Dual Actions of ROS on Insulin Signaling Figure 3. Effects of H2O2 on the expression of mRNAs encoding antioxidative enzymes in H4IIEC hepatocytes. H4IIEC cells were serum- starved overnight and treated with the indicated concentrations of H2O2 for 3 h. Levels of the mRNAs encoding GPX1, GPX3, and catalase were measured by real-time-PCR. Values represent the mean 6 S.D (n = 6). doi:10.1371/journal.pone.0027401.g003 concentrations of H2O2 reduce insulin-stimulated Akt phosphor- ylation, at least partly, by increasing the phosphorylation of JNK and serine residue of IRS-1. Effects of antioxidant N-acetyl-L-cysteine on H2O2-induced alterations of insulin signal transduction in H4IIEC hepatocytes Next, we determined whether antioxidant N-acetyl-L-cysteine (NAC) attenuates the dual actions of H2O2 on insulin signal transduction. We pretreated H4IIEC hepatocytes with 10 mM of NAC, which corresponds to the maximum concentration of NAC commonly used in in vitro experiments [6]. Pretreatment with NAC decreased H2O2 concentrations in the culture medium of the cells treated with H2O2 (Fig. 8). NAC at the concentration of 10 mM was enough to quench H2O2 at up to 50 mM. NAC canceled the signal-promoting action of low concentrations of H2O2 (Fig. 9A). In addition, although NAC impaired insulin-stimulated phosphor- ylation of Akt in the absence of H2O2, it canceled out further impairment of insulin signaling induced by 25 mM of H2O2 (Fig. 9B). Discussion In the current study, we used the membrane-permeable oxidant H2O2 to demonstrate that ROS can exert either positive or negative effects on insulin signal transduction, depending on the ROS concentration. Our data suggest that the overall concentra- tion of ROS, irrespective of the subcellular compartments in which they act, determines whether they enhance or impair insulin signal transduction. The observed negative effects of high H2O2 concentrations on insulin signal transduction are consistent with our previous report, which showed that mitochondria-derived ROS induce insulin resistance in H4IIEC hepatocytes treated with palmitate by activating JNK [6]. Many lines of evidence suggest that chronic accumulation of ROS has a causal role in the development of insulin resistance [4,5]. The activation of stress kinases such as JNK is thought to play a central role in the development of ROS- associated insulin resistance. The upstream molecules that regulate JNK phosphorylation in response to increases in cellular ROS levels include thioredoxin (TRX) and apoptosis signal-regulating kinase 1 (ASK1) [9,10]. ROS oxidizes TRX and consequently removes it from pre-existing TRX-ASK1 complexes, leading to the activation of ASK1 and JNK signaling. The results of our previous in vitro study indicated that using antioxidants to remove ROS and consequently suppress JNK activation has the potential to improve insulin sensitivity [6]. To date, however, the larger clinical intervention trials conducted to evaluate the potential of antioxidant supplements in preventing the development of diabetes have been unable to observe any positive effects [11,12,13]. These conflicting findings led us to hypothesize that the complete removal of ROS from cells does not necessarily improve insulin resistance, and to pay particular attention to the dose-dependent dual actions of ROS on insulin signaling. PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27401