Deoxynucleoside stress exacerbates the phenotype of a mouse model of mitochondrial neurogastrointestinal encephalopathy.

Balanced pools of deoxyribonucleoside triphosphate precursors are required for DNA replication, and alterations of this balance are relevant to human mitochondrial diseases including mitochondrial neurogastrointestinal encephalopathy. In this disease, autosomal recessive TYMP mutations cause severe reductions of thymidine phosphorylase activity; marked elevations of the pyrimidine nucleosides thymidine and deoxyuridine in plasma and tissues, and somatic multiple deletions, depletion and site-specific point mutations of mitochondrial DNA. Thymidine phosphorylase and uridine phosphorylase double knockout mice recapitulated several features of these patients including thymidine phosphorylase activity deficiency, elevated thymidine and deoxyuridine in tissues, mitochondrial DNA depletion, respiratory chain defects and white matter changes. However, in contrast to patients with this disease, mutant mice showed mitochondrial alterations only in the brain. To test the hypothesis that elevated levels of nucleotides cause unbalanced deoxyribonucleoside triphosphate pools and, in turn, pathogenic mitochondrial DNA instability, we have stressed double knockout mice with exogenous thymidine and deoxyuridine, and assessed clinical, neuroradiological, histological, molecular, and biochemical consequences. Mutant mice treated with exogenous thymidine and deoxyuridine showed reduced survival, body weight, and muscle strength, relative to untreated animals. Moreover, in treated mutants, leukoencephalopathy, a hallmark of the disease, was enhanced and the small intestine showed a reduction of smooth muscle cells and increased fibrosis. Levels of mitochondrial DNA were depleted not only in the brain but also in the small intestine, and deoxyribonucleoside triphosphate imbalance was observed in the brain. The relative proportion, rather than the absolute amount of deoxyribonucleoside triphosphate, was critical for mitochondrial DNA maintenance. Thus, our results demonstrate that stress of exogenous pyrimidine nucleosides enhances the mitochondrial phenotype of our knockout mice. Our mouse studies provide insights into the pathogenic role of thymidine and deoxyuridine imbalance in mitochondrial neurogastrointestinal encephalopathy and an excellent model to study new therapeutic approaches.

Gene therapy using a liver-targeted AAV vector restores nucleoside and nucleotide homeostasis in a murine model of MNGIE.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in TYMP, enconding thymidine phosphorylase (TP). TP deficiency results in systemic accumulation of thymidine and deoxyuridine, which interferes with mitochondrial DNA (mtDNA) replication and leads to mitochondrial dysfunction. To date, the only treatment available for MNGIE patients is allogeneic hematopoietic stem cell transplantation, which is associated with high morbidity and mortality. Here, we report that AAV2/8-mediated transfer of the human TYMP coding sequence (hcTYMP) under the control of a liver-specific promoter prevents the biochemical imbalances in a murine model of MNGIE. hcTYMP expression was restricted to liver, and a dose as low as 2 × 10(11) genome copies/kg led to a permanent reduction in systemic nucleoside levels to normal values in about 50% of treated mice. Higher doses resulted in reductions to normal or slightly below normal levels in virtually all mice treated. The nucleoside reduction achieved by this treatment prevented deoxycytidine triphosphate (dCTP) depletion, which is the limiting factor affecting mtDNA replication in this disease. These results demonstrate that the use of AAV to direct TYMP expression in liver is feasible as a potentially safe gene therapy strategy for MNGIE.

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.

A novel structural mechanism for redox regulation of uridine phosphorylase 2 activity.

Uridine phosphorylase (UPP) catalyzes the reversible conversion of uridine to uracil and ribose-1-phosphate and plays an important pharmacological role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil and capecitabine. Most vertebrate animals, including humans, possess two homologs of this enzyme (UPP1 & UPP2), of which UPP1 has been more thoroughly studied and is better characterized. Here, we report two crystallographic structures of human UPP2 (hUPP2) in distinctly active and inactive conformations. These structures reveal that a conditional intramolecular disulfide bridge can form within the protein that dislocates a critical phosphate-coordinating arginine residue (R100) away from the active site, disabling the enzyme. In vitro activity measurements on both recombinant hUPP2 and native mouse UPP2 confirm the redox sensitivity of this enzyme, in contrast to UPP1. Sequence analysis shows that this feature is conserved among UPP2 homologs and lacking in all UPP1 proteins due to the absence of a necessary cysteine residue. The state of the disulfide bridge has further structural consequences for one face of the enzyme that suggest UPP2 may have additional functions in sensing and initiating cellular responses to oxidative stress. The molecular details surrounding these dynamic aspects of hUPP2 structure and regulation provide new insights as to how novel inhibitors of this protein may be developed with improved specificity and affinity. As uridine is emerging as a promising protective compound in neuro-degenerative diseases, including Alzheimer's and Parkinson's, understanding the regulatory mechanisms underlying UPP control of uridine concentration is key to improving clinical outcomes in these illnesses.

Unbalanced deoxynucleotide pools cause mitochondrial DNA instability in thymidine phosphorylase-deficient mice.

Replication and repair of DNA require equilibrated pools of deoxynucleoside triphosphate precursors. This concept has been proven by in vitro studies over many years, but in vivo models are required to demonstrate its relevance to multicellular organisms and to human diseases. Accordingly, we have generated thymidine phosphorylase (TP) and uridine phosphorylase (UP) double knockout (TP(-/-)UP(-/-)) mice, which show severe TP deficiency, increased thymidine and deoxyuridine in tissues and elevated mitochondrial deoxythymidine triphosphate. As consequences of the nucleotide pool imbalances, brains of mutant mice developed partial depletion of mtDNA, deficiencies of respiratory chain complexes and encephalopathy. These findings largely account for the pathogenesis of mitochondrial neurogastrointestinal encephalopathy (MNGIE), the first inherited human disorder of nucleoside metabolism associated with somatic DNA instability.

Paracrine stimulation of endothelial cell motility and angiogenesis by platelet-derived deoxyribose-1-phosphate.

OBJECTIVE: Micromolar concentrations of the proangiogenic metabolite deoxyribose-1-phosphate (dRP) were detected in platelet supernatants by mass spectrometry. In this study, we assessed whether the release of dRP by platelets stimulates endothelial cell migration and angiogenesis. METHODS AND RESULTS: Protein-free supernatants from thrombin-stimulated platelets increased human umbilical vein endothelial cell migratory activity in transmigration and monolayer repair assays. This phenomenon was ablated by genetic silencing of dRP-generating uridine phosphorylase (UP) and thymidine phosphorylase (TP) or pharmacological inhibition of UP and restored by exogenous dRP. The stimulation of endothelial cell migration by platelet-derived dRP correlated with upregulation of integrin ß(3), which was induced in a reactive oxygen species-dependent manner, and was mediated by the activity of the integrin heterodimer α(v)ß(3). The physiological relevance of dRP release by platelets was confirmed in a chick chorioallantoic membrane assay, where the presence of this metabolite in platelet supernatants strongly induced capillary formation. CONCLUSIONS: Platelet-derived dRP stimulates endothelial cell migration by upregulating integrin ß(3) in a reactive oxygen species-dependent manner. As demonstrated by our in vivo experiments, this novel paracrine regulatory pathway is likely to play an important role in the stimulation of angiogenesis by platelets.

CD9, a tetraspanin target for cancer therapy?

In the present minireview, we intend to provide a brief history of the field of CD9 involvement in oncogenesis and in the metastatic process of cancer, considering its potential value as a tumor-associated antigenic target. Over the years, CD9 has been identified as a favorable prognostic marker or predictor of metastatic potential depending on the cancer type. To understand its implications in cancer beside its use as an antigenic biomarker, it is essential to know its physiological functions, including its molecular partners in a given cell system. Moreover, the discovery that CD9 is one of the most specific and broadly expressed markers of extracellular membrane vesicles, nanometer-sized entities that are released into extracellular space and various physiological body fluids and play a role in intercellular communication under physiological and pathological conditions, notably the establishment of cancer metastases, has added a new dimension to our knowledge of CD9 function in cancer. Here, we will discuss these issues as well as the possible cancer therapeutic implications of CD9, their limitations, and pitfalls.

Implications of the structure of human uridine phosphorylase 1 on the development of novel inhibitors for improving the therapeutic window of fluoropyrimidine chemotherapy.

BACKGROUND: Uridine phosphorylase (UPP) is a key enzyme of pyrimidine salvage pathways, catalyzing the reversible phosphorolysis of ribosides of uracil to nucleobases and ribose 1-phosphate. It is also a critical enzyme in the activation of pyrimidine-based chemotherapeutic compounds such a 5-fluorouracil (5-FU) and its prodrug capecitabine. Additionally, an elevated level of this enzyme in certain tumours is believed to contribute to the selectivity of such drugs. However, the clinical effectiveness of these fluoropyrimidine antimetabolites is hampered by their toxicity to normal tissue. In response to this limitation, specific inhibitors of UPP, such as 5-benzylacyclouridine (BAU), have been developed and investigated for their ability to modulate the cytotoxic side effects of 5-FU and its derivatives, so as to increase the therapeutic index of these agents. RESULTS: In this report we present the high resolution structures of human uridine phosphorylase 1 (hUPP1) in ligand-free and BAU-inhibited conformations. The structures confirm the unexpected solution observation that the human enzyme is dimeric in contrast to the hexameric assembly present in microbial UPPs. They also reveal in detail the mechanism by which BAU engages the active site of the protein and subsequently disables the enzyme by locking the protein in a closed conformation. The observed inter-domain motion of the dimeric human enzyme is much greater than that seen in previous UPP structures and may result from the simpler oligomeric organization. CONCLUSION: The structural details underlying hUPP1's active site and additional surfaces beyond these catalytic residues, which coordinate binding of BAU and other acyclouridine analogues, suggest avenues for future design of more potent inhibitors of this enzyme. Notably, the loop forming the back wall of the substrate binding pocket is conformationally different and substantially less flexible in hUPP1 than in previously studied microbial homologues. These distinctions can be utilized to discover novel inhibitory compounds specifically optimized for efficacy against the human enzyme as a step toward the development of more effective chemotherapeutic regimens that can selectively protect normal tissues with inherently lower UPP activity.

Uridine phosphorylase in breast cancer: a new prognostic factor?

Uridine phosphorylase (UPase) is an enzyme that converts the pyrimidine nucleoside uridine into uracil. Upon availability of ribose-1-phosphate, UPase can also catalyze the formation of nucleosides from uracil as well as from 5-fluorouracil, therefore involved in fluoropyrimidine metabolism. UPase gene expression is strictly controlled at the promoter level by oncogenes, tumor suppressor genes, and cytokines. UPase activity is usually elevated in various tumor tissues, including breast cancer, compared to matched normal tissues and this induction appears to contribute to the therapeutic efficacy of fluoropyrimidines in cancer patients. In this review, we will discuss in detail the role of UPase in the activation of fluoropyrimidines and its effect on the prognosis of breast cancer patients.

Uridine phosphorylase (-/-) murine embryonic stem cells clarify the key role of this enzyme in the regulation of the pyrimidine salvage pathway and in the activation of fluoropyrimidines.

We have reported the elevation of uridine phosphorylase (UPase) in many solid tumors and the presence of a variant phosphorolytic activity in breast cancer tissues (M. Liu et al., Cancer Res., 58: 5418-5424, 1998). To better understand the biological and pharmacological significance of these findings, we have developed an UPase gene knockout embryonic stem (ES) cell model by specific gene targeting techniques. In this cellular model, we establish the critical role of UPase as an important anabolic enzyme in 5-fluorouracil (5-FU) activation and pyrimidine salvage pathway regulation. It has long been known that UPase regulates the plasma concentration of uridine; however, little is known of the role of UPase in the activation and metabolism of 5-FU and its derivatives, mainly because of the lack of an appropriate model system. The experimental data indicate that the disruption of UPase activity in murine ES cells leads to a 10-fold increase in 5-FU IC(50) and a 2-3-fold reduction in its incorporation into nucleic acids, whereas no differences in toxicity is seen with other pyrimidine nucleoside analogues such as 5-fluorouridine, 2'-deoxy-5-fluorouridine, and 1-beta-D-arabinofuranosylcytosine compared with WT (wild-type) ES cells. Benzylacyclouridine can specifically prevent the WT ES cells from the sensitivity of 5-FU. Our data also shows the effect of UPase on the cytotoxicity of 5'-deoxy-5-fluorouridine (5'DFUR), a 5-FU prodrug. The IC(50) is increased almost 16-fold in the knockout cells compared with the wild type cells, demonstrating the role of UPase in catalyzing the conversion of 5'DFUR to 5-FU. These findings additionally elucidate the tumor-specific selectivity of capecitabine, the oral fluoropyrimidine prodrug approved for the treatment of metastatic breast and colorectal cancers. Not only do the knockout cells present a decreased incorporation of 5-FU into nucleic acids but also an increased reliance on the pyrimidine salvage pathway. The reduced dependence of UPase knockout cells on the pyrimidine de novo synthesis is reflected in the apparent resistance to phosphonacetyl-L-aspartic acid, a specific inhibitor of pyrimidine pathway, with a 5-fold elevation in its IC(50) in UPase-nullified cells compared with WT. In summary, we have successfully generated an UPase gene knockout cell model that presents reduced sensitivity to 5-FU, 5'DFUR, and phosphonacetyl-L-aspartic acid, although it does not affect the basic cellular physiology under normal tissue culture conditions. Considering the role of UPase in 5-FU metabolism and the elevated expression of this protein in cancer cells compared with paired normal tissues, additional investigation should be warranted to firmly establish the clinical role of UPase in the tumor selective activation of 5-FU and capecitabine.

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

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.

Homeostatic control of uridine and the role of uridine phosphorylase: a biological and clinical update.

Uridine, a pyrimidine nucleoside essential for the synthesis of RNA and bio-membranes, is a crucial element in the regulation of normal physiological processes as well as pathological states. The biological effects of uridine have been associated with the regulation of the cardio-circulatory system, at the reproduction level, with both peripheral and central nervous system modulation and with the functionality of the respiratory system. Furthermore, uridine plays a role at the clinical level in modulating the cytotoxic effects of fluoropyrimidines in both normal and neoplastic tissues. The concentration of uridine in plasma and tissues is tightly regulated by cellular transport mechanisms and by the activity of uridine phosphorylase (UPase), responsible for the reversible phosphorolysis of uridine to uracil. We have recently completed several studies designed to define the mechanisms regulating UPase expression and better characterize the multiple biological effects of uridine. Immunohistochemical analysis and co-purification studies have revealed the association of UPase with the cytoskeleton and the cellular membrane. The characterization of the promoter region of UPase has indicated a direct regulation of its expression by the tumor suppressor gene p53. The evaluation of human surgical specimens has shown elevated UPase activity in tumor tissue compared to paired normal tissue.

Vector targeting makes 5-fluorouracil chemotherapy less toxic and more effective in animal models of epithelial neoplasms.

PURPOSE: 5-fluorouracil (5-FU) has been combined in the past with other drugs for the combination chemotherapy for cancers of the breast, ovary, and colon. These drug regimens were limited by the fact that 5-FU fails to kill nondividing cancer cells at the doses that are safe to deliver. The goal of the present study is to test the feasibility of replacing 5-FU in established 5-FU combination chemotherapy with the Ad-LpCDIRESE1A/5-fluorocytosine (5-FC) system for the purpose of reducing toxicity and increasing efficacy. EXPERIMENTAL DESIGN: We have replaced 5-FU in the weekly combination of CPT-11, folinic acid (FA) and 5-FU chemotherapy by 5-FC and an adenoviral vector that carries the L-plastin (Lp) tumor-specific promoter-driven transcription unit encoding the cytosine deaminase gene linked to the E1A gene by an internal ribosomal entry site element. This combination is called "genetic combination therapy." The goal of using the vector was to decrease the toxicity to normal tissue and to increase the efficacy of therapy in the cancer cells by increasing the concentration of 5-FU sufficiently high that even nondividing cancer cells would be killed by 5-FU through its incorporation into mRNA and consequent inhibition of synthesis of functional proteins. We compared the in vivo efficacy of the genetic combination therapy with the conventional combination chemotherapy in a mouse colon cancer model. RESULTS: Both replication-competent and -noncompetent adenoviral vectors carrying an L-plastin-driven cytosine deaminase transcription unit when combined with 5-FC, CPT-11, and FA showed increased in vitro therapeutic activity that was significantly higher than that of the conventional chemotherapy combination. Tumor-bearing mice treated with the genetic combination therapy showed a statistically significant advantage in terms of increased response rate, response duration, survival, and reduced toxicity when compared with tumor-bearing mice treated with the conventional combination chemotherapy. CONCLUSIONS: Replacement of 5-FU in 5-FU-based combination chemotherapy with the Ad-LpCDIRESE1A vector and 5-FU reduces toxicity and increases efficacy. This is a concept that could be potentially applied widely for many forms of cancer treatment.

Phase I study of perillyl alcohol in patients with refractory malignancies.

We treated 21 patients in a dose-finding and pharmacokinetic study of the monoterpene perillyl alcohol with the drug given orally in 3 divided doses on a chronic basis. The average number of days that patients remained on study was 48 (range 11-172). Fatigue and low-grade nausea were dose limiting. Using this schedule, a starting dose of 1.6 g/m2 with escalation to 2.1 g/m2 as tolerated is recommended. Two major metabolites were detectable and the mean peak plasma concentrations were 383 microM for perillic acid and 27 microM for dihydroperillic acid. The peak plasma concentration and the metabolite half-life were 2 h and 1 h post ingestion for perillic acid, and 4 h and 2.4 h for dihydroperillic acid, respectively. Stabilization of disease was observed in one of the 16 patients evaluable for response. Many of the gastrointestinal side effects that were poorly tolerated on a chronic basis may be partly related to the soybean oil base used in the current formulation. Further development of perillyl alcohol on this schedule would be facilitated by reformulation of the capsule.

Magnetic resonance spectroscopic imaging of tumor metabolic markers for cancer diagnosis, metabolic phenotyping, and characterization of tumor microenvironment.

Cancer cells display heterogeneous genetic characteristics, depending on the tumor dynamic microenvironment. Abnormal tumor vasculature and poor tissue oxygenation generate a fraction of hypoxic tumor cells that have selective advantages in metastasis and invasion and often resist chemo- and radiation therapies. The genetic alterations acquired by tumors modify their biochemical pathways, which results in abnormal tumor metabolism. An elevation in glycolysis known as the "Warburg effect" and changes in lipid synthesis and oxidation occur. Magnetic resonance spectroscopy (MRS) has been used to study tumor metabolism in preclinical animal models and in clinical research on human breast, brain, and prostate cancers. This technique can identify specific genetic and metabolic changes that occur in malignant tumors. Therefore, the metabolic markers, detectable by MRS, not only provide information on biochemical changes but also define different metabolic tumor phenotypes. When combined with the contrast-enhanced Magnetic Resonance Imaging (MRI), which has a high sensitivity for cancer diagnosis, in vivo magnetic resonance spectroscopic imaging (MRSI) improves the diagnostic specificity of malignant human cancers and is becoming an important clinical tool for cancer management and care. This article reviews the MRSI techniques as molecular imaging methods to detect and quantify metabolic changes in various tumor tissue types, especially in extracranial tumor tissues that contain high concentrations of fat. MRI/MRSI methods have been used to characterize tumor microenvironments in terms of blood volume and vessel permeability. Measurements of tissue oxygenation and glycolytic rates by MRS also are described to illustrate the capability of the MR technology in probing molecular information non-invasively in tumor tissues and its important potential for studying molecular mechanisms of human cancers in physiological conditions.

Age-related differences in vincristine toxicity and biodistribution in wild-type and transporter-deficient mice.

The impact of mouse multidrug resistance genes mdrla/b and mrpl on age-related differences in the toxicity and biodistribution of vincristine (VCR) was evaluated in wild-type, mrpl(-/-), mdrla/b(-/-), and combined mdrla/b(-/-), mrpl(-/-) weanling and adult mice given a single IP dose of VCR ranging from 0.0625 to 6 mg/kg. Weanling mice of all four genotypes were more sensitive than adult animals as determined by survival rate, average time of death, and pathologic findings. Wild-type animals were the least sensitive and combined mdrla/b(-/-), mrpl(-/-) mice the most sensitive to VCR toxicity. Mdrla/b(-/-) and mrpl(-/-) genotypes exhibited intermediate sensitivities, with mdrla/b(-/-) mice being more sensitive than mrpl(-/-) animals to the vinca alkaloid. Administration of [3H]VCR to wild-type and mdrla/b(-/-), mrpl(-/-) animals revealed relatively greater accumulation of radioactive VCR equivalents in weanlings over adults in several tissues, with weanling mdrla/b(-/-), mrpl(-/-) lung and heart exhibiting the greatest enhanced accumulation of 26- and 15-fold over adults, respectively. A similar cardiopulmonary differential accumulation of VCR was not observed in wild-type weanlings to adults. Semiquantitative RT-PCR expression analyses of ABC transporter genes in weanling and adult tissues of wild-type and combined mdrla/b(-/-), mrpl(-/-) mice did not reveal major age-related differences in these ABC transporters that would explain the relatively greater toxicity observed in weanling mice. However, the greater cardiopulmonary accumulation of VCR equivalents seen in the combined mdrla/b(-/-), mrpl(-/-) weanlings over that of adults underscores the potential for unique organ and age-related toxicities of this agent in the setting of transporter deficiency.

Uridine prevents tamoxifen-induced liver lipid droplet accumulation.

BACKGROUND: Tamoxifen, an agonist of estrogen receptor, is widely prescribed for the prevention and long-term treatment of breast cancer. A side effect of tamoxifen is fatty liver, which increases the risk for non-alcoholic fatty liver disease. Prevention of tamoxifen-induced fatty liver has the potential to improve the safety of long-term tamoxifen usage. METHODS: Uridine, a pyrimidine nucleoside with reported protective effects against drug-induced fatty liver, was co-administered with tamoxifen in C57BL/6J mice. Liver lipid levels were evaluated with lipid visualization using coherent anti-Stokes Raman scatting (CARS) microscopy, biochemical assay measurement of triacylglyceride (TAG), and liquid chromatography coupled with mass spectrometry (LC-MS) measurement of membrane phospholipid. Blood TAG and cholesterol levels were measured. Mitochondrial respiration of primary hepatocytes in the presence of tamoxifen and/or uridine was evaluated by measuring oxygen consumption rate with an extracellular flux analyzer. Liver protein lysine acetylation profiles were evaluated with 1D and 2D Western blots. In addition, the relationship between endogenous uridine levels, fatty liver, and tamoxifen administration was evaluated in transgenic mice UPase1-/-and UPase1-TG. RESULTS: Uridine co-administration prevented tamoxifen-induced liver lipid droplet accumulation in mice. The most prominent effect of uridine co-administration with tamoxifen was the stimulation of liver membrane phospholipid biosynthesis. Uridine had no protective effect against tamoxifen-induced impairment to mitochondrial respiration of primary hepatocytes or liver TAG and cholesterol export. Uridine had no effect on tamoxifen-induced changes to liver protein acetylation profile. Transgenic mice UPase1-/-with increased pyrimidine salvage activity were protected against tamoxifen-induced liver lipid droplet accumulation. In contrast, UPase1-TG mice with increased pyrimidine catabolism activity had intrinsic liver lipid droplet accumulation, which was aggravated following tamoxifen administration. CONCLUSION: Uridine co-administration was effective at preventing tamoxifen-induced liver lipid droplet accumulation. The ability of uridine to prevent tamoxifen-induced fatty liver appeared to depend on the pyrimidine salvage pathway, which promotes biosynthesis of membrane phospholipid.

Uridine affects liver protein glycosylation, insulin signaling, and heme biosynthesis.

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 UPase1-/- mice with long-term elevation of endogenous uridine level. UPase1-/- mice exhibit activation of the liver HRI-eIF-2α-ATF4 heme-deficiency stress response pathway. UPase1-/- 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 UPase1-/- mice. UPase1-/- 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.

Uridine prevents fenofibrate-induced fatty liver.

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.

Autocrine amplification of integrin αIIbß3 activation and platelet adhesive responses by deoxyribose-1-phosphate.

Using direct injection mass spectrometry (DIMS) we discovered that deoxyribose-1-phosphate (dRP) is released by platelets upon activation. Interestingly, the addition of exogenous dRP to human platelets significantly increased platelet aggregation and integrin αIIbß3 activation in response to thrombin. In parallel, genetically modified platelets with double genetic deletion of thymidine phosphorylase and uridine phosphorylase were characterised by reduced release of dRP, impaired aggregation and decreased integrin αIIbß3 activation in response to thrombin. In vitro platelet adhesion onto fibrinogen and collagen under physiological flow conditions was potentiated by treatment of human platelets with exogenous dRP and impaired in transgenic platelets with reduced dRP release. Human and mouse platelets responded to dRP treatment with a sizeable increase in reactive oxygen species (ROS) generation and the pre-treament with the antioxidant apocynin abolished the effect of dRP on aggregation and integrin activation. Experiments directly assessing the activation of the small G protein Rap1b and protein kinase C suggested that dRP increases the basal levels of activity of these two pivotal platelet-activating pathways in a redox-dependent manner. Taken together, we present evidence that dRP is a novel autocrine amplifier of platelet activity, which acts on platelet redox levels and modulates integrin αIIbß3.