Carbamoyl phosphate synthase 1 (CPS1) is the rate-limiting enzyme in the first step of the urea cycle and an indispensable enzyme in the metabolism of human liver. However, CPS1 epigenetic regulation involves promoter analysis and the role of liver-enriched transcription factors (LETFs), which is not fully elucidated. In this work, the promoter region of hCPS1 gene was cloned, and its activity was investigated. An LETF, hepatocyte nuclear factor 3-beta (HNF3β), was found to promote the transcriptional expression of CPS1 in liver-derived cell lines. In addition, dual-luciferase reporter assay shows that the essential binding sites of the HNF3β may exist in the oligonucleotide -70 nt to +73 nt. Two putative binding sites are available for HNF3β. Mutation analysis results show that the binding site 2 of HNF3β was effective, and the transcriptional activity of CPS1 promoter significantly decreased after mutation. Electrophoretic mobile shift assay (EMSA) and ChIP assay confirmed that HNF3β can interact with the binding site in the CPS1 promoter region of -70 nt to +73 nt promoter region in vivo and in vitro to regulate the transcription of CPS1. Moreover, HNF3β overexpression enhanced the transcription of CPS1 and consequently improved the mRNA and protein levels of CPS1, whereas the knockdown of HNF3β showed the opposite effects. Finally, urea production in cells was measured, and ammonia detoxification improved significantly in cells after transfection with HNF3β. HNF3β plays a vital role in regulation of CPS1 gene and could promote the metabolism of ammonia by regulating CPS1 expression.

Tetrapods and fish have adapted distinct carbamoyl-phosphate synthase (CPS) enzymes to initiate the ornithine urea cycle during the detoxification of nitrogenous wastes. We report evidence that in the ureotelic subgenus of extremophile fish Oreochromis Alcolapia, CPS III has undergone convergent evolution and adapted its substrate affinity to ammonia, which is typical of terrestrial vertebrate CPS I. Unusually, unlike in other vertebrates, the expression of CPS III in Alcolapia is localized to the skeletal muscle and is activated in the myogenic lineage during early embryonic development with expression remaining in mature fish. We propose that adaptation in Alcolapia included both convergent evolution of CPS function to that of terrestrial vertebrates, as well as changes in development mechanisms redirecting CPS III gene expression to the skeletal muscle.

N-carbamoyl-l-glutamate (NCG), the N-acetyl-l-glutamate analogue used to treat N-acetylglutamate synthase deficiency, has been proposed as potential therapy of carbamoyl phosphate synthetase 1 deficiency (CPS1D). Previous findings in five CPS1D patients suggest that NCG-responsiveness could be mutation-specific. We report on a patient with CPS1D, homozygous for the novel p.(Pro1211Arg) CPS1 mutation, who presented at 9 days of life with hyperammonemic coma which was successfully treated with emergency measures. He remained metabolically stable on merely oral NCG, arginine, and modest protein restriction. Ammonia scavengers were only added after poor dietary compliance following solid food intake at age 1 year. The patient received a liver transplantation at 3.9 years of age, having normal cognitive, motor, and quality of life scores despite repeated but successfully treated episodes of hyperammonemia. Studies using recombinantly produced mutant CPS1 confirmed the partial nature of the CPS1D triggered by the p.(Pro1211Arg) mutation. This mutation decreased the solubility and yield of CPS1 as expected for increased tendency to misfold, and reduced the thermal stability, maximum specific activity (V max; ~2-fold reduction), and apparent affinity (~5-fold reduction) for ATP of the purified enzyme. By increasing the saturation of the NAG site in vivo, NCG could stabilize CPS1 and minimize the decrease in the effective affinity of the enzyme for ATP. These observations, together with prior experience, support the ascertainment of clinical responsiveness to NCG in CPS1 deficient patients, particularly when decreased stability or lowered affinity for NAG of the mutant enzyme are suspected or proven.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are potential resources for the regeneration of defective organs, including the liver. However, some obstacles must be overcome before this becomes reality. Undifferentiated cells that remain following differentiation have teratoma-forming potential. Additionally, practical applications require a large quantity of differentiated cells, so the differentiation process must be economical. Here we describe a DNA microarray-based global analysis of the gene expression profiles of differentiating human pluripotent stem cells. We identified differences and commonalities among six human pluripotent stem cell lines: the hESCs KhES1, KhES2, KhES3, and H1, and the iPSCs 201B7 and 243G1. Embryoid bodies (EBs) formed without requiring supplementation with inducing factors. EBs also expressed some liver-specific metabolic genes including the ammonia-metabolizing enzymes glutamine synthetase and carbamoyl-phosphate synthase 1. Real-time PCR analysis revealed hepatocyte-like differentiation of EBs treated with ammonia in Lanford medium. Analysis of DNA microarray data suggested that hepatocyte-like cells were the most abundant population in ammonia-treated cells. Furthermore, expression levels of undifferentiated pluripotent stem cell markers were drastically reduced, suggesting a reduced teratoma-forming capacity. These results indicate that treatment of EBs with ammonia in Lanford medium may be an effective inducer of hepatic differentiation in absence of expensive inducing factors.

Urea cycle is an important metabolic process that initiates in liver mitochondria and converts ammonia to urea. The impairment of ammonia detoxification, both primary and secondary causes, lead to hyperammonemia, a life-threatening condition affecting to the brain. Current treatments are not enough effective. In addition, our recent proteomics study in hypercholesterolemic rat model demonstrated that sericin enhances hepatic nitrogenous waste removal through carbamoyl-phosphate synthase 1 (CPS-1), aldehyde dehydrogenase-2 (ALDH-2), and uricase proteins. However, the underlining mechanisms regard to this property is not clarified yet. Therefore, the present study aims to examine the effect of sericin on urea cycle enzyme genes (CPS-1 and ornithine transcarbamylase; OTC) and proteins (mitogen-activated protein kinase; MAPK, caspase recruitment domain-containing protein 9; CARD-9, Microtubule-associated protein light chain 3; LC-3), which relate to urea production and liver homeostasis in hepatic cell line (HepG2) and hypercholesterolemic rat treated with or without sericin. qRT-PCR, immunohistochemistry, and electron microscopy techniques were performed. In vitro study determined that high dose of sericin at 1 mg/ml increased liver detoxification enzyme (Cytochrome P450 1A2; CYP1A2 and ALDH-2) and urea cycle enzyme (CPS-1 and OTC) genes. Both in HepG2 cell and rat liver mitochondria, sericin significantly downregulated CARD-9 (apoptotic protein) expression while upregulated MAPK (hepatic homeostasis protein) and LC-3 (autophagic protein) expressions. Hence, it might be concluded that sericin promotes ammonia detoxification by both increases urea cycle enzyme genes and enhances hepatic autophagy in associated with CARD-9/MAPK pathway (as shown by their own negative relationship). This study presents another beneficial property of sericin to develop an upcoming candidate for ammonia toxicity alleviation and liver function improvement.

The molecular mechanisms of uric acid (UA)-induced liver injury has not been clearly elucidated. In this study, we aimed to investigate the effect and action mechanisms of UA in liver injury. We analyzed the damaging effect of UA on mouse liver and L02 cells and subsequently performed metabolomics studies on L02 cells to identify abnormal metabolic pathways. Finally, we verified transcription factors that regulate related metabolic enzymes. UA directly activated the hepatic NLRP3 inflammasome and Bax apoptosis pathway invivo and invitro. Related metabolites in the arginine biosynthesis pathway (or urea cycle), l-arginine and l-argininosuccinate were decreased, and ammonia was increased in UA-stimulated L02 cells, which was mediated by carbamoyl phosphate synthase 1 (CPS1), argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) downregulation. UA upregulated hypoxia inducible factor-1alpha (HIF-1α) invivo and invitro, and HIF-1α inhibition alleviated the UA-induced ASS downregulation and hepatocyte injury. In conclusion, UA upregulates HIF-1α and inhibits urea cycle enzymes (UCEs). This leads to liver injury, with evidence of hepatocyte inflammation, apoptosis and oxidative stress.

Sirt5, localized in the mitochondria, is a member of sirtuin family of NAD⁺-dependent deacetylases. Sirt5 was shown to deacetylate and activate carbamoyl phosphate synthase 1. Most recently, Sirt5 was reported to be the predominant protein desuccinylase and demalonylase in the mitochondria because the ablation of Sirt5 enhanced the global succinylation and malonylation of mitochondrial proteins, including many metabolic enzymes. In order to determine the physiological role of Sirt5 in metabolic homeostasis, we generated a germline Sirt5 deficient (Sirt5⁻/⁻) mouse model and performed a thorough metabolic characterization of this mouse line. Although a global protein hypersuccinylation and elevated serum ammonia during fasting were observed in our Sirt5⁻/⁻ mouse model, Sirt5 deficiency did not lead to any overt metabolic abnormalities under either chow or high fat diet conditions. These observations suggest that Sirt5 is likely to be dispensable for the metabolic homeostasis under the basal conditions.

The present research draws a map of the characteristic carbonylation of proteins in rats fed high-caloric diets with the aim of providing a new insight of the pathogenesis of metabolic diseases derived from the high consumption of fat and refined carbohydrates. Protein carbonylation was analyzed in plasma, liver and skeletal muscle of Sprague-Dawley rats fed a high-fat, high-sucrose (HFHS) diet by a proteomics approach based on carbonyl-specific fluorescence-labeling, gel electrophoresis and mass spectrometry. Oxidized proteins along with specific sites of oxidative damage were identified and discussed to illustrate the consequences of protein oxidation. The results indicated that long-term HFHS consumption increased protein oxidation in plasma and liver; meanwhile, protein carbonyls from skeletal muscle did not change. The increment of carbonylation by HFHS diet was singularly selective on specific target proteins: albumin from plasma and liver, and hepatic proteins such as mitochondrial carbamoyl-phosphate synthase (ammonia), mitochondrial aldehyde dehydrogenase, argininosuccinate synthetase, regucalcin, mitochondrial adenosine triphosphate synthase subunit beta, actin cytoplasmic 1 and mitochondrial glutamate dehydrogenase 1. The possible consequences that these specific protein carbonylations have on the excessive weight gain, insulin resistance and nonalcoholic fatty liver disease resulting from HFHS diet consumption are discussed.

We recently demonstrated that in addition to its protective effect on pancreatic and adrenal biosynthesis, antioxidant properties of sericin decrease blood cholesterol levels and improve the liver mitochondrial architecture. However, little is known about the detailed mechanisms underlying these effects. Using proteomics and electron microscopy, we identified mitochondrial proteins that play important roles in the preservation of the mitochondrial ultrastructure and cholesterol-lowering properties of sericin. Our results showed that sericin maintains the mitochondrial architecture during conditions of high blood cholesterol by regulating apoptotic (NADH-ubiquinone oxidoreductase 75 kDa subunit) and autophagic (mitochondrial elongation factor Tu and prohibitin-2) proteins as well as energy maintenance proteins [haloacid dehalogenase-like hydrolase domain-containing protein 3, succinate dehydrogenase (ubiquinone) flavoprotein subunit, ATP synthase-α subunit precursor, enoyl-CoA hydratase domain-containing protein 3 and electron transfer flavoprotein subunit-α]. Sericin also exerts anti-oxidative properties via aconitate hydratase and Chain A, crystal structure of rat carnitine palmitoyltrasferase 2 proteins. Together, these activities may reduce hepatocytic triglyceride deposition, thereby decreasing steatosis, as demonstrated by the modulatory effects on ornithine aminotransferase, mitochondrial aspartate aminotransferase, acyl-CoA synthase, hydroxyacyl-CoA dehydrogenase and D-beta-hydroxybutyrate dehydrogenase. Sericin activity further balanced nitrogenous waste detoxification, characterised by carbamoyl-phosphate synthase (ammonia), aldehyde dehydrogenase and uricase, or folate biosynthesis via sarcosine dehydrogenase and dimethyl glycine dehydrogenase. These results suggest that sericin maintains the hepatic mitochondrial architecture through apoptotic, autophagic, energy maintenance and anti-oxidative mitochondrial proteins for alleviating hepatic steatosis and promoting liver function under conditions of hypercholesterolaemia.

In many pharmaceuticals, a hydrogen atom or hydroxyl group is replaced by a fluorine to increase bioavailability and biostability. The fate of fluorine released from fluorine-containing drugs is not well investigated. The aim of this study was to examine possible fluorination of proteins in rat liver and brain after administration of the fluorinated drug cinacalcet. We assigned 18 Wistar rats to a control group (n = 6) and a group treated with cinacalcet (2 mg kg-1/body weight, 5 days/week), divided into 7 day (n = 6) and 21 day (n = 6) treatment subgroups. Fluorinated proteins were identified using a free proteomics approach; chromatographic separation and analysis by high-resolution mass spectrometry; peptide/protein identification using the Mascot search algorithm; manual verification of an experimentally generated MS/MS spectrum with the theoretical MS/MS spectrum of identified fluorinated peptides. Three fluorinated proteins (spectrin beta chain; carbamoyl-phosphate synthase [ammonia], mitochondrial; 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 1) were identified in the liver and four (spectrin beta chain, dihydropyrimidinase-related protein 4, prominin-2, dihydropyrimidinase-related protein 4) in the brain tissue after 21 days of cinacalcet treatment, but not in the control group. Introduction of fluorine into an organism by administration of fluorinated drugs results in tissue-specific fluorination of proteins.

Cancer cells hijack and remodel existing metabolic pathways for their benefit. Argininosuccinate synthase (ASS1) is a urea cycle enzyme that is essential in the conversion of nitrogen from ammonia and aspartate to urea. A decrease in nitrogen flux through ASS1 in the liver causes the urea cycle disorder citrullinaemia. In contrast to the well-studied consequences of loss of ASS1 activity on ureagenesis, the purpose of its somatic silencing in multiple cancers is largely unknown. Here we show that decreased activity of ASS1 in cancers supports proliferation by facilitating pyrimidine synthesis via CAD (carbamoyl-phosphate synthase 2, aspartate transcarbamylase, and dihydroorotase complex) activation. Our studies were initiated by delineating the consequences of loss of ASS1 activity in humans with two types of citrullinaemia. We find that in citrullinaemia type I (CTLN I), which is caused by deficiency of ASS1, there is increased pyrimidine synthesis and proliferation compared with citrullinaemia type II (CTLN II), in which there is decreased substrate availability for ASS1 caused by deficiency of the aspartate transporter citrin. Building on these results, we demonstrate that ASS1 deficiency in cancer increases cytosolic aspartate levels, which increases CAD activation by upregulating its substrate availability and by increasing its phosphorylation by S6K1 through the mammalian target of rapamycin (mTOR) pathway. Decreasing CAD activity by blocking citrin, the mTOR signalling, or pyrimidine synthesis decreases proliferation and thus may serve as a therapeutic strategy in multiple cancers where ASS1 is downregulated. Our results demonstrate that ASS1 downregulation is a novel mechanism supporting cancerous proliferation, and they provide a metabolic link between the urea cycle enzymes and pyrimidine synthesis.

Knowledge of the Michaelis-Menten parameters and their meaning in different circumstances is an essential prerequisite to understanding enzyme function and behaviour. The published literature contains an abundance of values reported for many enzymes. The problem concerns assessing the appropriateness and validity of such material for the purpose to which it is to be applied. This review considers the evaluation of such data with particular emphasis on the assessment of its fitness for purpose.

Flucloxacillin is a β-lactam antibiotic associated with a high incidence of drug-induced liver injury. Although expression of HLA-B*57:01 is associated with increased susceptibility, little is known of the pathological mechanisms involved in the induction of the clinical phenotype. Irreversible protein modification is suspected to drive the reaction through the provision of flucloxacillin-modified peptides that are presented to T-cells by the protein encoded by the risk allele. In this study, we have shown that flucloxacillin binds to multiple proteins within human primary hepatocytes, including major hepatocellular proteins (hemoglobin and albumin) and mitochondrial proteins. Inhibition of membrane transporters multidrug resistance-associated protein 2 (MRP2) and P-glycoprotein (P-gp) appeared to reduce the levels of covalent binding. A diverse range of proteins with different functions was found to be targeted by flucloxacillin, including adaptor proteins (14-3-3), proteins with catalytic activities (liver carboxylesterase 1, tRNA-splicing endonuclease subunit Sen2, All-trans-retinol dehydrogenase ADH1B, Glutamate dehydrogenase 1 mitochondrial, Carbamoyl-phosphate synthase [ammonia] mitochondrial), and transporters (hemoglobin, albumin, and UTP-glucose-1-phosphate uridylyltransferase). These flucloxacillin-modified intracellular proteins could provide a potential source of neoantigens for HLA-B*57:01 presentation by hepatocytes. More importantly, covalent binding to critical cellular proteins could be the molecular initiating events that lead to flucloxacillin-induced cholestasis Data are available via ProteomeXchange with identifier PXD038581.

The urea cycle (UC) removes the excess nitrogen and ammonia generated by nitrogen-containing compound composites or protein breakdown in the human body. Research has shown that changes in UC enzymes are not only related to tumorigenesis and tumor development but also associated with poor survival in hepatocellular, breast, and colorectal cancers (CRC), etc. Cytoplasmic ornithine, the intermediate product of the urea cycle, is a specific substrate for ornithine decarboxylase (ODC, also known as ODC1) for the production of putrescine and is required for tumor growth. Polyamines (spermidine, spermine, and their precursor putrescine) play central roles in more than half of the steps of colorectal tumorigenesis. Given the close connection between polyamines and cancer, the regulation of polyamine metabolic pathways has attracted attention regarding the mechanisms of action of chemical drugs used to prevent CRC, as the drug most widely used for treating type 2 diabetes (T2D), metformin (Met) exhibits antitumor activity against a variety of cancer cells, with a vaguely defined mechanism. In addition, the influence of metformin on the UC and putrescine generation in colorectal cancer has remained unclear. In our study, we investigated the effect of metformin on the UC and putrescine generation of CRC in vivo and in vitro and elucidated the underlying mechanisms. In nude mice bearing HCT116 tumor xenografts, the administration of metformin inhibited tumor growth without affecting body weight. In addition, metformin treatment increased the expression of monophosphate (AMP)-activated protein kinase (AMPK) and p53 in both HCT116 xenografts and colorectal cancer cell lines and decreased the expression of the urea cycle enzymes, including carbamoyl phosphate synthase 1 (CPS1), arginase 1 (ARG1), ornithine trans-carbamylase (OTC), and ODC. The putrescine levels in both HCT116 xenografts and HCT116 cells decreased after metformin treatment. These results demonstrate that metformin inhibited CRC cell proliferation via activating AMPK/p53 and that there was an association between metformin, urea cycle inhibition and a reduction in putrescine generation.

In this study, we investigated the differential pattern of protein expression in the liver of these mice to provide insights on why they have different responses to F.

Aspartame (L-aspartyl phenylalanine methyl ester) is a non-nutritive sweetener (NNS) approved for use in more than 6000 dietary products and pharmaceuticals consumed by the general public including adults and children, pregnant and nursing mothers. However a recent prospective study reported a doubling of the risk of being overweight amongst 1-year old children whose mothers consumed NNS-sweetened beverages daily during pregnancy. We have previously shown that chronic aspartame (ASP) exposure commencing in utero may detrimentally affect adulthood adiposity status, glucose metabolism and aspects of behavior and spatial cognition, and that this can be modulated by developmental N-methyl-D-aspartate receptor (NMDAR) blockade with the competitive antagonist CGP 39551 (CGP). Since glucose homeostasis and certain aspects of behavior and locomotion are regulated in part by the NMDAR-rich hypothalamus, which is part of the hypothalamic-pituitary-adrenal- (HPA) axis, we have elected to examine changes in hypothalamic and adrenal gene expression in response to ASP exposure in the presence or absence of developmental NMDAR antagonism with CGP, using Affymetrix microarray analysis.