Carbamoyl Phosphate Synthetase Research Articles

Integrated transcriptomic and metabolomic investigation of the genes and metabolites involved in swine follicular cyst formation.

Follicular cysts are a common reproductive disorder in mammals that is usually caused by stress. However, the pathogenesis of follicular cysts in sows remains unclear. To provide new insights into the mechanisms of follicular cyst formation in pigs, we conducted a combined transcriptomic and metabolomic analysis on theca interna and mural granulosa cells of follicular cysts and mature follicles. We identified 2,533 up-regulated and 1,355 down-regulated genes in follicular cysts, compared with mature follicles. These differentially expressed genes were mainly found in signaling pathways related to tumor formation and cortisol synthesis and secretion as shown by Ingenuity Pathway Analysis, which predicted 4,362 upstream regulatory factors. The combined gene expression and pathway analysis identified the following genes as potential biomarkers for porcine follicular cysts: cytochrome P450 family 2 subfamily C polypeptide 18, L-lactate dehydrogenase, carbamoyl-phosphate synthase, fibroblast growth factor 7, integrin binding sialoprotein, interleukin 23 receptor, prolactin receptor, epiregulin, interleukin 1 receptor type II, arginine vasopressin receptor 1A, fibroblast growth factor 10, claudin 7, G Protein Subunit Gamma 3, cholecystokinin B receptor and cytosolic phospholipase A2. Metabolomics analysis found significant differences in 87 metabolites, which were enriched in unsaturated fatty acid biosynthesis, and sphingolipid signaling pathways. These results provide valuable information on the molecular mechanisms of follicular cyst formation, which may facilitate the development of new therapeutics to prevent and treat follicular cysts.

Beyond protein synthesis: the emerging role of arginine in poultry nutrition and host-microbe interactions.

Arginine is a functional amino acid essential for various physiological processes in poultry. The dietary essentiality of arginine in poultry stems from the absence of the enzyme carbamoyl phosphate synthase-I. The specific requirement for arginine in poultry varies based on several factors, such as age, dietary factors, and physiological status. Additionally, arginine absorption and utilization are also influenced by the presence of antagonists. However, dietary interventions can mitigate the effect of these factors affecting arginine utilization. In poultry, arginine is utilized by four enzymes, namely, inducible nitric oxide synthase arginase, arginine decarboxylase and arginine: glycine amidinotransferase (AGAT). The intermediates and products of arginine metabolism by these enzymes mediate the different physiological functions of arginine in poultry. The most studied function of arginine in humans, as well as poultry, is its role in immune response. Arginine exerts immunomodulatory functions primarily through the metabolites nitric oxide (NO), ornithine, citrulline, and polyamines, which take part in inflammation or the resolution of inflammation. These properties of arginine and arginine metabolites potentiate its use as a nutraceutical to prevent the incidence of enteric diseases in poultry. Furthermore, arginine is utilized by the poultry gut microbiota, the metabolites of which might have important implications for gut microbial composition, immune regulation, metabolism, and overall host health. This comprehensive review provides insights into the multifaceted roles of arginine and arginine metabolites in poultry nutrition and wellbeing, with particular emphasis on the potential of arginine in immune regulation and microbial homeostasis in poultry.

CPS1 augments hepatic glucagon response through CaMKII/FOXO1 pathway.

Introduction: Elevated glucagon levels are a characteristic feature of type 2 diabetes. This abnormal increase in glucagon can lead to an accelerated rate of gluconeogenesis. Glucagon also stimulates hepatic metabolism of amino acids, particularly promoting the formation of urea. The specific role of carbamoyl phosphate synthetase 1 (CPS1), a rate-limiting enzyme in the urea cycle, in the development versus the persistence of glucagon-induced hyperglycemia has not been previously established. Methods: The study employed both in vivo and in vitro approaches to assess the impact of CPS1 modulation on glucagon response. CPS1 was knockdown or overexpression to evaluate its influence on hepatic gluconeogenesis. In addition, an in-silico strategy was employed to identify a potential CPS1 inhibitor. Results: Knockdown of CPS1 significantly reduced the glucagon response both in vivo and in vitro. Conversely, overexpression of CPS1 resulted in an overactive hepatic gluconeogenic response. Mechanistically, CPS1 induced the release of calcium ions from the endoplasmic reticulum, which in turn triggered the phosphorylation of CaMKII. The activation of CaMKII then facilitated the dephosphorylation and nuclear translocation of FOXO1, culminating in the enhancement of hepatic gluconeogenesis. Furthermore, cynarin, a natural CPS1 inhibitor derived from the artichoke plant, had the capacity to attenuate the hepatic glucagon response in a CPS1-dependent manner. Discussion: CPS1 played a pivotal role in mediating glucagon-induced hepatic gluconeogenesis. The discovery of cynarin as a natural inhibitor of CPS1 suggested its potential as a therapeutic agent for diabetes treatment.

Long-term follow-up of children with carbamoyl phosphate synthase 1 deficiency detected in newborn screening.

To investigate genotype-phenotype characteristics and long-term prognosis of neonatal carbamoyl phosphate synthetase 1 (CPS1) deficiency among children through newborn screening in Zhejiang province. The clinical and follow-up data of children with CPS1 deficiency detected through neonatal screening and confirmed by tandem mass spectrometry and genetic testing in Zhejiang Province Newborn Disease Screening Center from September 2013 to August 2023 were retrospectively analyzed. A total of 4 056 755 newborns were screened and 6 cases of CPS1 deficiency were diagnosed through phenotypic and genetic testing. Ten different variations of CPS1 genewere identified in genetic testing, including 2 known pathogenic variations (c.2359C>T and c.1549+1G>T) and 8 unreported variations (c.3405-1G>T, c.2372C>T, c.1436C>T, c.2228T>C, c.2441G>A, c.3031G>A, c.3075T>C and c.390-403del). All patients had decreased citrulline levels (2.72-6.21 μmol/L), and varying degrees of elevated blood ammonia. The patients received restricted natural protein intake (special formula), arginine and supportive therapy after diagnosis, and were followed-up for a period ranging from 9 months to 10 years. Three patients experienced hyperammonemia, and one patient each had attention deficit hyperactivity disorder, transient facial twitching and increased muscle tone. One patient died, while the other five surviving patients had normal scores of the Ages & Stages Questionnaires (ASQ) and Griffiths Development Scales up to the present time; 4 cases had combined height or weight lag and one case was normal in height and weight. Low citrulline levels and hyperammonemia are common in CPS1 deficiency patients in Zhejiang. Most gene variants identified were specific to individual families, and no hotspot mutations were found. Early diagnosis through newborn screening and following standardized treatment can significantly improve the prognosis of the patients.

First specific detection and validation of tomato wilt caused by Fusarium brachygibbosum using a PCR assay.

Tomato wilt is a widespread soilborne disease of tomato that has caused significant yield losses in many tomato growing regions of the world. Previously, it was reported that tomato wilt can be caused by many pathogens, such as Fusarium oxysporum, Ralstonia solanacearum, Ralstonia pseudosolanacearum, Fusarium acuminatum, and Plectosphaerella cucumerina. In addition, we have already reported that Fusarium brachygibbosum caused symptomatic disease of tomato wilt for the first time in China. The symptoms of tomato wilt caused by these pathogens are similar, making it difficult to distinguish them in the field. However, F. brachygibbosum specific identification method has not been reported. Therefore, it is of great importance to develop a rapid and reliable diagnostic method for Fusarium brachygibbosum to establish a more effective plan to control the disease. In this study, we designed F. brachygibbosum-specific forward primers and reverse primers with a fragment size of 283bp located in the gene encoding carbamoyl phosphate synthase arginine-specific large chain by whole genome sequence comparison analysis of the genomes of eight Fusarium spp.. We then tested different dNTP, Mg2+ concentrations, and annealing temperatures to determine the optimal parameters for the PCR system. We evaluated the specificity, sensitivity and stability of the PCR system based on the optimized reaction system and conditions. The PCR system can specifically identify the target pathogens from different fungal pathogens, and the lower detection limit of the target pathogens is at concentrations of 10 pg/uL. In addition, we can accurately identify F. brachygibbosum in tomato samples using the optimized PCR method. These results prove that the PCR method developed in this study can accurately identify and diagnose F. brachygibbosum.

Evolutionary history of a cold-adapted limnephilid caddisfly: Effects of climate change and topography on genetic structure

The distribution of organisms is influenced by complex factors such as the phylogenetic evolutionary histories of species, the physiological and ecological characteristics of organisms, climate, and geographical and geohistorical features. In this study, we focused on a caddisfly, Asynarchus sachalinensis (Trichoptera: Limnephilidae), which has adapted to cold habitats. From phylogeographic analyses based on the mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) and 16S rRNA regions and the nuclear DNA (nDNA) 18S rRNA, 28S rRNA, carbamoyl-phosphate synthetase (CAD), elongation factor-1 alpha (EF1-α), and RNA polymerase II (POLII) regions, two distinct genetic clades were detected. Clade I was shown to be widely distributed from Sakhalin to Honshu, whereas Clade II was only distributed within Honshu. The distributions of these clades overlapped in Honshu. The habitats were located at relatively lower altitudes for Clade I and higher altitudes for Clade II. The divergence time of these clades was estimated to be during the Pleistocene, indicating that repeated climatic changes facilitated distributional shifts. Haplotype network and demographic analyses based on the mtDNA COI region showed contrasting genetic structures in the two clades. It was indicated that the population sizes of Clade I had expanded rapidly in a recent period, whereas Clade II had maintained stable population sizes. The habitats of Clade II were typically isolated and scattered at high altitudes, resulting in restricted migration and dispersal because of their discontinuous “Sky Island” habitats. The habitats of Clade I were located at relatively low altitudes, and it was assumed that the populations were continuous, which resulted in a higher frequency of migration and dispersal between populations. Thus, differences in the spatial scale of the adapted habitats of each clade may have resulted in different patterns of population connectivity and fragmentation associated with repeated climatic changes during the Pleistocene. Our study provided new insight into the distributional patterns of cold-adapted aquatic insects in the Japanese Archipelago. Furthermore, the distributional shifts predicted by ecological niche modeling under future climatic change conditions were different for each clade. Therefore, different principles are required in the assessment of each clade to predict temporal changes in their distributions.

TMET-17. LEVERAGING METABOLIC VULNERABILITIES INDUCED BY TERT EXPRESSION FOR GLIOBLASTOMA THERAPY IN VIVO

Abstract Telomerase reverse transcriptase (TERT) is essential for telomere maintenance and tumor immortality. Although TERT is an attractive therapeutic target, targeting TERT directly is hampered by toxicity to stem cells that also rely on TERT for proliferation and the lag period before telomere attrition is observed. Targeting metabolic reprogramming associated with TERT is an alternate strategy. Our studies indicate that TERT is essential for redox homeostasis in glioblastoma cells (GBM; GBM6 and U251). Specifically, TERT increases levels of glutathione (GSH) via upregulation of the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate-limiting enzyme in de novo GSH synthesis. Inhibiting GCLC using buthionine sulfoximine (BSO) or siRNA reduces synthesis of 13C-GSH from [U-13C]-glutamine and inhibits viability of GBM6 and U251 cells. However, GCLC inhibition causes a compensatory increase in synthesis of glutamate, aspartate, and pyrimidine nucleotides from [U-13C]-glutamine via upregulation of two key glutamine-utilizing enzymes i.e., glutaminase (GLS), the enzyme that generates glutamate from glutamine, and carbamoyl phosphate synthetase II (CPSII), the enzyme that converts glutamine to carbamoyl phosphate during pyrimidine biosynthesis. We then examined the therapeutic potential of inhibiting GLS and CPSII in combination with GCLC in GBMs. 6-diazo-5-oxy-L-norleucin (DON), a potent inhibitor of glutamine-utilizing enzymes including GLS and CPSII, synergizes with BSO in inhibiting viability of GBM6 and U251 cells. This effect is specific to TERT-expressing GBM cells because the combination of DON and BSO does not affect viability of normal astrocytes or astrocytomas that use the ALT pathway for telomere maintenance. Importantly, in vivo stable isotope tracing confirmed that combined treatment with JHU-083 (a brain-penetrant prodrug of DON) and BSO abrogates synthesis of GSH, aspartate, and pyrimidine nucleotides from [U-13C]-glutamine and induces apoptotic death in mice bearing intracranial GBM6 tumors.Collectively, our studies highlight the therapeutic potential of targeting metabolic vulnerabilities induced by TERT expression in GBMs in vivo.

Abstract 14779: Whole Exome Sequencing Study in Pediatric Postural Orthostatic Tachycardia Syndrome Unveils a Path Forward From Genes to Drugs

Introduction: Postural orthostatic tachycardia syndrome (POTS) is more common in Caucasian individuals with a positive family history, while the risk in females is ~4-times of that in males. To date, the etiology of POTS is poorly understood. Hypothesis: Integrative sequencing and translational approaches to identify its genetic underpinning may better understand its pathogenesis and molecular mechanisms for the development of novel precision-based therapies. Methods: We adopted exome sequencing to evaluate the etiology and molecular mechanisms of POTS. We examined a unique collection of 87 unrelated pediatric POTS cases (61 females and 26 males, 6~21 years old), using exome sequencing. Non-relationship between individuals were validated by genome wide genotyping and identity by descent (IBD) analysis. Results: All the individuals were classified as European by principal component analysis (Figure 1). We identified 184 pathogenic (P) or likely pathogenic (LP) variants (in 162 genes). Sixty-eight (78%) cases have at least one P/LP variant. Each P/LP mutation is supported by a minimum of two databases, including ClinVar, InterVar or HGMD_Pro_2023.1. Over-representation analysis (ORA) by the DisGeNET approach showed the enriched gene sets of muscle hypotonia and epilepsy/seizures with statistical significance (FDR<E-10). Clinically, among the 87 WES patients, 41 (47%) suffered from chronic fatigue, and 13 (15%) patients have the symptoms of epilepsy, seizure or convulsion. A number of drug targets in these gene sets are highlighted, including vitamin D receptor, carbamoyl-phosphate synthase 1, methylmalonyl-CoA mutase, androgen receptor, and phenylalanine hydroxylase. Conclusions: Our results suggests that chronic fatigue and epilepsy/seizure symptoms are comorbid symptoms in POTS patients, resulting from common genetic defects. Future precision-based therapies targeting these specific genetic defects may lead to improved outcome in POTS.

Sericin enhances ammonia detoxification by promotes urea cycle enzyme genes and activates hepatic autophagy in relation to CARD-9/MAPK pathway

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.

Cyclin D1 extensively reprograms metabolism to support biosynthetic pathways in hepatocytes

Cell proliferation requires metabolic reprogramming to accommodate biosynthesis of new cell components, and similar alterations occur in cancer cells. However, the mechanisms linking the cell cycle machinery to metabolism are not well defined. Cyclin D1, along with its main partner cyclin-dependent kinase 4 (Cdk4), is a pivotal cell cycle regulator and driver oncogene that is overexpressed in many cancers. Here, we examine hepatocyte proliferation to define novel effects of cyclin D1 on biosynthetic metabolism. Metabolomic studies reveal that cyclin D1 broadly promotes biosynthetic pathways including glycolysis, the pentose phosphate pathway, and purine and pyrimidine nucleotide synthesis in hepatocytes. Proteomic analyses demonstrate that overexpressed cyclin D1 binds to numerous metabolic enzymes including those involved in glycolysis and pyrimidine synthesis. In the glycolysis pathway, cyclin D1 activates aldolase and GAPDH, and these proteins are phosphorylated by cyclin D1/Cdk4 in vitro. De novo pyrimidine synthesis is particularly dependent on cyclin D1. Cyclin D1/Cdk4 phosphorylates the initial enzyme of this pathway, CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase), and metabolomic analysis indicates that cyclin D1 depletion markedly reduces the activity of this enzyme. Pharmacologic inhibition of Cdk4 along with the downstream pyrimidine synthesis enzyme DHODH (dihydroorotate dehydrogenase) synergistically inhibits proliferation and survival of hepatocellular carcinoma cells. These studies demonstrate that cyclin D1 promotes a broad network of biosynthetic pathways in hepatocytes, and this model may provide insights into potential metabolic vulnerabilities in cancer cells.

Effects of Ambient Ammonia‐Nitrogen Exposure on Uric Acid and Urea Metabolic Pathways and Tissue Distribution in the Swimming Crab Portunus trituberculatus

AbstractAmmonia‐nitrogen (ammonia‐N) is an important threat factor to the development of the crustacean breeding industry; however, information on nitrogen metabolism remains limited. In this study, effects of ammonia‐N on the metabolic pathways and tissue distribution of nitrogenous wastes were investigated in swimming crabs Portunus trituberculatus. The results showed that ammonia content in the hemolymph of crabs exposed to 1‐ and 5‐mg/L NH4Cl (ammonia‐N concentrations were 1.36 and 5.31 mg/L, respectively) significantly (P < 0.05) increased with time. The expression of xanthine oxidase was significantly up‐regulated in gills and muscles but was significantly down‐regulated in the hepatopancreas of crabs exposed to 1‐ and 5‐mg/L NH4Cl after 6–12 h. The expression of uricase was significantly up‐regulated in gills and muscles at 3 h, whereas it was significantly down‐regulated in hepatopancreas during 6–12 h but up‐regulated again after 24 h. Moreover, the expression levels of carbamoyl phosphate synthetase III and arginase were significantly up‐regulated in gills and muscles but significantly down‐regulated in the hepatopancreas of crabs exposed to 5‐mg/L NH4Cl after 6–12 h. Furthermore, under 5‐mg/L NH4Cl stress, trace amounts of uric acid were detected, which significantly increased in hepatopancreas and hemolymph at 3 h, in muscles at 6–12 h, and in gills at 6–24 h. In addition, similar to hemolymph ammonia, urea was also significantly increased after NH4Cl exposure. Moreover, uric acid was mainly stored in muscles and urea was primarily synthesized in the hepatopancreas. This study provides a theoretical basis for further study on the nitrogen metabolism mechanism in crustaceans.

Phylogenetic relationships of the woodlouse flies (Diptera: Rhinophorinae) and the cluster flies (Diptera: Polleniidae).

Phylogenetic relationships within the oestroid subclades Rhinophorinae (Calliphoridae) and Polleniidae were reconstructed for the first time, applying a Sanger sequencing approach using the two protein-coding nuclear markers CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase; 1794 bp) and MCS (molybdenum cofactor sulfurase; 2078 bp). Three genera of Polleniidae and nineteen genera of Rhinophorinae were analyzed together with a selection of taxa representing the major lineages of Oestroidea (non-rhinophorine Calliphoridae, Oestridae, Sarcophagidae, Tachinidae). The selected markers provide good resolution and moderate to strong support of the distal branches, but weak support for several deeper nodes. Polleniidae (cluster flies) emerge as monophyletic and their sister-group relationship to Tachinidae is confirmed. Morinia Robineau-Desvoidy as currently circumscribed emerges as paraphyletic with regard to Melanodexia Williston, and Pollenia Robineau-Desvoidy is the sister taxon of the Morinia-Melanodexia clade. We propose a classification with two subfamilies, Moriniinae Townsend (including Morinia, Melanodexia, and Alvamaja Rognes), and Polleniinae Brauer & Bergenstamm (including Pollenia, Dexopollenia Townsend, and Xanthotryxus Aldrich). Anthracomyza Malloch and Nesodexia Villeneuve are considered as Oestroidea incertae sedis pending further study. Rhinophorinae (woodlouse flies) emerge as monophyletic and sister to a clade composed of (Ameniinae + (Ameniinae + Phumosiinae)), and a tribal classification is proposed with the subfamily divided into Rhinophorini Robineau-Desvoidy, 1863 and Phytonini Robineau-Desvoidy, 1863 (the Stevenia-group and the Phyto-group of authors, respectively). Oxytachina Brauer & Bergenstamm, 1891, stat. rev. is resurrected to contain nine Afrotropical rhinophorine species currently assigned to genus Rhinomorinia Brauer & Bergenstamm, 1891: Oxytachina approximata (Crosskey, 1977) comb. nov., O. atra (Bischof, 1904) comb. nov., O. bisetosa (Crosskey, 1977) comb. nov., O. capensis (Brauer & Bergenstamm, 1893) comb. nov., O. scutellata (Crosskey, 1977) comb. nov., O. setitibia (Crosskey, 1977) comb. nov., O. verticalis (Crosskey, 1977) comb. nov., O. vittata Brauer & Bergenstamm, 1891, and O. xanthocephala (Bezzi, 1908) comb. nov.

Identification of virulence-associated factors in Vibrio parahaemolyticus with special reference to moonlighting protein: a secretomics study.

Vibrio parahaemolyticus causes seafood-borne gastroenteritis infection in human which can even lead to death. The pathogenic strain of V. parahaemolyticus secretes different types of virulence factors thatare directly injected into the host cell by a different type of secretion system which helps bacteria to establish its own ecological niche within the organism. Therefore, the aim of this study was to isolate the extracellular secreted proteins from the trh positive strain of V. parahaemolyticus and identify them using two-dimensional gel electrophoresis and MALDI-TOFMS/MS. Seventeen different cellular proteins viz, Carbamoyl-phosphate synthase, 5-methyltetrahydropteroyltriglutamate, tRNA-dihydrouridine synthase, Glycerol-3-phosphate dehydrogenase, Orotidine 5'-phosphate decarboxylase, Molybdenum import ATP-binding protein, DnaJ, DNA polymerase IV, Ribosomal RNA small subunit methyltransferase G, ATP synthase subunit delta and gamma, Ribosome-recycling factor, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, tRNA pseudouridine synthase B, Ditrans, polycis-undecaprenyl-diphosphate synthase, Oxygen-dependent coproporphyrinogen-III oxidase, and Peptide deformylase 2 were identified whichare mainly involved in different metabolic and biosynthetic pathways. Furthermore, the molecular function of the identified proteins were associated with catalytic activity, ligase activity, transporter, metal binding, and ATP synthase when they are intercellular. However, to understand the importance of these secreted proteins in the infection and survival of bacteria inside the host cell, pathogen-host protein-protein interactions (PPIs) were carried out which identified the association of eight secreted proteins with 41 human proteins involved in different cellular pathways, including ubiquitination degradation, adhesion, inflammation, immunity, and programmed cell death. The present study provides unreported strategies on host-cell environment's survival and adaptation mechanisms for the successful establishment of infections and intracellular propagation.

Feeding strategies for rearing replacement beef heifers in a grazing system under tropical conditions

Our objective was to evaluate the effects of pre- and post-weaning protein supplement on the productive response and nutritional and metabolic status of replacement beef heifers grazed on tropical pasture. Fifty-six Nellore heifers (7.7 ± 0.1 months age) with an average initial body weight (BW) of 237 ± 3.5 kg were distributed in a completely randomized design in 2 × 2 factorial arrangement. The four strategies were: US-ME, unsupplemented heifers at pre-weaning and supplemented with 5 g/kg BW at post-weaning; US-HI, unsupplemented heifers at pre-weaning and supplemented with 7.5 g/kg BW at post-weaning; ME-ME, heifers supplemented with 5 g/kg BW at pre-weaning and post-weaning; and ME-HI, heifers supplemented with 5 g/kg BW at pre-weaning and 7.5 g/kg BW at post-weaning. On average, crude protein (CP) intake and CP: digested organic matter (DOM) ratio were influenced (P < 0.05) by protein supplement in the post-weaning period, with a higher intake of CP and CP:DOM ratio in HI heifers than in ME heifers (average CP of 0.812 vs 0.665 kg/day and average DOM of 204 vs 174 g CP/kg). Nonetheless, the post-weaning protein supplement did not affect the intake of total dry matter (DM), pasture DM, organic matter (OM), neutral detergent fiber corrected for ash and protein residue (apNDF), non-fibrous carbohydrates, and DOM. The CP digestibility was highest (P = 0.005) for HI heifers than for ME heifers (average of 0.732 vs 0.645 g/g DM). The pre-weaning and post-weaning protein supplement did not affect the concentration of insulin-like growth factor–1 (IGF-1), progesterone, albumin, globulins, glucose, and triglycerides, and liver mRNA expression of carbamoyl phosphate synthetase-1 (CPS-1) and phosphoenolpyruvate carboxykinase (PEPCK). Finally, the pre-weaning and post-weaning protein supplement did not affect growth performance, carcass traits, and body growth in the post-weaning. Supplementation during pre-weaning does not bring any carry-over effect on the productive performance and metabolic status of heifers supplemented during post-weaning. However, supplementation in post-weaning with 5 or 7.5 g/kg BW allows a compensatory growth of heifers that are unsupplemented at pre-weaning. Therefore, the supply of 7.5 g/kg BW of protein supplement for beef heifers only during the post-weaning period is recommended.

Limited role for hyperammonemia in the progression of diet-induced nonalcoholic steatohepatitis.

To determine whether hyperammonemia has a direct impact on steatohepatitis in mice fed with a high-fat diet (HFD). Male C57BL/6 mice were divided into two groups receiving either chow diet or HFD. After 12-week NASH modeling, hyperammonemia was induced by intragastric administration of ammonium chloride solution (NH4 Cl) or liver-specific carbamoyl phosphate synthetase 1 (Cps1) knockdown. In vitro experiments were performed in HepG2 cells induced by free fatty acid (FFA) and NH4 Cl. NH4 Cl administration led to increased levels of plasma and hepatic ammonia in NASH mice. NH4 Cl-induced hyperammonemia did not influence liver histological changes in mice fed with HFD; however, elevated plasma cholesterol level, and an increasing trend of liver lipid content were observed. No significant effect of hyperammonemia on hepatic inflammation and fibrosis in NASH mice was found. In vitro cell experiments showed that NH4 Cl treatment failed to increase the lipid droplet content and the expressions of de novo lipogenesis genes in HepG2 cells induced by FFA. The knockdown of Cps1 in HFD-fed mice resulted in elevated plasma ammonia levels but did not cause histological change in the liver. Our study revealed a limited role of ammonia in aggravating the progression of NASH. Further studies are needed to clarify the role and mechanism of ammonia in NASH development.

Expression of enzymes involved in the urea cycle and muscle and mammary gland development of Holstein × Gyr heifers in a rotational grazing system supplemented with increasing protein levels

Studies evaluating the crude protein (CP) supplementation strategies across the year for grazing cattle and its association with the enzymes involved in the urea cycle and muscle and mammary gland developments are scarce. Thus, we aimed to evaluate the effect of supplementation with different levels of CP on the expression of genes involved in the urea cycle and muscle and mammary gland development of Holstein × Gyr crossbreed heifers grazing intensively managed Brachiaria decumbens throughout the year. Thirty-eight heifers with average initial BW of 172.5 ± 11.15 kg (mean ± SE) and 8.2 ± 0.54 mo of age were randomly assigned to 1 of 4 treatments: 3 protein supplements (SUP) fed at 5g/kg of body weight, plus a control group (CON, non-supplemented animals). The supplement CP levels evaluated were: 12, 24, and 36%. The study was divided into 4 seasons: rainy, dry, rainy-dry transition (RDT), and dry-rainy transition (DRT). On the penultimate day of each season, ultrasound images of the carcass and mammary gland were taken. Five animals from each treatment were randomly chosen on the last day of each season, and liver and muscle tissue biopsies were performed. The target genes were the mammalian target of rapamycin (mTOR) and adenosine monophosphate-activated protein kinase (AMPK) in the muscle samples. Carbamoyl phosphate synthetase (CPS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), arginosuccinate lyase (ASL), and arginase (ARG) were evaluated in the liver samples. Data were analyzed using PROC GLIMMIX of the SAS with repeated measures. We observed a greater rib eye area (cm2) and fat thickness (mm) in SUP animals than in non-supplemented animals. However, we did not observe differences among SUP levels for both variables. No effects of supplementation were detected on mammary gland development. Nevertheless, seasonal effects were observed, where the RDT and dry season had the most and least accumulated fat in the mammary gland. In muscle, we observed greater expression of AMPK in non-supplemented animals than SUP animals. On the other hand, no differences were observed in gene expression between SUP and non-supplemented animals and among SUP animals for mTOR. Season affected both AMPK and mTOR; heifers had a greater AMPK gene expression on rainy than RDT. For mTOR, we observed greater gene expression in RDT and DRT than in rainy. No differences were observed among RDT, dry, and DRT, and between dry and rainy seasons for mTOR. We observed greater CPS, ASL, and ARG gene expression in SUP animals than in non-supplemented animals. Among SUP animals, supplement CP linearly affected CPS. In conclusion, the supplementation strategy did not affect mammary gland development and mTOR expression in muscle tissue. However, we observed a seasonal effect on mammary gland development and AMPK and mTOR expression. The CP supplementation increased the rib eye area and fat thickness, directly affecting AMPK expression in the muscle. Moreover, the CP supplementation increased urea cycle enzyme expression, indicating greater urea production in the liver.

Hypoosmosis alters hepatocyte mitochondrial morphology and induces selective release of carbamoyl phosphate synthetase 1.

Carbamoyl phosphate synthetase 1 (CPS1) is the most abundant hepatocyte mitochondrial matrix protein. Hypoosmotic stress increases CPS1 release in isolated mouse hepatocytes without cell death. We hypothesized that increased CPS1 release during hypoosmosis is selective and associates with altered mitochondrial morphology. Both ex vivo and in vivo models were assessed. Mouse hepatocytes and livers were challenged with isotonic or hypoosmotic (35 mosM) buffer. Mice were injected intraperitoneally with water (10% body weight) with or without an antidiuretic. Mitochondrial and cytosolic fractions were isolated using differential centrifugation, then analyzed by immunoblotting to assess subcellular redistribution of four mitochondrial proteins: CPS1, ornithine transcarbamylase (OTC), pyrroline-5-carboxylate reductase 1 (PYCR1), and cytochrome c. Mitochondrial morphology alterations were examined using electron microscopy. Hypoosmotic treatment of whole livers or hepatocytes led to preferential or increased mitochondrial release, respectively, of CPS1 as compared with two mitochondrial matrix proteins (OTC/PYCR1) and with the intermembrane space protein, cytochrome c. Mitochondrial apoptosis-induced channel opening using staurosporine in hepatocytes led to preferential CPS1 and cytochrome c release. The CPS1-selective changes were accompanied by dramatic alterations in ultrastructural mitochondrial morphology. In mice, hypoosmosis/hyponatremia led to increased liver vascular congestion and increased CPS1 in bile but not blood, coupled with mitochondrial structural alterations. In contrast, isotonic increase of intravascular volume led to a decrease in mitochondrial size with limited change in bile CPS1 compared with hypoosmotic conditions and absence of the hypoosmosis-associated histological alterations. Taken together, hepatocyte CPS1 is selectively released in response to hypoosmosis/hyponatremia and provides a unique biomarker of mitochondrial injury.NEW & NOTEWORTHY Exposure of isolated mouse livers, primary cultured hepatocytes, or mice to hypoosmosis/hyponatremia conditions induces significant mitochondrial shape alterations accompanied by preferential release of the mitochondrial matrix protein CPS1, a urea cycle enzyme. In contrast, the intermembrane space protein, cytochrome c, and two other matrix proteins, including the urea cycle enzyme ornithine transcarbamylase, remain preferentially retained in mitochondria. Therefore, hepatocyte CPS1 manifests unique mitochondrial stress response compartmentalization and is a sensitive sensor of mitochondrial hypoosmotic/hyponatremic injury.

Citrulline in the management of patients with urea cycle disorders

BackgroundTreatment recommendations for urea cycle disorders (UCDs) include supplementation with amino acids involved in the urea cycle (arginine and/or citrulline, depending on the enzyme deficiency), to maximize ammonia excretion through the urea cycle, but limited data are available regarding the use of citrulline. This study retrospectively reviewed clinical and biological data from patients with UCDs treated with citrulline and/or arginine at a reference center since 1990. The aim was to describe the prescription, impact, and safety of these therapies. Data collection included patient background, treatment details, changes in biochemical parameters (plasma ammonia and amino acids concentrations), decompensations, and patient outcomes.ResultsOverall, 79 patients (median age at diagnosis, 0.9 months) received citrulline and/or arginine in combination with a restricted protein diet, most with ornithine transcarbamylase (n = 57, 73%) or carbamoyl phosphate synthetase 1 (n = 15, 19%) deficiencies. Most patients also received ammonium scavengers. Median follow-up was 9.5 years and median exposure to first treatment with arginine + citrulline, citrulline monotherapy, or arginine monotherapy was 5.5, 2.5, or 0.3 years, respectively. During follow-up, arginine or citrulline was administered at least once (as monotherapy or in combination) in the same proportion of patients (86.1%); the overall median duration of exposure was 5.9 years for arginine + citrulline, 3.1 years for citrulline monotherapy, and 0.6 years for arginine monotherapy. The most common switch was from monotherapy to combination therapy (41 of 75 switches, 54.7%). During treatment, mean ammonia concentrations were 35.9 µmol/L with citrulline, 49.8 µmol/L with arginine, and 53.0 µmol/L with arginine + citrulline. Mean plasma arginine concentrations increased significantly from the beginning to the end of citrulline treatment periods (from 67.6 µmol/L to 84.9 µmol/L, P < 0.05). At last evaluation, mean height and weight for age were normal and most patients showed normal or adapted behavior (98.7%) and normal social life (79.0%). Two patients (2.5%) experienced three treatment-related gastrointestinal adverse reactions.ConclusionsThis study underlines the importance of citrulline supplementation, either alone or together with arginine, in the management of patients with UCDs. When a monotherapy is considered, citrulline would be the preferred option in terms of increasing plasma arginine concentrations.

Underexpression of Carbamoyl Phosphate Synthetase I as Independent Unfavorable Prognostic Factor in Intrahepatic Cholangiocarcinoma: A Potential Theranostic Biomarker.

Intrahepatic cholangiocarcinoma (IHCC) is the second most common malignant neoplasm of the liver. In spite of the increasing incidence worldwide, it is relatively rare in Western countries. IHCC is relatively common in Eastern and Southeastern Asia. Patients with IHCC are usually diagnosed at an advanced stage, therefore, the clinical outcome is dismal. Dysregulation of urea cycle metabolic enzyme expression is found in different types of cancers. Nevertheless, a comprehensive evaluation of genes related to the urea cycle (i.e., GO:0000050) has not been conducted in IHCC. By performing a comparative analysis of gene expression profiles, we specifically examined genes associated with the urea cycle (GO:0000050) in a publicly accessible transcriptomic dataset (GSE26566). Interestingly, CPS1 was identified as the second most prominently down-regulated gene in this context. Tumor tissues of 182 IHCC patients who underwent curative-intent hepatectomy were enrolled. The expression level of CPS1 protein in our IHCC cohort was assessed by immunohistochemical study. Subsequent to that, statistical analyses were carried out to examine the expression of CPS1 in relation to various clinicopathological factors, as well as to assess its impact on survival outcomes. We noticed that lower immunoreactivity of CPS1 in IHCC was associated with tumor progression (pT status) with statistical significance (p = 0.003). CPS1 underexpression was not only negatively correlated to overall survival (OS), disease-free survival (DFS), local recurrence-free survival (LRFS) and metastasis-free survival (MeFS) in univariate analysis but also an independent prognosticator to forecast poorer clinical outcome for all prognostic indices (OS, DFS, LRFS and MeFs) in patients with IHCC (all p ≤ 0.001). These results support that CPS1 may play a crucial role in IHCC oncogenesis and tumor progression and serve as a novel prognostic factor and a potential diagnostic and theranostic biomarker.

Clinical and genetic analysis of a case of late onset carbamoyl phosphate synthase I deficiency caused by CPS1 mutation and literature review

BackgroundCarbamoyl phosphate synthetase I defect (CPS1D) is a rare disease with clinical case reports mainly in early neonates or adults, with few reports of first onset in late neonatal to childhood. We studied the clinical and genotypic characteristics of children with childhood onset CPS1D caused by two loci mutations (one of these is a rarely reported non-frame shift mutation) in the CPS1.Case presentationWe present a rare case of adolescent-onset CPS1D that had been misdiagnosed due to atypical clinical features, and further investigations revealed severe hyperammonemia (287µmol/L; reference range 11.2 ~ 48.2umol/L). MRI of the brain showed diffuse white matter lesions. Blood genetic metabolic screening showed elevated blood alanine (757.06umol/L; reference range 148.8 ~ 739.74umol/L) and decreased blood citrulline (4.26umol/L; reference range 5.45 ~ 36.77umol/L). Urine metabolic screening showed normal whey acids and uracil. Whole-exome sequencing revealed compound heterozygous mutations in the CPS1, a missense mutation (c.1145 C > T) and an unreported de novo non-frame shift mutation (c.4080_c.4091delAGGCATCCTGAT), respectively, which provided a clinical diagnosis.ConclusionA comprehensive description of the clinical and genetic features of this patient, who has a rare age of onset and a relatively atypical clinical presentation, will facilitate the early diagnosis and management of this type of late onset CPS1D and reduce misdiagnosis, thus helping to reduce mortality and improve prognosis. It also provides a preliminary understanding of the relationship between genotype and phenotype, based on a summary of previous studies, which reminds us that it may help to explore the pathogenesis of the disease and contribute to genetic counselling and prenatal diagnosis.