Effects of Hydrogenized Water on Intracellular Biomarkers for Antioxidants, Glucose Uptake, Insulin Signaling and SIRT 1 and Telomerase Activity

Robert Settineri^1, , Jin Ji², Chunlan Luo², Rita R. Ellithorpe³, Gonzalo Ferreira de Mattos⁴, Steven Rosenblatt⁵, James LaValle⁶, Antonio Jinenez⁷, Shigeo Ohta⁸ and Garth L. Nicolson⁹

¹Sierra Productions Research, Irvine, USA

²Brunswick Laboratories, Inc., Southborough, USA

³Tustin Longevity Center, Tustin, USA

⁴Laboratory of Ion Channels, School of Medicine, Universidad de la República, Montevideo, Uruguay

⁵Saint John’s Health Center, Santa Monica, USA

⁶Progressive Medical Center, Orange, USA

⁷Hope4Cancer Institute, Baja California

⁸Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nippon Medical School, Japan

⁹Department of Molecular Pathology, The Institute for Molecular Medicine, Huntington Beach, USA

Cite this paper:
Robert Settineri, Jin Ji, Chunlan Luo, Rita R. Ellithorpe, Gonzalo Ferreira de Mattos, Steven Rosenblatt, James LaValle, Antonio Jinenez, Shigeo Ohta and Garth L. Nicolson. Effects of Hydrogenized Water on Intracellular Biomarkers for Antioxidants, Glucose Uptake, Insulin Signaling and SIRT 1 and Telomerase Activity. American Journal of Food and Nutrition. 2016; 4(6):161-168. doi: 10.12691/ajfn-4-6-4

Abstract

Hydrogen has been shown in several clinical trials to be completely safe without adverse events and there are no warnings in the literature of its toxicity or adverse effects during long-term exposure. Molecular hydrogen has proven useful and convenient as a novel antioxidant and modifier of gene expression in many conditions where oxidative stress and changes in gene expression result in cellular damage. Our intracellular biomarker studies have shown that a hydrogenized water drink formula containing 2.6 ppm dissolved hydrogen was able to penetrate cellular membranes and function as an antioxidant in human liver cells (HePG2) utilizing the Cellular Antioxidant Assay (CAA). This assay uses the protection of a florescent probe as a marker for cellular damage by reactive oxygen species (ROS), such as peroxyl radical, and compares this to the known antioxidant standard, Quercetin. Using this system oxidative damage was reduced in a dose-dependent manner. One ml of hydrogenized water was found to possess antioxidant capacity equivalent to 0.05 µmole of quercetin. When examined in a human colon cell line (Caco-2 cells), hydrogenized water demonstrated a dose- and time-dependent permeability inhibition of an intracellular fluorescent glucose derivative (2-NBDG), indicating decreased glucose uptake. In another study, the impact of hydrogenized water on Akt phosphorylation (Ser473), a biomarker for insulin signaling, was monitored in human skeletal muscle cells. The hydrogenized water treatment markedly elevated the level of phosphorylation of Akt (Ser473) in a dose-dependent manner. The anti-aging effects of hydrogenized water were examined utilizing SIRT1 expression as a biomarker of aging in human umbilical cells (HUVECs). Hydrogenized water increased dose-dependent SIRT1 gene expression. Hydrogenized water also increased telomerase activity (an anti-aging biomarker in HUVEC cells) up to 148% when cells were treated with media containing 25% hydrogenized water formula. Increased telomerase activity caused by hydrogenized water may be able to protect telomeres from degradation, suggesting the possible use of hydrogenized water in therapeutic interventions of age-related diseases. These studies show that commercial hydrogenized water improved the levels or activities of a few intracellular biomarkers specific for antioxidant activity, glucose uptake, insulin signaling and SIRT 1 and telomerase activities. Industrial Relevance: The molecular hydrogen used in this study indicates that certain commercial sources of hydrogenized water can provide similar antioxidant and gene expression modifications seen in other sources of molecular hydrogen. The biomarkers evaluated here lend well to hydrogenized water’s biological activity relating to health conditions and aging.

Keywords:
Hydrogenized water bioassays antioxidant Caco-2 permeability assay glucose uptake insulin receptor SIRT 1 telomerase activity

This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

References:

[1]	Ohsawa I, Ishikawa M, Takahashi K, et al. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med. 2007; 13: 688-694.
[2]	Shen M, Zhang H, Yu C, et al. A review of experimental studies of hydrogen as a new therapeutic agent in emergency and critical care medicine. Med Gas Res. 2014; 4:17.
[3]	Manaenko A, Lekic T, Ma Q, et al. Hydrogen inhalation ameliorated mast cell-mediated brain injury after intracerebral hemorrhage in mice. Crit Care Med. 2013; 41:1266-1275.
[4]	Kawamura T, Wakabayashi N, Shigemura N, et al. Hydrogen gas reduces hyperoxic lung injury via the Nrf2 pathway in vivo. Am J Physiol Lung Cell Mol Physiol. 2013; 304:L646-656.
[5]	Ghoshal UC. How to interpret hydrogen breath tests. J Neurogastroenterol Motil. 2011. 17: 312-317.
[6]	Ishibashi T, Sato B, Rikitake M, et al. Consumption of water containing a high concentration of molecular hydrogen reduces oxidative stress and disease activity in patients with rheumatoid arthritis: an open-label pilot study. Med Gas Res. 2012; 2(1): 27.
[7]	Amitani H, Asakawa A, Cheng K, et al. Hydrogen improves glycemic control in type1 diabetic animal model by promoting glucose uptake into skeletal muscle. PLoS ONE. 2013; 8: e53913.
[8]	Gharib B, Hanna S, Abdallahi OM, et al. Anti-inflammatory properties of molecular hydrogen: investigation on parasite-induced liver inflammation. C. R. Acad. Sci. III. 2001; 324: 719-724.
[9]	Lio A, Ito M, Itoh T, et al. Molecular hydrogen attenuates fatty acid uptake and lipid accumulation through downregulating CD36 expression in HepG2 cells. Med Gas Res. 2013; 3(1):6.
[10]	Zhang Y, Sun Q, He B, et al. Anti-inflammatory effect of hydrogen-rich saline in a rat model of regional myocardial ischemia and reperfusion. Int J Cardiol. 2011; 148: 91-95.
[11]	Song G, Li M, Sang H, et al. Hydrogen-rich water decreases serum LDL-cholesterol levels and improves HDL function in patients with potential metabolic syndrome. J Lipid Res. 2013; 54(7): 1884-1893.
[12]	Nicolson G, Ferreira de Mattos G, Settineri R, Costa C, Ellithorpe R, Rosenblatt S, La Valle J, Jimenez A, Ohta S. Clinical Effects of Hydrogen Administration: From Animal and Human Diseases to Exercise Medicine. Int. J. Clin. Med. 2016; 7(1):1-45.
[13]	Ohta S. Molecular hydrogen as a preventive and therapeutic medical gas: initiation, development and potential of hydrogen medicine. Pharm Ther. 2014; 144: 1-11.
[14]	Wolfe, K. L. and R. H. Liu. Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements. J Agricultural and Food Chemistry. 2007; 55(22): 8896-8907.
[15]	Zou, C., Y. Wang and Z. Shen. “2-NBDG as a fluorescent indicator for direct glucose uptake measurement. J Biochemical and Biophysical Methods. 2005; 64(3): 207-215.
[16]	Shi, Y., H. Yan, P. Frost, J. Gera and A. Lichtenstein. “Mammalian target of rapamycin inhibitors activate the AKT kinase in multiple myeloma cells by up-regulating the insulin-like growth factor receptor/insulin receptor substrate-1/phosphatidylinositol 3-kinase cascade. Molecular Cancer Therapeutics. 2005; 4(10): 1533-1540.
[17]	Brooks, C. L. and W. Gu. “How does SIRT1 affect metabolism, senescence and cancer. Nature Reviews Cancer. 2009; 9(2): 123-128.
[18]	Vaziri, H. and S. Benchimol. “Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span. Current Biology. 1998; 8(5): 279-282.
[19]	McDowell A, Thompson S, et al. Antioxidant activity of Puha (Sonchus oleraceus L.) as assessed by the cellular antioxidant activity (CAA) assay. Phytotherapy Research 2011; 25(12): 1876-1882.
[20]	Brunswick laboratories Inc., Brunswick Laboratories Database for ORAC 6.0 and Cellular antioxidant Assay (CAA), Available: http://www.brunswicklabs.com/tech-library/orac-database-preface [Accessed May 20, 2016].
[21]	Siissalo et al. Effect of cell differentiation and passage number on the expression of efflux proteins in wild type and vinblastine-induced Caco-2 cell lines, Eur J Pharm Biopharm. 2007 67(2): 548-54.
[22]	Hubatsch et al. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers, Nat Protoc. 2007; 2(9): 2111-9.
[23]	Calcagno et al. , Comparison of drug transporter levels in normal colon, colon cancer, and Caco-2 cells: impact on drug disposition and discovery. 2006; Mol Pharm. 3(1): 87-9.
[24]	Cozzone, D, Froejdoe, S, Disse, E, et al. Isoform-specific defects of insulin stimulation of Akt/proteinkinase B (PKB) in skeletal muscle cells from type 2 diabetic patients. Diebetologia. 2008; 51(3): 512-521.
[25]	Kobayashi, M, Matsuda, Y, Iwai, H, et al., Coffee Improves Insulin-Stimulated Akt Phosphorylation in Liver and Skeletal Muscle in Diabetic KK-A(y) Mice, J Nutritional Science and Vitaminology. 2012; 58(6): 408-414.
[26]	Kamimura N, Ichimiya H, Iuchi K, Ohta S. Molecular hydrogen stimulates the gene expression of transcriptional coactivator PGC-1α to enhance fatty acid metabolism. NPJ Aging and Mechanisms of Disease. 2016; 2: 16008.
[27]	Allard, J, Perez, E, Zou, S, et al., Dietary activators of Sirt1, Molecular and Cellular Endocrinology. 2009; 299(1): 58-63.
[28]	Polychronopoulou, S, Koutroumba, P, Telomere length and telomerase activity: Variations with advancing age and potential role in childhood malignancies, J OF Pediatric Hematology Oncology. 2004; 26(6): 342-350.