Disruption of uridine homeostasis links liver pyrimidine metabolism to lipid accumulation.

We report in this study an intrinsic link between pyrimidine metabolism and liver lipid accumulation utilizing a uridine phosphorylase 1 transgenic mouse model UPase1-TG. Hepatic microvesicular steatosis is induced by disruption of uridine homeostasis through transgenic overexpression of UPase1, an enzyme of the pyrimidine catabolism and salvage pathway. Microvesicular steatosis is also induced by the inhibition of dihydroorotate dehydrogenase (DHODH), an enzyme of the de novo pyrimidine biosynthesis pathway. Interestingly, uridine supplementation completely suppresses microvesicular steatosis in both scenarios. The effective concentration (EC(50)) for uridine to suppress microvesicular steatosis is approximately 20 µM in primary hepatocytes of UPase1-TG mice. We find that uridine does not have any effect on in vitro DHODH enzymatic activity. On the other hand, uridine supplementation alters the liver NAD(+)/NADH and NADP(+)/NADPH ratios and the acetylation profile of metabolic, oxidation-reduction, and antioxidation enzymes. Protein acetylation is emerging as a key regulatory mechanism for cellular metabolism. Therefore, we propose that uridine suppresses fatty liver by modulating the liver protein acetylation profile. Our findings reveal a novel link between uridine homeostasis, pyrimidine metabolism, and liver lipid metabolism.

Differential expression of uridine phosphorylase in tumors contributes to an improved fluoropyrimidine therapeutic activity.

Abrogation of uridine phosphorylase (UPase) leads to abnormalities in pyrimidine metabolism and host protection against 5-fluorouracil (5-FU) toxicity. We elucidated the effects on the metabolism and antitumor efficacy of 5-FU and capecitabine (N(4)-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) in our UPase knockout (UPase(-/-)) model. Treatment with 5-FU (85 mg/kg) or capecitabine (1,000 mg/kg) five days a week for four weeks caused severe toxicity and structural damage to the intestines of wild-type (WT) mice, but not in UPase(-/-) animals. Capecitabine treatment resulted in a 70% decrease in blood cell counts of WT animals, with only a marginal effect in UPase(-/-) mice. UPase expressing colon 38 tumors implanted in UPase(-/-) mice revealed an improved therapeutic efficacy when treated with 5-FU and capecitabine because of the higher maximum tolerated dose for fluoropyrimidines achievable in UPase(-/-) mice. (19)F-MRS evaluation of capecitabine metabolism in tumors revealed similar activation of the prodrug in UPase(-/-) mice compared with WT. In WT mice, approximately 60% of capecitabine was transformed over three hours into its active metabolites, whereas 80% was transformed in tumors implanted in UPase(-/-) mice. In UPase(-/-) mice, prolonged retention of 5'dFUR allowed a proportional increase in tumor tissue. The similar presence of fluorinated catabolic species confirms that dihydropyrimidine dehydrogenase activity was not altered in UPase(-/-) mice. Overall, these results indicate the importance of UPase in the activation of fluoropyrimidines, the effect of uridine in protecting normal tissues, and the role for tumor-specific modulation of the phosphorolytic activity in 5-FU or capecitabine-based chemotherapy.

Uridine protects cortical neurons from glucose deprivation-induced death: possible role of uridine phosphorylase.

We previously reported that uridine blocked glucose deprivation-induced death of immunostimulated astrocytes by preserving ATP levels. Uridine phosphorylase (UPase), an enzyme catalyzing the reversible phosphorylation of uridine, was involved in this effect. Here, we tried to expand our previous findings by investigating the uridine effect on the brain and neurons using in vivo and in vitro ischemic injury models. Orally administrated uridine (50-200 mg/kg) reduced middle cerebral artery occlusion (1.5 h)/reperfusion (22 h)-induced infarct in mouse brain. Additionally, in the rat brain subjected to the same ischemic condition, UPase mRNA and protein levels were up-regulated. Next, we employed glucose deprivation-induced hypoglycemia in mixed cortical cultures of neurons and astrocytes as an in vitro model. Cells were deprived of glucose and, two hours later, supplemented with 20 mM glucose. Under this condition, a significant ATP loss followed by death was observed in neurons but not in astrocytes, which were blocked by treatment with uridine in a concentration-dependent manner. Inhibition of cellular uptake of uridine by S-(4-nitrobenzyl)-6-thioinosine blocked the uridine effect. Similar to our in vivo data, UPase expression was up-regulated by glucose deprivation in mRNA as well as protein levels. Additionally, 5-(phenylthio)acyclouridine, a specific inhibitor of UPase, prevented the uridine effect. Finally, the uridine effect was shown only in the presence of astrocytes. Taken together, the present study provides the first evidence that uridine protects neurons against ischemic insult-induced neuronal death, possibly through the action of UPase.

Profiles of pyrimidine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers.

In order to obtain general metabolic profiles of pyrimidine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers was investigated. The activities of key enzymes in potato tuber extracts were also studied. The following results were obtained. Of the intermediates in de novo pyrimidine biosynthesis, [(14)C]carbamoylaspartate was converted to orotic acid and [2-(14)C]orotic acid was metabolized to nucleotides and RNA. UMP synthase, a bifunctional enzyme with activities of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23), exhibited high activity. The rates of uptake of pyrimidine ribo- and deoxyribonucleosides by the disks were high, in the range 2.0-2.8 nmol (g FW)(-1) h(-1). The pyrimidine ribonucleosides, uridine and cytidine, were salvaged exclusively to nucleotides, by uridine/cytidine kinase (EC 2.7.1.48) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Cytidine was also salvaged after conversion to uridine by cytidine deaminase (EC 3.5.4.5) and the presence of this enzyme was demonstrated in cell-free tuber extracts. Deoxycytidine, a deoxyribonucleoside, was efficiently salvaged. Since deoxycytidine kinase (EC 2.7.1.74) activity was extremely low, non-specific nucleoside phosphotransferase (EC 2.7.1.77) probably participates in deoxycytidine salvage. Thymidine, which is another pyrimidine deoxyribonucleoside, was degraded and was not a good precursor for nucleotide synthesis. Virtually all the thymidine 5'-monophosphate synthesis from thymidine appeared to be catalyzed by phosphotransferase activity, since little thymidine kinase (EC 2.7.1.21) activity was detected. Of the pyrimidine bases, uracil, but not cytosine, was salvaged for nucleotide synthesis. Since uridine phosphorylase (EC 2.4.2.3) activity was not detected, uracil phosphoribosyltransferase (EC 2.4.2.9) seems to play the major role in uracil salvage. Uracil was degraded by the reductive pathway via beta-ureidopropionate, but cytosine was not degraded. The activities of the cytosine-metabolizing enzymes observed in other organisms, pyrimidine nucleoside phosphorylase (EC 2.4.2.2) and cytosine deaminase (EC 3.5.4.1), were not detected in potato tuber extracts. Operation of the de novo synthesis of deoxyribonucleotides via ribonucleotide reductase and of the salvage pathway of deoxycytidine was demonstrated via the incorporation of radioactivity from both [2-(14)C]cytidine and [2-(14)C]deoxycytidine into DNA. A novel pathway converting deoxycytidine to uracil nucleotides was found and deoxycytidine deaminase (EC 3.5.4.14), an enzyme that may participate in this pathway, was detected in the tuber extracts.

Cloning and expression of human uridine phosphorylase.

Using a mouse cDNA probe we have identified a human uridine phosphorylase cDNA clone from the cDNA library of a human colorectal tumor cell line, HCT116. The recombinant human uridine phosphorylase expressed in COS-7 cells demonstrated specific enzyme activity with uridine as the substrate; this activity was inhibited by the competitive inhibitor 2,2'-anhydro-5-ethyluridine. Northern blot analysis with the cDNA as a probe demonstrated high levels of mRNA expression in several tumor cell lines but very low level in normal cell, WI-38. The expression of uridine phosphorylase mRNA in HCT-116 cells was further enhanced by treating the cells with vitamin D3 and the inflammatory cytokines: tumor necrosis factor alpha, interleukin 1 alpha and interferon gamma.

Inhibition of 5'-deoxy-5-fluorouridine phosphorolysis by acyclopyrimidinenucleosides in intestinal tissue homogenates.

This study examined the inhibitory effect of acyclopyrimidinenucleosides on 5'-deoxy-5-fluorouridine (5'-DFUR) phosphorolysis in intestinal tissue derived from rabbit, rat, mouse, and human. 5-Bromoacyclouridine, 5-fluoroacyclouridine, acyclouridine, and 5-nitroacyclouridine showed little or only moderate effect, but acyclothymidine [5-methyl-1-(2'-hydroxyethoxymethyl)uracil] showed strong inhibitory effect on 5'-DFUR phosphorolysis in intestinal tissue homogenates derived from human. In the absence of inhibitor (acyclothymidine), the Vmax of 5'-DFUR phosphorolysis was 2.66 mumol/min and the Km was 0.57 mM in human intestinal homogenates. The Vmax was unaltered by increased inhibitor concentration. The maximal inhibitory effect of acyclothymidine on 5'-DFUR phosphorolysis in rat homogenates was over 90%. The Ki/Km was 0.63 in human, 2.14 in rabbit, 1.09 x 10(-2) in rat, and 1.71 x 10(-2) in mouse. These data show that acyclothymidine is a competitive inhibitor of 5'-DFUR phosphorolysis, and that it can inhibit not only uridine phosphorylase but also thymidine phosphorylase.

Enzymes of pyrimidine metabolism in Mycoplasma mycoides subsp. mycoides.

The major pathways of ribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides have been proposed from studies on its use of radioactive purines and pyrimidines. To interpret more fully the observed pattern of pyrimidine usage, cell extracts of this organism have been assayed for several enzymes associated with the salvage synthesis of pyrimidine nucleotides. M. mycoides possessed uracil phosphoribosyltransferase, uridine phosphorylase, uridine (cytidine) kinase, uridine 5'-monophosphate kinase, and cytidine 5'-triphosphate synthetase. No activity for phosphorolysis of cytidine was detected, and no in vitro conditions were found to give measurable deamination of cytidine. Of the two potential pathways for incorporation of uridine, our data suggest that this precursor would largely undergo initial phosphorolysis to uracil and ribose-1-phosphate. Conversely, cytidine is phosphorylated directly to cytidine 5'-monophosphate in its major utilization, although conversion of cytidine to uracil, uridine, and uridine nucleotide has been observed in vivo, at least when uracil is provided in the growth medium. Measurements of intracellular nucleotide contents and their changes on additions of pyrimidine precursors have allowed suggestions as to the operation of regulatory mechanisms on pyrimidine nucleotide biosynthesis in M. mycoides in vivo. With uracil alone or uracil plus uridine as precursors of pyrimidine ribonucleotides, the regulation of uracil phosphoribosyltransferase and cytidine 5'-triphosphate synthetase is probably most important in determining the rate of pyrimidine nucleotide synthesis. When cytidine supplements uracil in the growth medium, control of cytidine kinase activity would also be important in this regard.