Hydrogen Improves Glycemic Control in Type1 Diabetic Animal Model by Promoting Glucose Uptake into Skeletal Muscle

et al. (2013) Hydrogen Improves Glycemic Control in Type1 Diabetic Animal Model by Promoting Glucose Uptake into Skeletal Muscle. PLoS ONE 8(1): e53913. doi:10.1371/journal.pone.0053913 Hydrogen Improves Glycemic Control in Type1 Diabetic Animal Model by Promoting Glucose Uptake into Skeletal Muscle Haruka Amitani 0 Akihiro Asakawa 0 Kaichun Cheng 0 Marie Amitani 0 Kaori Kaimoto 0 Masako Nakano 0 Miharu Ushikai 0 Yingxiao Li 0 Minglun Tsai 0 Jiang-Bo Li 0 Mutsumi Terashi 0 Huhe Chaolu 0 Ryozo Kamimura 0 Akio Inui 0 Victor Sanchez-Margalet, Virgen Macarena University Hospital, School of Medicine, Spain 0 1 Department of Psychosomatic Internal Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan, 2 Institute of Laboratory Animals, Kagoshima University Graduate School of Medical and Dental Sciences , Kagoshima , Japan Hydrogen (H2) acts as a therapeutic antioxidant. However, there are few reports on H2 function in other capacities in diabetes mellitus (DM). Therefore, in this study, we investigated the role of H2 in glucose transport by studying cultured mouse C2C12 cells and human hepatoma Hep-G2 cells in vitro, in addition to three types of diabetic mice [Streptozotocin (STZ)-induced type 1 diabetic mice, high-fat diet-induced type 2 diabetic mice, and genetically diabetic db/db mice] in vivo. The results show that H2 promoted 2-[14C]-deoxy-D-glucose (2-DG) uptake into C2C12 cells via the translocation of glucose transporter Glut4 through activation of phosphatidylinositol-3-OH kinase (PI3K), protein kinase C (PKC), and AMP-activated protein kinase (AMPK), although it did not stimulate the translocation of Glut2 in Hep G2 cells. H2 significantly increased skeletal muscle membrane Glut4 expression and markedly improved glycemic control in STZ-induced type 1 diabetic mice after chronic intraperitoneal (i.p.) and oral (p.o.) administration. However, long-term p.o. administration of H2 had least effect on the obese and non-insulin-dependent type 2 diabetes mouse models. Our study demonstrates that H2 exerts metabolic effects similar to those of insulin and may be a novel therapeutic alternative to insulin in type 1 diabetes mellitus that can be administered orally. - Funding: This research is partially funded by the Young Researcher Overseas Visits Program for Vitalizing Brain Circulation of the Japan Society for the Promotion of Science. The authors gratefully acknowledge VANA Co., Ltd. (Yamanashi, Japan) for the generous gift of natural hydrogen water. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Diabetes mellitus (DM) is characterized by abnormal insulin secretion, derangements in carbohydrate-lipid metabolism, and chronic hyperglycemia. The total number of people with DM is predicted to rise from 171 million in 2000 to 366 million in 2030 [1]. Therefore, various therapies, including alternative medicine, are being developed for DM. Insulin-like growth factor 1 (IGF-1) exerts metabolic effects similar to those of insulin [2,3]. Although IGF-1 was once thought to have the same effect as insulin, recently a relationship between IGF-1 and diabetic retinopathy has been suggested [4]. Indeed, the only treatment for progressed DM is insulin therapy. Insulin initiates its action by binding its specific cell-surface receptor, insulin receptor (IR), in the peripheral tissues such as skeletal muscle and liver under normal conditions. IR is a heterotetrameric protein that consists of two extracellular asubunits and two transmembrane b-subunits connected by disulfide bridges. Insulin signaling is initiated following binding of insulin to the extracellular a-subunits. This results in autophosphorylation of the b-subunits and activation of the tyrosine kinase domain of the intracellular part of the b subunits [5]. The active tyrosine kinase of IR phosphorylates IR substrate (IRS) proteins. Then, the phosphorylated IRS proteins recruit and activate phosphatidylinosisitol 3-kinase (PI3K). IR, IRS, and PI3K are considered critical nodes in insulin signaling [6]. The activated PI3K activates its downstream effectors, such as Akt and protein kinase C (PKC), by increasing the production of its lipid product, phosphatidylinositol-3,4,5-(PO4)3, which leads to glucose uptake. Hydrogen (H2) is the lightest gas molecule. The numerous strains of intestinal bacteria, primarily in the large intestine, produce H2, and approximately 14% of H2 is absorbed in the colon and released from the lungs [7]. The hydrogen breath test has become popular in clinical practice because it is useful to assess abnormal pathophysiology, such as bacterial overgrowth in the small intestine, and to diagnose lactose or fructose malabsorption [8]. Gharib et al reported the antioxidant effect of H2 using mice with parasite-induced liver inflammation [9]. Ohsawa et al recently reported that hydrogen acts as a therapeutic anti-oxidant by selectively reducing hydroxyl radicals (?OH) [10]. H2 affects ischemia-reperfusion injury [10,11], atherosclerosis [12], Parkinsons disease [13], acute pancreatitis [14], and type 1 allergic reaction [15]. Currently, there are more than 100 reports related to antioxidant effects of H2. Although several studies have described the antioxidant effect of H2 on DM [16,17], few studies have reported on other functions of H2 in DM. Therefore, in this study, we investigated the role of H2 in glucose homeostasis by studying cultured mouse C2C12 cells and human hepatoma Hep G2 cells in vitro, as well as three types of diabetic mice (STZ-induced diabetic mice, high-fat diet induced diabetic mice, and genetically diabetic db/db mice) in vivo. Materials and Methods Cell Culture The C2C12 cells and Hep G2 cells were obtained from Bioresource Collection and Research Center (Food Industry Research and Development Institute, Hsinchu City, Taiwan). The cells were plated at 56104 cells/dish in 35-mm-diameter culture dishes in Dulbeccos modified Eagles medium (DMEM) (Gibco BRL, Gaithersburg, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL) and 1% antibiotic solution (penicillin G sodium 10,000 U/ml and streptomycin sulfate 10 mg/ml). They were grown to 70% confluence at 37uC in humidified atmosphere containing 5% CO2. To induce fusion, confluent cells were exposed to DMEM supplemented with 10% horse serum instead of FBS. The cells fused into multinucleated myotubes after a further 710 days in culture. The medium was changed 24 h prior to experimental manipulations. H2-containing Water and Saline High-content (saturated) H2 water and saline (HHW and HHS, respectively: 0.8 mM each) were prepared by dissolving H2 in pure water or saline under high pressure (0.4 MPa) for 24 hours. Lowcontent H2 water (LHW: 0.08 mM) was prepared by dissolving H2 in pure water under low pressure (0.1 (...truncated)