Concentration-dependent Dual Effects of Hydrogen Peroxide on Insulin Signal Transduction in H4IIEC Hepatocytes Satoshi Iwakami1,2., Hirofumi Misu1., Takashi Takeda1,2, Makoto Sugimori1, Seiichi Matsugo2,3, Shuichi Kaneko 1 , Toshinari Takamura1* 1 Department of Disease Control and Homeostasis, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan, 2 Division of Material Engineering, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Japan, 3 College of Science and Engineering, School of Natural System Bioengineering Course, Kanazawa University, Kanazawa, Japan Abstract Background: Oxidative stress induced by the accumulation of reactive oxygen species (ROS) has a causal role in the development of insulin resistance, whereas ROS themselves function as intracellular second messengers that promote insulin signal transduction. ROS can act both positively and negatively on insulin signaling, but the molecular mechanisms controlling these dual actions of ROS are not fully understood. Methodology/Principal Findings: Here, we directly treated H4IIEC hepatocytes with hydrogen peroxide (H2O2), a representative membrane-permeable oxidant and the most abundant ROS in cells, to identify the key factors determining whether ROS impair or enhance intracellular insulin signaling. Treatment with high concentrations of H2O2 (25–50 mM) for 3 h reduced insulin-stimulated Akt phosphorylation, and increased the phosphorylation of both JNK and its substrate c-Jun. In contrast, lower concentrations of H2O2 (5–10 mM) enhanced insulin-stimulated phosphorylation of Akt. Moreover, lower concentrations suppressed PTP1B activity, suggesting that JNK and phosphatases such as PTP1B may play roles in determining the thresholds for the diametrical effects of H2O2 on cellular insulin signaling. Pretreatment with antioxidant N- acetyl-L-cysteine (10 mM) canceled the signal-promoting action of low H2O2 (5 mM), and it canceled out further impairment of insulin of insulin signaling induced by high H2O2 (25 mM). Conclusions/Significance: Our results demonstrate that depending on its concentration, H2O2 can have the positive or negative effect on insulin signal transduction in H4IIEC hepatocytes, suggesting that the concentration of intracellular ROS may be a major factor in determining whether ROS impair or enhance insulin signaling. Citation: Iwakami S, Misu H, Takeda T, Sugimori M, Matsugo S, et al. (2011) Concentration-dependent Dual Effects of Hydrogen Peroxide on Insulin Signal Transduction in H4IIEC Hepatocytes. PLoS ONE 6(11): e27401. doi:10.1371/journal.pone.0027401 Editor: Dhyan Chandra, Roswell Park Cancer Institute, United States of America Received September 29, 2010; Accepted October 16, 2011; Published November 15, 2011 Copyright: 2011 Iwakami et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ttakamura@m-kanazawa.jp . These authors contributed equally to this work. Introduction Insulin resistance is an underlying problem in people with type 2 diabetes and metabolic syndrome [1]. In an insulin-resistant state, impaired insulin action promotes hepatic glucose production and reduces the uptake of glucose by peripheral tissues, resulting in systemic hyperglycemia. In addition to type 2 diabetes and metabolic syndrome, the development of various other diseases such as non-alcoholic steatohepatitis [2] and atherosclerosis [3] involves insulin resistance. It is commonly assumed that combating insulin resistance is a viable therapeutic strategy in several kinds of diseases, although the molecular mechanisms underlying insulin resistance are not fully understood. Oxidative stress induced by the accumulation of reactive oxygen species (ROS) has a causal role in the development of insulin resistance. Using models in which cells were treated with tumor- necrosis factor a and glucocorticoids, Houstis et al. showed that increased ROS levels are an important trigger for insulin resistance in numerous contexts [4]. Activation of stress kinases such as C-Jun N-terminal kinase (JNK) and IkB kinase contributes to insulin resistance associated with oxidative stress [5]. In a previous report, we demonstrated that treatment with palmitate, a C16:0 saturated fatty acid, induces insulin resistance in H4IIEC hepatocytes by stimulating the generation of ROS in the mitochondria and thereby, the activation of JNK [6]. The administration of antioxidants such as N-acetylcysteine and a-tocopherol partially rescued cells from palmitate-induced insulin resistance, suggesting that antioxidative therapy may be useful in attenuating insulin resistance in patients with type 2 diabetes or metabolic syndrome. A growing body of evidence suggests that ROS function as intracellular second messengers to promote signaling by hormones, including insulin. Goldstein et al. have shown that insulin-induced endogenous hydrogen peroxide enhances proximal and distal insulin signaling, at least partly through the oxidative inhibition of protein tyrosine phosphatase 1B (PTP1B), which negatively regulates insulin PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27401