Dual Actions of ROS on Insulin Signaling Figure 6. Effects of H2O2 on JNK activation in H4IIEC hepatocytes. (A) H4IIEC cells were serum-starved overnight and then treated with the indicated concentrations of H2O2. Total cell lysates were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with the indicated antibodies. Signals were detected chemilu- minescence. Representative blots are shown. (B) Quantitative data from densitometric analysis of p-JNK signals from three independent experiments were normalized to the values for total JNK and are expressed as mean fold increases over control 6 S.D. ** p,0.01, versus untreated control. doi:10.1371/journal.pone.0027401.g006 a tyrosine residue of the insulin receptor [20]. The structure of the active site and the low pKa of the thiol group of the invariant catalytic cysteine residue render PTP1B highly susceptible to reversible oxidation by H2O2 [21]. Oxidation of its active site cysteine residue abolishes PTP1B’s nucleophilic properties, causing it to be inactivated. Goldstein et al. showed that an insulin-stimulated endogenous H2O2 burst positively regulated insulin signaling, at least partly through oxidative inhibition of PTP1B [7]. However, early papers showed that compounds of vanadium inhibit several forms of phosphatases including PTP1B [22,23]. Thus, additional studies are needed to determine whether low concentrations of H2O2 enhance insulin signaling in a PTP1B- specific manner. Figure 7. Effects of JNK knockdown on H2O2-induced alter- ations of insulin signal transduction in H4IIEC hepatocytes. H4IIEC cells were transfected with siRNA specific to JNK1 or negative control. 24 h later, the cells were serum-starved overnight. Then the cells were treated with the indicated concentrations of H2O2 for 3 h, 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. (A) Protein levels of total JNK, phosphorylated INK, and phosphorylated serine residue of IRS-1 in the cells transfected with JNK. (B) Protein levels of phosphorylated Akt in the cells stimulated with insulin. doi:10.1371/journal.pone.0027401.g007 Peak IR phosphorylation was observed in the cells treated with the lower concentrations of H2O2 compared with Akt phosphor- ylation (Fig. 2). These results suggest that the action mechanisms of low concentrations of H2O2 are different on the phosphorylation of Akt and IR. Several kinds of phosphatases are known to inactivate insulin signaling cascade by dephosphorylating different kinds of downstream effectors of insulin. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) dephosphorylates phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) to terminate PI3 kinase signaling, resulting in a selective inhibition of PI3 kinase/ Akt signaling, although PTP1B dephosphorylates tyrosine residue of insulin receptor, resulting in a total inhibition of insulin signaling. PTEN is also inactivated by H2O2–induced oxidation as well as PTP1B [24]. The different sensitivities of H2O2 to the phosphorylation of Akt and IR might be derived from the difference in threshold levels of H2O2 for the inactivation of various phosphatases, such as PTEN and PTP1B that target Akt and IR, respectively. Our results identify JNK and phosphatases such as PTP1B as candidate molecules that determine the thresholds for the diametrical effects of H2O2 on cellular insulin signaling. We propose that the action of H2O2 on cellular insulin signaling is determined by the net balance between inactivation of PTP1B (the PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27401