Chronic Uridine Administration Induces Fatty Liver and Pre-Diabetic Conditions in Mice

RESEARCH ARTICLE Chronic Uridine Administration Induces Fatty Liver and Pre-Diabetic Conditions in Mice Yasuyo Urasaki1, Giuseppe Pizzorno2, Thuc T. Le1* 1 Department of Biomedical Sciences, College of Medicine, Roseman University of Health Sciences, 10530 Discovery Drive, Las Vegas, Nevada, 89135, United States of America, 2 Desert Research Institute, 10530 Discovery Drive, Las Vegas, Nevada, 89135, United States of America * Abstract OPEN ACCESS Citation: Urasaki Y, Pizzorno G, Le TT (2016) Chronic Uridine Administration Induces Fatty Liver and Pre-Diabetic Conditions in Mice. PLoS ONE 11 (1): e0146994. doi:10.1371/journal.pone.0146994 Uridine is a pyrimidine nucleoside that exerts restorative functions in tissues under stress. Short-term co-administration of uridine with multiple unrelated drugs prevents drug-induced liver lipid accumulation. Uridine has the ability to modulate liver metabolism; however, the precise mechanism has not been delineated. In this study, long-term effects of uridine on liver metabolism were examined in both HepG2 cell cultures and C57BL/6J mice. We report that uridine administration was associated with O-GlcNAc modification of FOXO1, increased gluconeogenesis, reduced insulin signaling activity, and reduced expression of a liver-specific fatty acid binding protein FABP1. Long-term uridine feeding induced systemic glucose intolerance and severe liver lipid accumulation in mice. Our findings suggest that the therapeutic potentials of uridine should be designed for short-term acute administration. Editor: Yanqiao Zhang, Northeast Ohio Medical University, UNITED STATES Received: September 17, 2015 Accepted: December 26, 2015 Published: January 20, 2016 Copyright: © 2016 Urasaki 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. Data Availability Statement: All relevant data are within the paper. Funding: This study is supported by start-up funds from Roseman University of Health Sciences to TTL. The funder 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. Introduction Uridine is a biologically active pyrimidine with multiple therapeutic potentials. Uridine reduces cytotoxicity on non-cancerous cells due to the administration of anti-cancer drug 5-fluorouracil [1]. Uridine mitigates lipodystrophy associated with the usage of nucleoside reverse transcriptase inhibitors for HIV treatment [2]. Uridine is a nutrient critical for phosphatidylcholine biosynthesis and synapse formation [3]. Uridine improves neurophysiological functions in patients with diabetic neuropathy [4]. Uridine has also been shown to suppress hepatic steatosis induced by the usage of drugs in mice including zalcitabine [5], fenofibrate [6], and tamoxifen [7]. In addition, uridine triacetate (Xuriden), an orally active prodrug of uridine, has recently received an orphan drug designation by the FDA to treat hereditary orotic aciduria. Uridine has multi-targeted effects because it can be converted rapidly into other biologically active molecules [8]. Uridine is salvaged into pyrimidine nucleotides necessary for RNA and DNA synthesis [9]. Via cytidine triphosphate, uridine promotes membrane phospholipid biosynthesis. Via uridine triphosphate, uridine promotes the formation of uridine diphosphate glucose (UDPG) and uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), which are substrates for glycogen biosynthesis and protein O-linked glycosylation, respectively. Uridine PLOS ONE | DOI:10.1371/journal.pone.0146994 January 20, 2016 1 / 12 Long-Term Effects of Uridine catabolism produces acetyl-CoA, a substrate for protein lysine acetylation. Most interestingly, de novo pyrimidine biosynthesis is coupled to mitochondrial respiratory chain [10]. The protective function of uridine on mitochondrial functions is thought to be mediated by its conversion to other pyrimidine intermediates [11]. While the therapeutic potentials of uridine have been well-observed, its side effects on the biological systems have not been fully characterized. Recently, our lab reported that short-term uridine administration induced insulin resistance in the liver of C57BL/6J mice. Our data were consistent with several other independent observations, where a relationship between plasma uridine concentration and systemic insulin resistance was reported in both humans and rodents [12–14]. In this study, we further investigate the effects of long-term uridine supplementation on liver lipid and glucose metabolism. Materials and Methods Animal models C57BL/6J mice (male, 10–12 weeks old, Jackson Lab, Bar Harbor, Maine) were divided into four groups: Control mice fed with a lean diet for 5 days (C57BL/6J), mice fed with a lean diet supplemented with uridine for 5 days (C57BL/6J+U), control mice fed with a lean diet for 16 weeks (C57BL/6J+LD), and mice fed with a lean diet supplemented with uridine for 16 weeks (C57BL/6J+LDU). Lean diet was PicoLab Mouse Diet 20 (Cat. No. 5058, LabDiet, Brentwood, MO) that provided 4.6 kcal/g (22% kcal from fat, 23% kcal from protein, and 55% kcal from carbohydrates). For uridine-supplemented diet, uridine was thoroughly mixed with ground pellets at a daily dosage of 400 mg/kg. Mice were not fasted prior to terminal liver tissue collection in early mornings. Liver tissues were collected following the perfusion procedures described previously [15]. All animal studies were performed in conformity with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and with the approval of the Animal Care and Use Committees at Nevada Cancer Institute, Desert Research Institute, and Touro University Nevada. Glucose tolerance test Mice were fasted for 5 hours, then given d-glucose at 0.75 g/kg via intraperitoneal injection. Blood was drawn from the tail vein at 30-minute intervals for two hours after glucose injection and assayed with a glucose meter (Cat. No. 7151G, Bayer, Leverkusen, Germany). HepG2 cell cultures HepG2 cells were cultured in RPMI1640 media (Cat. No. 11875–093, ThermoFisher Scientific, Waltham, MA) with 10% fetal bovine serum, penicillin-streptomycin, and MEM non-essential amino acids (Cat. No. 25-025-Cl, Corning Life Science, Tewksbury, MA). For cells receiving treatments, cells were incubated with 100 μM of uridine and/or 100 μM of PUGNAc for 48 hours prior to the collection of total cell extracts. A deglycosylation enzyme mix (Cat. No. P6039S, New England Biolabs, Ipswich, MA) was employed to reverse the action of uridine in 100 μg of total cell extract. Deglycosylation was performed according to manufacturer’s protocols under non-denaturing condition. Identification of FOXO1 glycosylation with 2D Western bl (...truncated)