events are leading to cytoprotection mechanisms that act avoiding oxidative stress and ROS deleterious effects.