Endogenous and exogenous uridine protects liver against fenofibrate-induced steatosis.

<p>(<b>A</b>) CARS images of liver tissues of C57bl/6, <i>UPase1</i>^-/-, and <i>UPase1</i>-TG mice in the presence of uridine, fenofibrate, or both uridine and fenofibrate. (<b>B</b>) Quantitative analysis of liver lipid level using ImageJ-assisted analysis of CARS images. Liver lipid level is normalized to 1 for control untreated C57bl/6 mice and correspondingly for <i>UPase1</i>^-/- and <i>UPase1</i>-TG mice or treatment conditions. (<b>C</b>) Liver triacylglyceride (TAG) level determined with biochemical assays. Error bars are standard deviations across 9 mice analyzed per animal or treatment group. *P<0.05 versus untreated control.</p

Differential short- and long-term effects of uridine on protein expression level.

<p>(<b>A</b>) Reduced expression of FABP1 following chronic uridine feeding. (<b>B</b>) Quantitative analysis of Western blot data on FABP1 expression level. (<b>C</b>) Expression level of PEPCK, MnSOD, and FABP1 are unchanged following 5 days of feeding with uridine supplemented diet. (<b>D</b>) Quantitative analysis of Western blot data on protein expression level in (<b>C</b>). Error bars are standard deviation values across three mice per animal group. Asterisks indicate p-value <0.01 versus control. (E) Liver FOXO1 distribution as a function of pI detected with cIEF immunoassay. Peak chemiluminescence was normalized to 1. (<b>F</b>) Quantitative area under the curve analysis (pI 7.5–8.5) of glycosylated FOXO1 isoform as a function of liver tissues. Error bars are standard deviation values across 9 measurements.</p

2D Western blots of acetylated proteins in liver total cell extracts.

<p>White circles mark the acetylated protein spots presence in untreated samples. Yellow circles mark the acetylated protein spots presence in fenofibrate treated samples but not in untreated WT samples. Cyan circles mark the acetylated protein spots presence in uridine and fenofibrate treated samples but not in untreated samples or samples treated with fenofibrate alone. 2D Western blots were performed by Applied Biomics.</p

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

<div><p>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-2α-ATF4 heme-deficiency 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 <i>UPase1</i>^−/− mice with long-term elevation of endogenous uridine level. <i>UPase1</i>^−/− mice exhibit activation of the liver HRI-eIF-2α-ATF4 heme-deficiency stress response pathway. <i>UPase1</i>^−/− mice also exhibit impaired ability for insulin-stimulated blood glucose removal. However, other short-term effects of exogenous uridine in C57BL/6J mice are not conserved in <i>UPase1</i>^−/− mice. <i>UPase1</i>^−/− 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.</p></div

Evaluation of the effects of perturbations to uridine homeostasis on liver insulin signaling activity and blood glucose utilization.

<p>(<b>A</b>) Western blots of proteins participating in liver insulin signaling. (<b>B</b>) Quantitative analysis of protein expression and phosphorylation levels of Western blot data presented in (<b>A</b>). (<b>C</b>) Glucose tolerance test (GTT) to evaluate blood glucose levels as a function of time after glucose administration. (<b>D</b>) Integrated blood glucose level as a function of time after glucose administration. (<b>E</b>) Insulin tolerance test (ITT) to evaluate blood glucose level as a function of time after insulin administration. (<b>F</b>) Integrated blood glucose level as a function of time after insulin administration. Error bars are standard deviations across liver samples of 9 mice or blood samples of 12 mice. Areas under the curve were calculated using the trapezoidal rule. Asterisks indicate P<0.05 versus untreated control C57BL/6J mice.</p

Uridine Prevents Fenofibrate-Induced Fatty Liver

<div><p>Uridine, a pyrimidine nucleoside, can modulate liver lipid metabolism although its specific acting targets have not been identified. Using mice with fenofibrate-induced fatty liver as a model system, the effects of uridine on liver lipid metabolism are examined. At a daily dosage of 400 mg/kg, fenofibrate treatment causes reduction of liver NAD⁺/NADH ratio, induces hyper-acetylation of peroxisomal bifunctional enzyme (ECHD) and acyl-CoA oxidase 1 (ACOX1), and induces excessive accumulation of long chain fatty acids (LCFA) and very long chain fatty acids (VLCFA). Uridine co-administration at a daily dosage of 400 mg/kg raises NAD⁺/NADH ratio, inhibits fenofibrate-induced hyper-acetylation of ECHD, ACOX1, and reduces accumulation of LCFA and VLCFA. Our data indicates a therapeutic potential for uridine co-administration to prevent fenofibrate-induced fatty liver.</p></div

Glycosylation of FOXO1 detected with 2D Western blot and capillary isoelectric focusing (cIEF) immunoassay.

<p>(<b>A</b>) Total liver protein was evaluated with 2D WB using an antibody that recognizes FOXO1. (<b>B</b>) Chemluminescence intensity as a function of pI of the 2D WB shown in (<b>A</b>). Note an additional FOXO1 protein spot at high pI value was detected in the liver sample of C57BL/6J+LDU mice but not in C57BL/6J+LD mice. (<b>C</b>) Liver FOXO1 distribution as a function of pI detected with cIEF immunoassay. Peak chemiluminescence was normalized to 1. Peaks from pI 7.5 to 8.5 are due to FOXO1 glycosylation in the liver tissue of C57BL/6J+LDU mice. (<b>D</b>) Quantitative area under the curve analysis (pI 7.5–8.5) of glycosylated FOXO1 isoform as a function of liver tissues. Error bars are standard deviation values across 9 measurements. Asterisk indicates p-value < 0.01 versus C57BL/6J+LD.</p

Bioenergetics of primary hepatocytes.

<p>(<b>A</b>) An example of the mitochondrial function profiles of primary hepatocytes evaluated with a stress test kit. (<b>B</b>) Oxygen consumption rates (OCR) as a function of control and experimental C57bl/6 primary hepatocyte cultures. Error bars are standard deviations of 12 repeated measurements.</p

Evaluation of blood and liver lipids and liver NAD⁺/NADH and NADP⁺/NADPH ratios.

<p>(<b>A</b>) Blood level of triacylglyceride (TAG), cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) in control and treated C57bl/6 mice. (<b>B–D</b>) LC-MS analysis of liver (<b>B</b>) free fatty acids (FFA), (<b>C</b>) TAG, and (<b>D</b>) very long chain fatty acids (VLCFA). All data present in <b>A–D</b> are average of 3 mice analyzed per treatment group. (<b>E–F</b>) Liver (<b>E</b>) NAD⁺/NADH and (<b>F</b>) NADP⁺/NADPH ratios measured with biochemical assays. Error bars are standard deviations across 9 mice evaluated per treatment group. *P<0.05 versus untreated control.</p

Glucose levels in tumors are lower than those of normal tissues of the same tissue sites.

<p>Five pairs of matched tumor and normal tissue specimens from Biochain (B) and Origene (O) were analyzed for glucose and protein content. The amount of glucose was normalized with the total protein concentration. Each sample was assayed in quadruplicate.</p