Figure 8. Time course of extracellular H2O2 concentration following its administration to H4IIEC hepatocytes pretreated with N-acetyl-L-cysteine. H4IIEC cells were serum-starved and treated with 10 mM of N-acetyl-L-cysteine overnight. Then, the cells were incubated with the indicated concentrations of H2O2 for the indicated periods of time, and the concentration of H2O2 in the culture medium was measured by ferrous oxidation of xylenol orange (FOX) assay. doi:10.1371/journal.pone.0027401.g008 enhancive factor for insulin signaling) and activation of JNK (the inhibitory factor for insulin signaling) (Fig. 10). For example, 25– 50 mM of H2O2 significantly decreased PTP1B activity, whereas these concentrations of H2O2 dramatically increased JNK phosphorylation simultaneously. As a result of the summation of these opposite two factors, insulin-induced Akt phosphorylation was impaired by 25–50 mM of H2O2 (Figure 2B), suggesting that the increase in JNK activation has a greater influence on net insulin signaling than the inactivation of PTP1B in these concentrations of H2O2. On the other hands, 10 mM of H2O2 induced a dramatic decrease in PTP1B activity and a moderate increase in JNK phosphorylation, resulting in an enhancement of insulin-stimulated Akt phosphorylation. These results suggest that the decrease in PTP1B activity has a greater influence than the increase in JNK phosphorylation in the cells treated with 10 mM of H2O2. Early in vitro studies indicated that in the presence of H2O2, the pKa values and reaction rates for PTP1B were lower than those for TRX, an upstream regulator of JNK [25], suggesting that PTP1B may be more susceptible than TRX to oxidation by H2O2 in vivo. Thus, the dual actions of H2O2 on insulin signal transduction in H4IIEC hepatocytes may stem from the difference in susceptibility to H2O2 between PTP1B and TRX, although our study leaves open the possibility that other molecules in addition to PTP1B and TRX may participate in the dual effects of H2O2. Moreover, because we tested only the membrane-permeable ROS H2O2, we cannot exclude the possibility that factors other than concentration, such as intracellular localization, duration, or type of ROS, may influence the action of ROS on insulin signaling. Further investigations are required to elucidate the detailed Dual Actions of ROS on Insulin Signaling molecular mechanisms underlying the dual effects of H2O2 on insulin signal transduction. One limitation of our study is that our experiments were performed only in H4IIEC hepatocytes. At present, there are few reports demonstrating the dual action of ROS on insulin signaling in other cell lines. However, Loh et al. showed that mouse embryonic fibroblasts lacking Gpx1, an antioxidative enzyme involved in the removal of H2O2, exhibit enhanced insulin- induced Akt phosphorylation and PTEN oxidation, and normal tyrosine phosphorylation of the IR [8]. Thus, the positive regulation of insulin signaling by ROS may not be exclusive to H4IIEC hepatocytes. However, in the current study, we treated differentiated C2C12 myotubes with many concentrations of H2O2, but we could not find the dual effects of H2O2 on insulin signaling (data not shown). This result suggests that H2O2-induced dual effects on insulin signaling might be hepatocytes-specific, but additional studies on other insulin target cells such as adipocytes are needed to confirm that low concentrations of H2O2 enhance insulin signal transduction. In conclusion, the current study demonstrates that depending on its concentration, H2O2 can exert either a positive or negative effect on insulin signal transduction in H4IIEC hepatocytes, and suggests that the intracellular ROS concentrations and different susceptibilities of signaling molecules to ROS are major determi- nants of impaired or enhanced insulin signaling. Further investigations into the dual effects of ROS on insulin signaling may lead to the development of novel antioxidative therapies to selectively combat the oxidative stresses that cause insulin resistance. Materials and Methods Materials Antibodies against phospho-IGF-1 receptor (Tyr1131), phos- pho-insulin receptor (Tyr1146), insulin receptor b, Akt, phospho- Akt (Ser473), SAPK/JNK, phospho-SAPK/JNK (Thr183/ Tyr185), phospho-GSK-3 (Ser21/9), and phosphor-serine636 of IRS-1 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against GSK-3 and phospho-c-Jun were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human recombinant insulin was purchased from Sigma–Aldrich (St. Louis, MO, USA). Hydrogen peroxide, TiSO4, xylenol orange, ammonium ferrous sulfate, and sorbitol were obtained from Wako Chemical Co., Ltd. (Osaka, Japan). PTP1B inhibitor came from Merck (Tokyo, Japan). The rat hepatoma cell line H4IIEC was purchased from the American Type Culture Collection (Manassas, VA). Cell Harvesting and Western Blot Analysis Cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen), penicillin (100 U/mL), and streptomycin (0.1 mg/mL; Invitrogen) at 37uC in a humidified atmosphere containing 5% CO2. The cells were cultured to 60–70% confluence in 12-well plates. Next, the cells were serum-starved overnight and then treated with H2O2 for a total of 3 h, with medium changes every 30 min. After treatment, they were stimulated with recombinant human insulin (1 ng/mL) for 15 min, washed with ice-cold phosphate-buffered saline, and then lysed in RIPA buffer (Millipore, Billerica, MA) containing a CompleteTM Protease Inhibitor Cocktail Tablet, EDTA-free (Roche Diagnostics) and PhosSTOP Phosphatase Inhibitor Cocktail Tablets (Roche Diagnostics). The lysates were sonicated using a Bioruptor sonicator (Cosmo Bio, Tokyo, Japan), and insoluble materials were removed by centrifugation. The resulting PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27401