Figure 4. Effects of H2O2 on PTP1B activity in H4IIEC hepatocytes. (A) H4IIEC cells were serum-starved overnight and then treated with the indicated concentrations of H2O2. After snap-freezing in liquid N2, cells were disrupted through scraping into ice-cold RIPA buffer containing a protease inhibitor cocktail, followed by brief sonication and clearing of the resulting lysates by centrifugation at 15,000 rpm for 15 min. PTP1B activity was determined using a CycLexH Protein Tyrosine Phosphatase 1B (PTP1B) Fluorometric Assay Kit. Fluorescence values from three independent experiments were normalized to total protein concentrations and are expressed as mean fold increases over control 6 S.D. ** p,0.01, versus untreated control. (B) Protein levels of PTP1B were measured by Western blotting. doi:10.1371/journal.pone.0027401.g004 The most surprising finding from our study is that H2O2 can exert both positive and negative effects on insulin signaling within a relatively narrow concentration range of 5–50 mM. Banerjee et al. reported blood levels of H2O2 to be approximately 1.762.5 mM in healthy humans [14], suggesting that our finding of enhanced insulin signaling with 5–10 mM H2O2 reflects a physiological, rather than pharmacological, action of H2O2. H2O2 is reported to have insulin-mimetic actions in insulin target cells [15,16]. However, in these earlier studies, cells were treated with millimolar concentrations of H2O2 for short periods of time. To our knowledge, the present study is the first to demonstrate that mild oxidative stress, such as that caused by 5–10 mM H2O2, enhances intracellular insulin-mediated signaling. Recent reports have proposed that cells repeatedly exposed to sublethal stresses can develop stress resistance and display increased survival rates, as a result of a process called hormesis. The initial evidence for this novel concept was obtained using model organisms such as nematodes [17] and rats [18]. More recently, Ristow et al. extended the concept of hormesis to humans. They showed that exercise-induced low-grade oxidative stress improves insulin Dual Actions of ROS on Insulin Signaling Figure 5. Effects of vanadate on insulin-stimulated Akt phosphorylation in H4IIEC hepatocytes. H4IIEC cells were serum-starved overnight. Then the cells were treated with the indicated concentrations of H2O2 for 3 h. Then, the cells were treated with 1mM of sodium orthovanadate for 30 min, and stimulated with insulin (1 ng/ mL) for 15 min. Total cell lysates were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed using the indicated antibodies. doi:10.1371/journal.pone.0027401.g005 sensitivity in skeletal muscle [19]. However, few reports detailing the dual effects of H2O2 on insulin signaling in insulin target cells have been published. The cellular insulin signaling might be maintained by continuous exposure of the cells to low levels of ROS, while insulin resistance develops when cellular H2O2 levels rise above a certain threshold. We show that pretreatment with NAC had a tendency to reduce insulin signaling in both H2O2-treated and untreated cells (Fig. 9). In addition, we found that the cells treated with NAC and H2O2 showed lower levels of ROS than those in the absence of H2O2 (Fig. 8), suggesting that the current concentrations of NAC had a potent anti-oxidative capacity. Goldstein et al. have reported that endogenous H2O2 production stimulated by insulin is needed for the subsequent insulin signaling by inactivating phosphatases such as PTP1B [7]. Hence, we speculate that pretreatment with NAC inhibited insulin signaling generally by removing insulin-induced endogenous H2O2 production required for the suppression of phosphatase activity due to its strong anti-oxidative capacity. In our experimental system, it was hard to determine the minimum threshold concentrations of which H2O2 exerts promotive effects on insulin signaling, because anti-oxidative capacity in the cells seemed to vary at individual experiments depending on only a modest variation of the viability or numbers of the cells cultured. However, we found that 5–10 mM were the concentrations of which H2O2 certainly promotes insulin signal transduction in our cell lines, because these concentrations of H2O2 not only increased insulin-stimulated Akt phosphorylation reproducibly (Fig. 2), but also suppressed PTP1B activity significantly (Fig. 4). Thus, we used 5–10 mM of H2O2 to certainly increase insulin signaling in the following experiments. Our findings indicate that the enhancement of insulin signaling by low concentrations of H2O2 is accompanied by the suppression of PTP1B activity, and is rescued by the co-administration with vanadate, a non-specific inhibitor for phosphatases. We showed that vanadate further increased insulin signaling in the cells treated with 5 mM of H2O2, but not in those treated with 10 mM of H2O2 (Fig. 5). These findings seem to be reasonable, because our results showed that 5 mM of H2O2 decreased PTP1B activity by 40%, whereas 10 mM of H2O2 decreased it by approximately 90% (Fig. 4). Thus, we speculate that co-administration with vanadate was unable to further enhance insulin signaling in the cells where PTP1B activity was almost completely suppressed by 10 mM of H2O2. These results suggest that low concentrations of H2O2 enhance insulin signaling, at least partly, by inhibiting phospha- tases such as PTP1B. PTP1B, a protein tyrosine phosphatase (PTP), negatively regulates insulin signaling by dephosphorylating PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27401