Expression Of Urea Cycle Enzymes Research Articles

From the Inside Out: Exposing the Roles of Urea Cycle Enzymes in Tumors and Their Micro and Macro Environments.

Catabolic pathways change in anabolic diseases such as cancer to maintain metabolic homeostasis. The liver urea cycle (UC) is the main catabolic pathway for disposing excess nitrogen. Outside the liver, the UC enzymes are differentially expressed based on each tissue's needs for UC intermediates. In tumors, there are changes in the expression of UC enzymes selected for promoting tumorigenesis by increasing the availability of essential UC substrates and products. Consequently, there are compensatory changes in the expression of UC enzymes in the cells that compose the tumor microenvironment. Moreover, extrahepatic tumors induce changes in the expression of the liver UC, which contribute to the systemic manifestations of cancer, such as weight loss. Here, we review the multilayer changes in the expression of UC enzymes throughout carcinogenesis. Understanding the changes in UC expression in the tumor and its micro and macro environment can help identify biomarkers for early cancer diagnosis and vulnerabilities that can be targeted for therapy.

P53 inhibits the Urea cycle and represses polyamine biosynthesis in glioma cell lines.

Glioma is the most common malignant tumor of the central nervous system. The urea cycle (UC) is an essential pathway to convert excess nitrogen and ammonia into the less toxic urea in humans. However, less is known about the functional significance of the urea cycle in glioma. p53 functions as a tumor suppressor and modulates several cellular functions and disease processes. In the present study, we aimed to explore whether p53 influences glioma progression by regulating the urea cycle. Here, we demonstrated the inhibitory impact of p53 on the expression of urea cycle enzymes and urea genesis in glioma cells. The level of polyamine, a urea cycle metabolite, was also regulated by p53 in glioma cells. Carbamoyl phosphate synthetase-1 (CPS1) is the first key enzyme involved in the urea cycle. Functionally, we demonstrated that CPS1 knockdown suppressed glioma cell proliferation, migration and invasion. Mechanistically, we demonstrated that the expression of ornithine decarboxylase (ODC), which determines the generation of polyamine, was regulated by CPS1. In addition, the impacts of p53 knockdown on ODC expression, glioma cell growth and aggressive phenotypes were significantly reversed by CPS1 inhibition. In conclusion, these results demonstrated that p53 inhibits polyamine metabolism by suppressing the urea cycle, which inhibits glioma progression.

The roles and mechanisms of urea cycle enzymes in tumor

<p indent="0mm">The urea cycle (UC) is the production of urea from ammonia, a metabolic waste product, through a series of enzymatic reactions. It plays an important role in maintaining amino acid levels and ammonia homeostasis. The complete UC process takes place almost exclusively in the liver. While the UC is dysregulated in cancer, tumor cells adapt to their environment and regulate biosynthesis by regulating the expression of urea cycle enzymes. Meanwhile, the shortcomings of tumor are exposed by the dysregulation of the UC. Understanding the evolvability of the UC pathway in cancer provides the basis for cancer diagnosis and treatment. Here we review the differential regulation of UC enzymes in tumorigenesis and the reprogramming of UC in tumor cells. Also, this review summarizes the relationship between UC and tumor microenvironment and cancer therapy.

Abstract IA02: Regulating amino acid metabolism can improve response to immunotherapy

Abstract Aberrant expression of urea cycle enzymes occurs in multiple types of cancer. We have previously demonstrated that the urea cycle enzyme ASS1 is downregulated in different tumor types to increase the availability of its substrate aspartate for pyrimidine synthesis, supporting cell proliferation. Therapeutically, we demonstrated that high pyrimidine to purine ratio improves response to immunotherapy. Interestingly, in several prevalent types of cancer ASS1 expression is upregulated for a yet unknown metabolic benefit that associates with poor patients’ prognosis. We now find that ASS1 expression in cancer provides a survival advantage in nutrient deficiency states by generating NO that activates gluconeogenesis. The increase in gluconeogenic flux increases serine levels and leads to a nucleotide imbalance promoting pyrimidine to purine transversion mutations. Consequently, cancers with high ASS1 expression are less immunogenic. Reversing the nucleotide imbalance to favor pyrimidines leads to better response to immunotherapy. Our data establish ASS1 expression as a metabolic survival mechanism under nutrient deprivation that may also serve as a strong determinant of the immune response. Importantly, our findings suggest that we can metabolically regulate tumor mutations to improve patients’ responsiveness to immunotherapy. Citation Format: Ayelet Erez, Eytan Ruppin. Regulating amino acid metabolism can improve response to immunotherapy [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr IA02.

AMP-activated protein kinase signaling regulated expression of urea cycle enzymes in response to changes in dietary protein intake.

Abundance of urea cycle enzymes in the liver is regulated by dietary protein intake. Although urea cycle enzyme levels rise in response to a high-protein (HP) diet, signaling networks that sense dietary protein intake and trigger changes in expression of urea cycle genes have not been identified. The aim of this study was to identify signaling pathway(s) that respond to changes in protein intake and regulate expression of urea cycle genes in mice and human hepatocytes. Mice were adapted to either HP or low-protein diets followed by isolation of liver protein and mRNA and integrated analysis of the proteomic and transcriptomic data. HP diet led to increased expression of mRNA and enzymes in amino acid degradation pathways and decreased expression of mRNA and enzymes in carbohydrate and fat metabolism, which implicated adenosine monophosphate-activated protein kinase (AMPK) as a possible regulator. Primary human hepatocytes, treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) an activator of AMPK, were used to test whether AMPK regulates expression of urea cycle genes. The abundance of carbamoylphosphate synthetase 1 and ornithine transcarbamylase mRNA increased in hepatocytes treated with AICAR, which supports a role for AMPK signaling in regulation of the urea cycle. Because AMPK is either a target of drugs used to treat type-2 diabetes, these drugs might increase the expression of urea cycle enzymes in patients with partial urea cycle disorders, which could be the basis of a new therapeutic approach.

Rewiring urea cycle metabolism incancer to support anabolism.

Cancer cells reprogramme metabolism to maximize the use of nitrogen and carbon for the anabolic synthesis of macromolecules that are required during tumour proliferation and growth. To achieve this aim, one strategy is to reduce catabolism and nitrogen disposal. The urea cycle (UC) in the liver is the main metabolic pathway to convert excess nitrogen into disposable urea. Outside the liver, UC enzymes are differentially expressed, enabling the use of nitrogen for the synthesis of UC intermediates that are required to accommodate cellular needs. Interestingly, the expression of UC enzymes is altered in cancer, revealing a revolutionary mechanism to maximize nitrogen incorporation into biomass. In this Review, we discuss the metabolic benefits underlying UC deregulation in cancer and the relevance of these alterations for cancer diagnosis and therapy.

Sensitivity of Colorectal Cancer to Arginine Deprivation Therapy is Shaped by Differential Expression of Urea Cycle Enzymes

Tumors deficient in the urea cycle enzymes argininosuccinate synthase-1 (ASS1) and ornithine transcarbamylase (OTC) are unable to synthesize arginine and can be targeted using arginine-deprivation therapy. Here, we show that colorectal cancers (CRCs) display negligible expression of OTC and, in subset of cases, ASS1 proteins. CRC cells fail to grow in arginine-free medium and dietary arginine deprivation slows growth of cancer cells implanted into immunocompromised mice. Moreover, we report that clinically-formulated arginine-degrading enzymes are effective anticancer drugs in CRC. Pegylated arginine deiminase (ADI-PEG20), which degrades arginine to citrulline and ammonia, affects growth of ASS1-negative cells, whereas recombinant human arginase-1 (rhArg1peg5000), which degrades arginine into urea and ornithine, is effective against a broad spectrum of OTC-negative CRC cell lines. This reflects the inability of CRC cells to recycle citrulline and ornithine into the urea cycle. Finally, we show that arginase antagonizes chemotherapeutic drugs oxaliplatin and 5-fluorouracil (5-FU), whereas ADI-PEG20 synergizes with oxaliplatin in ASS1-negative cell lines and appears to interact with 5-fluorouracil independently of ASS1 status. Overall, we conclude that CRC is amenable to arginine-deprivation therapy, but we warrant caution when combining arginine deprivation with standard chemotherapy.

FRI-229 - Role of Toll-like receptor 4 (TLR4) in regulating severity of hyperammonemia and gene expression of urea cycle enzymes

FRI-229 - Role of Toll-like receptor 4 (TLR4) in regulating severity of hyperammonemia and gene expression of urea cycle enzymes

Role of Bioflavonoid Quercetin on Expression of Urea Cycle Enzymes, Astrocytic and Inflammatory Markers in Hyperammonemic Rats.

This study evaluates the role of quercetin on the expression of urea cycle enzymes, astrocytic, neuronal and inflammatory markers in hyperammonemic rats. Hyperammonemia (provoked by intraperitonial injections of (ammonium chloride-100mg/kg b.w for 56days), showed diminished expression of urea cycle enzymes [carbamyl phosphate synthetase-1 (CPS-1), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS) and arginase (ARG)] in liver and decreased expression of neuronal and astrocytic markers-glutamine synthase (GS) and phosphate activated glutaminase (PAG) in brain and increased expression of brain inflammatory markers such as interleukin 6 (IL6), inducible nitric oxide synthase (iNOS) and nuclear transcription factor kappa B (NF-κB) (by western blot analysis) and exhibited downregulated expression of soluble guanylate cyclase (sGC), glial fibrillary acidic protein (GFAP) in brain and ASS in liver investigated (by RT-PCR). Oral treatment of quercetin (50mg/kg b.w) to hyperammonemic rats (1) increased the expression of urea cycle enzymes (CPS-1, OTC, ASS and ARG), neuronal and astrocytic markers (GS and PAG) (2) decreased the expression of IL6, iNOS and NF-κB and (3) upregulated mRNA expression of SGC, GFAP and ASS. Our results specify that quercetin's antihyperammonemic effects could be through its, anti-inflammatory, neuroprotective and hepatoprotective effects.

Chronotherapeutic effect of fisetin on expression of urea cycle enzymes and inflammatory markers in hyperammonaemic rats

BackgroundElevated blood ammonia leads to hyperammonaemia that affects vital central nervous system (CNS) functions. Fisetin, a naturally occurring flavonoid, exhibits therapeutic benefits, such as anti-cancer, anti-diabetic, anti-oxidant, anti-angiogenic, neuroprotective and neurotrophic effects. MethodsIn this study, the chronotherapeutic effect of fisetin on ammonium chloride (AC)-induced hyperammonaemic rats was investigated, to ascertain the time point at which the maximum drug effect is achieved. The anti-hyperammonaemic potential of fisetin (50mg/kg b.w. oral) was analysed when administered to AC treated (100mg/kg b.w. i.p.) rats at 06:00, 12:00, 18:00 and 00:00h. Amelioration of pathophysiological conditions by fisetin at different time points was measured by analysing the levels of expression of liver urea cycle enzymes (carbamoyl phosphate synthetase-I (CPS-I), ornithine transcarbamoylase (OTC) and argininosuccinate synthetase (ASS)), nuclear transcription factor kappaB (NF-κB p65), brain glutamine synthetase (GS) and inducible nitric oxide synthase (iNOS) by Western blot analysis. ResultsFisetin increased the expression of CPS-I, OTC, ASS and GS and decreased iNOS and NF-κB p65 in hyperammonaemic rats. Fisetin administration at 00:00h showed more significant effects on the expression of liver and brain markers, compared with other time points. ConclusionsFisetin could exhibit anti-hyperammonaemic effect owing to its anti-oxidant and cytoprotective influences. The temporal variation in the effect of fisetin could be due to the (i) chronopharmacological, chronopharmacokinetic properties of fisetin and (ii) modulations in the endogenous circadian rhythms of urea cycle enzymes, brain markers, redox enzymes and renal clearance during hyperammonaemia by fisetin. However, future studies in these lines are necessitated.

Aberrant expression and distribution of enzymes of the urea cycle and other ammonia metabolizing pathways in dogs with congenital portosystemic shunts.

The detoxification of ammonia occurs mainly through conversion of ammonia to urea in the liver via the urea cycle and glutamine synthesis. Congenital portosystemic shunts (CPSS) in dogs cause hyperammonemia eventually leading to hepatic encephalopathy. In this study, the gene expression of urea cycle enzymes (carbamoylphosphate synthetase (CPS1), ornithine carbamoyltransferase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase (ARG1)), N-acetylglutamate synthase (NAGS), Glutamate dehydrogenase (GLUD1), and glutamate-ammonia ligase (GLUL) was evaluated in dogs with CPSS before and after surgical closure of the shunt. Additionally, immunohistochemistry was performed on urea cycle enzymes and GLUL on liver samples of healthy dogs and dogs with CPSS to investigate a possible zonal distribution of these enzymes within the liver lobule and to investigate possible differences in distribution in dogs with CPSS compared to healthy dogs. Furthermore, the effect of increasing ammonia concentrations on the expression of the urea cycle enzymes was investigated in primary hepatocytes in vitro. Gene-expression of CPS1, OTC, ASL, GLUD1 and NAGS was down regulated in dogs with CPSS and did not normalize after surgical closure of the shunt. In all dogs GLUL distribution was localized pericentrally. CPS1, OTC and ASS1 were localized periportally in healthy dogs, whereas in CPSS dogs, these enzymes lacked a clear zonal distribution. In primary hepatocytes higher ammonia concentrations induced mRNA levels of CPS1. We hypothesize that the reduction in expression of urea cycle enzymes, NAGS and GLUD1 as well as the alterations in zonal distribution in dogs with CPSS may be caused by a developmental arrest of these enzymes during the embryonic or early postnatal phase.

Subzero nonfreezing storage of rat hepatocytes using UW solution and 1,4-butanediol. II- functional testing on rewarming and gene expression of urea cycle enzymes

In the present study we have analyzed the viability and metabolic competence of isolated rat hepatocytes subjected first, to subzero nonfreezing storage (up to 120 h at -4 °C) in modified University of Wisconsin (UW) solution with 8% 1,4-butanediol, and then to a normothermic rewarming step (KHR media, 37 °C, up to 120 min, carbogen atmosphere). Results were compared with hepatocytes stored up to 120 h at 0°C in modified UW solution and with freshly isolated hepatic cells. We have found that only cell suspensions stored in subzero nonfreezing conditions were able to finish the rewarming period with a viability comparable with the control group. Also, we have investigated the enzyme activities and the relative expression at messenger RNAs levels of two of the Urea cycle (UC) enzymes: Carbamyl phosphate synthetase I (CPSI) and ornithine transcarbamylase (OTC), during 60 min of rewarming. Results were compared with the ammonium removal efficiency of the three groups.In conclusion: These data indicated that hepatocytes preserved under cold or subzero conditions up to 120 h followed by 60 min of rewarming, maintain UC enzymes at levels similar to freshly isolated hepatocytes, allowing their use in bioartificial liver devices.

Growth hormone and growth hormone secretagogue effects on nitrogen balance and urea synthesis in steroid treated rats

Objectives Growth hormone (GH) reduces the catabolic side effects of steroid treatment via effects on the amino-nitrogen metabolism. Ipamorelin is a synthetic peptide with GH releasing properties. We wished to study the metabolic effects of Ipamorelin and GH on selected hepatic measures of α-amino-nitrogen conversion during steroid-induced catabolism. Design Five groups of rats were included: (1) free-fed controls (2) pair-fed controls (3) prednisolone (delcortol, 4 mg × kg −1 × day −1) (4) prednisolone and GH (1 mg × kg −1 × day −1) (5) prednisolone and Ipamorelin (0.5 mg × kg −1 × day −1). After seven days the hepatic capacity of urea-N synthesis (CUNS) was determined in parallel with measurements of liver mRNA levels of urea cycle enzymes, whole-body N-balance, and N-contents of various organs. Results Compared to pair-fed controls, prednisolone increased CUNS ( p < 0.01) as well as the expression of urea cycle genes ( p < 0.01), and decreased N-balance ( p < 0.01) as well as organ N-contents ( p < 0.05). Compared to prednisolone treated animals, co-administration of GH reduced CUNS by 33% ( p < 0.01), normalized urea cycle gene expression, improved N-balance 2.5-fold, and normalized or improved organ N-contents. In prednisolone treated rats Ipamorelin reduced CUNS by 20% ( p < 0.05), decreased the expression of urea cycle enzymes, neutralised N-balance, and normalized or improved organ N-contents. Conclusion Accelerated nitrogen wasting in the liver and other organs caused by prednisolone treatment was counteracted by treatment with either GH or its secretagogue Ipamorelin, though at the doses given less efficiently by the latter. This functional study of animals confirms that the GH secretagogue exerts GH related metabolic effects and may be useful in the treatment of steroid-induced catabolism.

Changes of activity and mRNA expression of urea cycle enzymes in the liver of developing Holstein calves1

Urea is an important reutilizable nitrogen source for the ruminant and is mainly synthesized through the urea cycle in the liver. The cycle is undertaken by 5 enzymes: carbamoyl phosphate synthetase (CPS), ornithine transcarbamoylase (OTC), arginino-succinate synthetase (AS), argininosuccinate lyase (AL), and arginase. The purpose of this study was to investigate changes in the activity of the enzymes and mRNA expression, given that previous observations have indicated an increase in plasma urea concentrations with age in Holstein calves. First, plasma concentrations of metabolites and hormones were determined in calves at 1, 3, 8, 13, and 19 wk of age (n = 4, weaned at 6 wk of age). The plasma concentration of urea drastically increased after weaning (P < 0.001). The plasma concentration of glucose was lowest at 8 wk. The plasma concentration of IGF-I gradually increased with age, although those of NEFA, glucagon, and cortisol decreased (P < 0.001). Concentrations of triglyceride, alpha-amino nitrogen, growth hormone, and insulin did not change significantly with age of the calf. Next, using the liver tissues taken from calves at 2, 13, and 19 wk of age (n = 4 to 6 at each time point, weaned at 6 wk of age), we measured the activity and mRNA expression of the enzymes by biochemical methods and quantitative reverse transcription-PCR, respectively. The activities of CPS (P < 0.001), OTC (P = 0.001), and AS (P = 0.015) increased with age, whereas AL (P = 0.003) decreased. Although mRNA expression was decreased with age for AL (P = 0.002) and arginase (P = 0.007), no significant change was observed for CPS, OTC, or AS mRNA expression. We conclude that the increased urea production in the liver may be explained not only by an increase in the activities of the urea cycle enzymes, but also by increased ammonia production by rumen fermentation and gluconeogenesis from amino acids around weaning time.

A proteomic analysis of liver mitochondria during acute endotoxemia

Accumulating evidence indicates that mitochondrial function is impaired in vital organs during sepsis. In addition to oxidative phosphorylation, mitochondria participate in diverse cellular functions ranging from protein and lipid metabolism to programmed cell death. We analyzed liver mitochondrial protein expression patterns (i.e., proteomics) during acute endotoxemia to discover novel insights into mitochondrial responses to acute systemic inflammation. A normotensive endotoxemia model was employed in which altered mitochondrial morphology occurs under conditions minimizing the potentially confounding effects of tissue hypoxia and acidosis. University medical research laboratory. Random-source, adult, male cats. Hemodynamic resuscitation and maintenance of acid-base balance and tissue oxygen availability were provided to preserve baseline homeostatic conditions. Treatment groups received isotonic saline vehicle (control; n = 5) or endotoxin (lipopolysaccharide, LPS, at 3.0 mg/kg intravenously; n = 5]. Liver samples were obtained 4 h posttreatment, and mitochondrial proteins were isolated and quantitatively compared using two-dimensional gel electrophoresis. Differentially expressed proteins (> 1.5-fold change relative to controls) were identified using mass spectrometry. Among over 500 protein spots that were separated 14 were differentially expressed in mitochondria of LPS-treated animals relative to matching controls. Spectrometric analyses demonstrated increased expression of urea cycle enzymes, heat shock protein (HSP) 60 and manganese superoxide dismutase, whereas expression of HSP70, F(1)-ATPase and key enzymes regulating lipid metabolism was reduced. Considering the known functions of each of the proteins exhibiting altered expression, it is likely that the observed changes in liver mitochondrial protein expression are reflective of significant changes in mitochondrial function in response to endotoxemia.

31. Immunological Response to and Long Term Expression of Urea Cycle Enzymes Delivered by Helper-Dependent Adenoviral Vectors

31. Immunological Response to and Long Term Expression of Urea Cycle Enzymes Delivered by Helper-Dependent Adenoviral Vectors

Regulation of enzymes of the urea cycle and arginine metabolism.

The urea cycle is comprised of five enzymes but also requires other enzymes and mitochondrial amino acid transporters to function fully. The complete urea cycle is expressed in liver and to a small degree also in enterocytes. However, highly regulated expression of several enzymes present in the urea cycle occurs also in many other tissues, where these enzymes are involved in synthesis of nitric oxide, polyamines, proline and glutamate. Glucagon, insulin, and glucocorticoids are major regulators of the expression of urea cycle enzymes in liver. In contrast, the "urea cycle" enzymes in nonhepatic cells are regulated by a wide range of pro- and antiinflammatory cytokines and other agents. Regulation of these enzymes is largely transcriptional in virtually all cell types. This review emphasizes recent information regarding roles and regulation of urea cycle and arginine metabolic enzymes in liver and other cell types.

GH decreases hepatic amino acid degradation after small bowel resection in rats without enhancing bowel adaptation.

Growth hormone (GH) treatment in short bowel syndrome is controversial, and the mechanisms of a possible positive effect remain to be elucidated. Rats were randomly subjected to either an 80% jejunoileal resection or sham operation and were given either placebo (NaCl) or biosynthetic rat GH (brGH). The in vivo capacity of urea nitrogen synthesis (CUNS) and the expression of urea cycle enzymes were measured and related to changes in body weight and adaptive growth in ileal segments on days 7 and 14. Ileal segments were examined by unbiased stereological techniques. brGH treatment decreased CUNS among the resected rats by 19% (P<0.05) and 36% (P<0.05) on days 7 and 14, respectively. The mRNA levels of urea cycle enzyme genes were not influenced by brGH treatment. brGH treatment did not increase the adaptive growth in the ileal segments. In conclusion, we found that GH treatment decreased the accelerated postoperative hepatic amino acid degradation in experimental short bowel syndrome without enhancing the morphological intestinal adaptation.

Expression of carbamoyl phosphate synthetase I and ornithine transcarbamoylase genes in Chinese hamster ovary dhfr-cells decreases accumulation of ammonium ion in culture media

Ammonium ion accumulation in mammalian cell culture media causes toxicity which inhibits cell growth and productivity. To reduce the level of the accumulated ammonium ion, carbamoyl phosphate synthetase I (CPS I) and ornithine transcarbamoylase (OTC) were used, which catalyze the first and second steps of the urea cycle in the liver. To examine the effects of overexpressed CPS I and OTC genes on the concentration of the ammonium ion in culture media, the two genes were introduced into Chinese hamster ovary (CHO) dhfr-cells. The CPS I expressing cell lines (CPS I-CHO) and both CPS I and OTC expressing cell lines (CPS I/OTC-CHO) were confirmed at the mRNA level and analyzed in terms of the cell growth and the accumulation of ammonium ion in culture media. The accumulation of ammonium ion was ≈25–33% less in CPS I/OTC-CHO than in either CPS I-CHO or the vector-control cell lines. Interestingly however, the cell growth was ≈15–30% faster in both CPS I-CHO and CPS I/OTC-CHO than in the control cell lines. Forced expression of urea cycle enzymes in the CHO cells revealed that both the expression of CPS I and OTC can reduce the accumulation of ammonium ion in the culture media.

Acute systemic and local inflammation decreases hepatic expression of urea cycle enzymes

Acute systemic and local inflammation decreases hepatic expression of urea cycle enzymes