action [7]. More recently, Loh et al. reported that mice lacking glutathione peroxidase 1 (Gpx1), a key enzyme involved in the removal of ROS, are protected from high-fat diet-induced insulin resistance, providing causal evidence for the enhancement of insulin signaling by ROS in vivo [8]. These early reports indicate that ROS can act both positively and negatively on insulin signaling. However, the molecular mechanisms regulating the dual actions of ROS on insulin signaling are not fully understood. In the current study, to identify the key factors determining whether ROS impair or enhance intracellular insulin signaling, we directly treated H4IIEC hepatocytes with hydrogen peroxide (H2O2), a representative membrane-permeable oxidant and the most abundant ROS in the cell. Our results demonstrate that H2O2 has dual effects on insulin signal transduction in H4IIEC hepatocytes and that these roles depend on the H2O2 concentra- tion used, suggesting that the intracellular concentration of ROS themselves may be a major factor in determining whether ROS impair or enhance insulin signaling. Results Time course of extracellular H2O2 concentration following its administration to H4IIEC hepatocytes We treated H4IIEC hepatocytes with H2O2, a representative membrane-permeable ROS. Exogenous H2O2 is time-dependent- ly reduced and neutralized by intracellular antioxidant enzymes. To assess how long the H2O2 remained effective, we measured H2O2 concentrations in the culture medium over a specific time course after its administration (Fig. 1). The H2O2 concentration in the culture medium returned to basal level within 30 min of its administration to H4IIEC hepatocytes. Therefore, when cells were Figure 1. Time course of extracellular H2O2 concentration following its administration to H4IIEC hepatocytes. H4IIEC cells were incubated with the indicated concentrations of H2O2 for the indicated periods of time, and then the concentration of H2O2 in the culture medium was measured by ferrous oxidation of xylenol orange (FOX) assay. doi:10.1371/journal.pone.0027401.g001 Dual Actions of ROS on Insulin Signaling treated with H2O2 for a total of 3 h in subsequent experiments, the H2O2-containing medium was replaced every 30 min. Dual effects of H2O2 on insulin-stimulated phosphorylation of Akt and GSK-3 We examined the effects of H2O2 on insulin signaling in H4IIEC hepatocytes (Fig. 2). H4IIEC3 cells were treated with a range of H2O2 concentrations for 3 h and then stimulated with insulin for 15 min. As expected, insulin-stimulated serine phosphorylation of Akt and GSK-3a was inhibited at high concentrations of H2O2. Insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR) was unaffected by high H2O2 concentrations. In contrast, lower concentrations of H2O2 enhanced insulin-stimulated phosphoryla- tion of Akt and GSK-3a, without affecting the overall levels of the proteins. We found that 5–10 mM were the concentrations of which H2O2 certainly promotes insulin-induced Akt phosphorylation in our cell lines. Thus, we used 5–10 mM of H2O2 to certainly increase insulin signaling in the following experiments. Effects of H2O2 treatment on the expression of genes encoding antioxidant enzymes We hypothesized that weak oxidative stress caused by low concentrations of H2O2 may trigger a preconditioning response by inducing antioxidant enzymes such as catalase and Gpx. We assayed the expression of the genes encoding catalase, Gpx1, and Gpx3 in H4IIEC hepatocytes 3 h after treatment with various concentrations of H2O2 (Fig. 3). Treatment with H2O2 at concentrations in the range 5–50 mM did not alter the expression of any of these three genes. Attenuation of PTP1B activity contributes to H2O2-induced enhancement of insulin signaling To clarify the mechanism by which low concentrations of H2O2 enhance insulin signaling, we measured the activity of PTP1B, a negative regulator of insulin signaling, in H2O2-treated cells. PTP1B activity was dose-dependently suppressed by H2O2 (Fig. 4A), even at H2O2 concentrations as low as 5 mM. Overall PTP1B protein levels were unaffected by H2O2 treatment (Fig 4B). To further determine whether phosphatases mediate the insulin-promotive effect induced by low concentrations of H2O2, we treated the cells with vanadate, a non-specific inhibitor for phosphatases such as PTP1B (Fig. 5). Coadministration with vanadate increased insulin-stimulated Akt phosphorylation in the cells treated with 5 mM of H2O2. However, it did not further increase insulin-stimulated Akt phosphorylation in the presence of 10 mM of H2O2 (Fig. 5). Activation of JNK contributes to H2O2-induced insulin resistance To examine the mechanism by which high concentrations of H2O2 impair insulin signaling, we measured the levels of phospho- JNK in H2O2-treated cells. JNK is a stress-activated protein kinase that phosphorylates IRS-1 and -2 at serine residues in response to increases in cellular ROS levels. JNK-induced serine phosphor- ylation of IRS impairs IRS tyrosine phosphorylation, resulting in the inhibition of insulin receptor-mediated signaling. Treatment with H2O2 increased the phosphorylation of both JNK and its direct target, c-Jun dose-dependently (Fig. 6). To further determine whether JNK mediates insulin resistance induced by high concentrations of H2O2, we transfected H4IIEC hepatocytes with siRNA specific for JNK1 (Fig. 7). Knockdown of JNK partly suppressed serine phosphorylation of IRS-1 induced by high concentrations of H2O2 (Figure 7A). As a result, insulin resistance induced by high concentrations of H2O2 was partly rescued by silencing of JNK (Fogiure 7B). These results suggest that high PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27401