Uridine Affects Liver Protein Glycosylation, Insulin Signaling, and Heme Biosynthesis

Citation: Urasaki Y, Pizzorno G, Le TT ( Uridine Affects Liver Protein Glycosylation, Insulin Signaling, and Heme Biosynthesis Yasuyo Urasaki 0 Giuseppe Pizzorno 0 Thuc T. Le 0 Yan Chen, Institute for Nutritional Sciences, China 0 1 Nevada Cancer Institute, Las Vegas, Nevada, United States of America, 2 Desert Research Institute , Las Vegas, Nevada , United States of America Purines and pyrimidines are complementary bases of the genetic code. The roles of purines and their derivatives in cellular signal transduction and energy metabolism are well-known. In contrast, the roles of pyrimidines and their derivatives in cellular function remain poorly understood. In this study, the roles of uridine, a pyrimidine nucleoside, in liver metabolism are examined in mice. We report that short-term uridine administration in C57BL/6J mice increases liver protein glycosylation profiles, reduces phosphorylation level of insulin signaling proteins, and activates the HRI-eIF-2a-ATF4 hemedeficiency stress response pathway. Short-term uridine administration is also associated with reduced liver hemin level and reduced ability for insulin-stimulated blood glucose removal during an insulin tolerance test. Some of the short-term effects of exogenous uridine in C57BL/6J mice are conserved in transgenic UPase12/2 mice with long-term elevation of endogenous uridine level. UPase12/2 mice exhibit activation of the liver HRI-eIF-2a-ATF4 heme-deficiency stress response pathway. UPase12/2 mice also exhibit impaired ability for insulin-stimulated blood glucose removal. However, other shortterm effects of exogenous uridine in C57BL/6J mice are not conserved in UPase12/2 mice. UPase12/2 mice exhibit normal phosphorylation level of liver insulin signaling proteins and increased liver hemin concentration compared to untreated control C57BL/6J mice. Contrasting short-term and long-term consequences of uridine on liver metabolism suggest that uridine exerts transient effects and elicits adaptive responses. Taken together, our data support potential roles of pyrimidines and their derivatives in the regulation of liver metabolism. - Funding: This work was partially supported by the Nevada INBRE Program of the National Center for Research Resources (P20RR-016464, TTL), the American Cancer Society (IRG-08-062-04, TTL), and the Vons Breast Cancer Research Award (TTL and GP). 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. Uridine is a pyrimidine nucleoside that has the ability to affect liver energy metabolism. Uridine is produced via several reversible reactions including de-phosphorylation of a uridine monophosphate, de-amination of a cytidine, or combination of a uracil and a ribose-1-phosphate [1]. Uridine homeostasis is regulated in part by uridine phosphorylase, an enzyme that catalyzes the reversible conversion of uridine to uracil [2]. UPase1-TG mice with ubiquitous genetic knock-in of a gene encoding for UPase1 exhibit depleted plasma and liver uridine concentration [3]. UPase1-TG mice also exhibit intrinsic liver lipid accumulation [3,4]. In contrast, UPase12/2 mice with ubiquitous genetic knock-out of a gene encoding for UPase1 exhibit elevated plasma and liver uridine concentration [5,6]. UPase12/2 mice are protected against fatty liver condition induced by a number of drugs with different acting mechanisms [79]. Dietary uridine supplementation is able to suppress intrinsic fatty liver in UPase1-TG mice or drug-induced fatty liver in C57BL/6J mice [3,7,9]. Clearly, uridine exerts protective effects against liver lipid accumulation; however, the underlying mechanisms have not been delineated. Uridine is a versatile molecule that exerts multi-targeted effects because it can be used to produce other biologically active molecules. Via the pyrimidine salvage pathway, uridine can be salvaged into uridine triphosphate (UTP) and cytidine triphosphate (CTP) [1]. Combination of UTP with glucose-1-phosphate produces uridine diphosphate glucose (UDPG), which is a basic building block for glycogen biosynthesis [10]. Combination of UTP with N-acetylglucosamine (GlcNAc) produces UDP-GlcNAc, which is a donor substrate for protein glycosylation [11]. Combination of CTP with phosphocholine produces cytidine diphosphocholine (CDPC), which is an essential molecule for membrane phospholipid biosynthesis [12,13]. On the other hand, uridine catabolism produces b-alanine and acetyl-CoA [1]. AcetylCoA is an important molecule in cellular energy metabolism and in the biosynthesis of the neurotransmitter acetylcholine [14,15]. Acetyl-CoA is also a donor substrate for protein lysine acetylation, a mode of nutrient-sensitive protein post-translational modification [16,17]. Therefore, uridine has the ability to affect a wide range of biological processes. In recent years, clinical data from several independent labs revealed a positive correlation between plasma uridine concentration and insulin resistance in humans [18,19]. This correlation has also been reported in rodents [11]. However, the mechanistic link between uridine and insulin signaling activity has not been elucidated. In this study, we screen for the effects of uridine on liver metabolism with specific focuses on glucose utilization and insulin signaling activity. C57BL/6J mice are fed with uridine supplemented diet for 5 days to evaluate short-term effects of uridine. Long-term effects of uridine are evaluated in transgenic The effects of uridine salvage into UTP on liver glycogen and protein glycosylation were evaluated in C57BL/6J mice. Consistent with previous findings in skeletal muscles [11], dietary uridine supplementation at a daily dosage of 400 mg/kg for 5 days increased liver glycogen content by more than 2 folds (Figure 1A). To evaluate liver protein glycosylation profiles, total liver extracts were used for 2D Western blots, where proteins were separated by both charges and molecular weights (Figure 1B). Anti-O-GlcNAc monoclonal antibody was used to detect glycosylated liver proteins. Selective protein spots were excised and identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Tables S1 & S2). 2D Western blots revealed that uridine supplementation increased O-linked glycosylation of 10 protein spots. Of particular interest are the changes to several O-linked glycosylated protein spots with molecular weight of 60 kD (Figure 1C, spots 9, 10, & 30). Interestingly, MALDI-TOF-MS analysis identified the presence of an ER protein disulfide isomerase A3 (PDI) following uridine administration in protein spots 9, 10, and 30. In the liver of control mice, only O-linked glycosylated protein spots 9 and 10 were presence. However, three O-linked glycosylated protein spots 9, 10, and 30 were observed in the liver of mice treated with uridine (...truncated)