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Carbamoyl phosphate synthetase 1 (CPS1) is primarily expressed in hepatocytes as a highly abundant mitochondrial matrix enzyme that catalyses the first step of the urea cycle that leads to renal nitrogen disposal. CPS1 is a member of the CPS family that manifests broad evolutionary expression from bacteria to humans. CPS1 expression and enzyme activity are highly regulated transcriptionally and post-translationally. Its autosomal recessive mutation leads to CPS1 deficiency, which causes encephalopathy and coma, typically neonatally, due to severe hyperammonaemia. CPS1 is physiologically secreted, apically, into bile likely via mitochondria-derived vesicles. Normally absent from serum, it is released by basolateral mistargeting and cellular injury and becomes readily detectable in serum during acute liver failure (ALF). Injury-triggered CPS1 release into blood, or media in cultured hepatocytes, is selective as compared with other mitochondrial proteins. This, coupled with its abundance and short (1-2 hours) serum half-life, renders it a prognostic serum biomarker, particularly in human acetaminophen-related ALF. Its rapid turnover is explained by its non-enzymatic role as an immune modulator via its uptake by circulating monocytes leading to differentiation of anti-inflammatory cells that home to, and protect, the injured liver. CPS1 also plays a growing role in several cancers, by CPS1 upregulation or downregulation, particularly via metabolic reprogramming which alters the tumour microenvironment and impacts cancer growth and progression. Therefore, CPS1 has multiple enzymatic and non-enzymatic touch points spanning a wide range of cellular and extracellular functions and roles, with important physiological, homoeostatic, genetic disease, diagnostic and potential therapeutic clinical implications. Show less

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. Show less

Carbamoyl phosphate synthetase 1 (CPS1) is a highly abundant mitochondrial urea cycle enzyme that is expressed primarily in hepatocytes. CPS1 is constitutively and physiologically secreted into bile but is released into the bloodstream upon acute liver injury (ALI). Given its abundance and known short half-life, we tested the hypothesis that it may serve as a prognostic serum biomarker in the setting of acute liver failure (ALF). CPS1 levels were determined using enzyme-linked immunosorbent assay and immunoblotting of sera collected by the ALF Study Group (ALFSG) from patients with ALI and ALF (103 patients with acetaminophen and 167 non-acetaminophen ALF etiologies). A total of 764 serum samples were examined. The inclusion of CPS1 was compared with the original ALFSG Prognostic Index by area under the receiver operating characteristic curve analysis. CPS1 values for acetaminophen-related patients were significantly higher than for non-acetaminophen patients (P < .0001). Acetaminophen-related patients who received a liver transplant or died within 21 days of hospitalization exhibited higher CPS1 levels than patients who spontaneously survived (P = .01). Logistic regression and area under the receiver operating characteristic analysis of CPS1 enzyme-linked immunosorbent assay values improved the accuracy of the ALFSG Prognostic Index, which performed better than the Model for End-Stage Liver Disease, in predicting 21-day transplant-free survival for acetaminophen- but not non-acetaminophen-related ALF. An increase of CPS1 but not alanine transaminase or aspartate transaminase, when comparing day 3 with day 1 levels was found in a higher percentage of acetaminophen transplanted/dead patients (P < .05). Serum CPS1 determination provides a new potential prognostic biomarker to assess patients with acetaminophen-induced ALF. Show less

Carbamoyl phosphate synthetase-1 (CPS1) is the major mitochondrial urea cycle enzyme in hepatocytes. It is released into mouse and human blood during acute liver injury, where is has a short half-life. The function of CPS1 in blood and the reason for its short half-life in serum are unknown. We show that CPS1 is released normally into mouse and human bile, and pathologically into blood during acute liver injury. Other cytoplasmic and mitochondrial urea cycle enzymes are also found in normal mouse bile. Serum, bile, and purified CPS1 manifest sedimentation properties that overlap with extracellular vesicles, due to the propensity of CPS1 to aggregate despite being released primarily as a soluble protein. During liver injury, CPS1 in blood is rapidly sequestered by monocytes, leading to monocyte M2-polarization and homing to the liver independent of its enzyme activity. Recombinant CPS1 (rCPS1), but not control r-transferrin, increases hepatic macrophage numbers and phagocytic activity. Notably, rCPS1 does not activate hepatic macrophages directly; rather, it activates bone marrow and circulating monocytes that then home to the liver. rCPS1 administration prevents mouse liver damage induced by Fas ligand or acetaminophen, but this protection is absent in macrophage-deficient mice. Moreover, rCPS1 protects from acetaminophen-induced liver injury even when given therapeutically after injury induction. In summary, CPS1 is normally found in bile but is released by hepatocytes into blood upon liver damage. We demonstrate a nonenzymatic function of CPS1 as an antiinflammatory protective cytokine during acute liver injury. Show less

Alcohol liver disease (ALD) is one of the major chronic liver diseases worldwide, ranging from fatty liver, alcoholic hepatitis, cirrhosis, and potentially, hepatocellular carcinoma. Epidemiological studies suggest a potential link between ALD and impaired circadian rhythms, but the role of hepatic circadian proteins in the pathogenesis of ALD remains unknown. Here we show that the circadian clock protein BMAL1 in hepatocytes is both necessary and sufficient to protect mice from ALD. Ethanol diet-fed mice with liver-specific knockout (Bmal1-LKO) or depletion of Bmal1 develop more severe liver steatosis and injury as well as a simultaneous suppression of both de novo lipogenesis and fatty acid oxidation, which can be rescued by the supplementation of synthetic PPARα ligands. Restoring de novo lipogenesis in the liver of Bmal1-LKO mice by constitutively active AKT not only elevates hepatic fatty acid oxidation but also alleviates ethanol-induced fatty liver and liver injury. Furthermore, hepatic over-expression of lipogenic transcription factor ChREBP, but not SREBP-1c, in the liver of Bmal1-LKO mice also increases fatty acid oxidation and partially reduces ethanol-induced fatty liver and liver injury. Conclusion: we identified a protective role of BMAL1 in hepatocytes against ALD. The protective action of BMAL1 during alcohol consumption depends on its ability to couple ChREBP-induced de novo lipogenesis with PPARα-mediated fatty oxidation. (Hepatology 2018). Show less

Epidemiologic and animal studies implicate overconsumption of fructose in the development of nonalcoholic fatty liver disease, but the molecular mechanisms underlying fructose-induced chronic liver diseases remain largely unknown. Here, we have presented evidence supporting the essential function of the lipogenic transcription factor carbohydrate response element-binding protein (ChREBP) in mediating adaptive responses to fructose and protecting against fructose-induced hepatotoxicity. In WT mice, a high-fructose diet (HFrD) activated hepatic lipogenesis in a ChREBP-dependent manner; however, in Chrebp-KO mice, a HFrD induced steatohepatitis. In Chrebp-KO mouse livers, a HFrD reduced levels of molecular chaperones and activated the C/EBP homologous protein-dependent (CHOP-dependent) unfolded protein response, whereas administration of a chemical chaperone or Chop shRNA rescued liver injury. Elevated expression levels of cholesterol biosynthesis genes in HFrD-fed Chrebp-KO livers were paralleled by an increased nuclear abundance of sterol regulatory element-binding protein 2 (SREBP2). Atorvastatin-mediated inhibition of hepatic cholesterol biosynthesis or depletion of hepatic Srebp2 reversed fructose-induced liver injury in Chrebp-KO mice. Mechanistically, we determined that ChREBP binds to nuclear SREBP2 to promote its ubiquitination and destabilization in cultured cells. Therefore, our findings demonstrate that ChREBP provides hepatoprotection against a HFrD by preventing overactivation of cholesterol biosynthesis and the subsequent CHOP-mediated, proapoptotic unfolded protein response. Our findings also identified a role for ChREBP in regulating SREBP2-dependent cholesterol metabolism. Show less

Several serum markers are used to assess hepatocyte damage, but they have limitations related to etiology specificity and prognostication. Identification of novel hepatocyte-specific biomarkers could provide important prognostic information and better pathogenesis classification. We tested the hypothesis that hepatocyte-selective biomarkers are released after subjecting isolated mouse hepatocytes to Fas-ligand-mediated apoptosis. Proteomic analysis of hepatocyte culture medium identified the mitochondrial matrix protein carbamoyl phosphate synthetase-1 (CPS1) among the most readily detected proteins that are released by apoptotic hepatocytes. CPS1 was also detected in mouse serum upon acute challenge with Fas-ligand or acetaminophen and in hepatocytes upon hypoosmotic stress, independent of hepatocyte caspase activation. Furthermore, CPS1 was observed in sera of mice chronically fed the hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Mouse CPS1 detectability was similar in serum and plasma, and its half-life was 126 ± 9 min. Immune staining showed that CPS1 localized to mouse hepatocytes but not ductal cells. Analysis of a few serum samples from patients with acute liver failure (ALF) due to acetaminophen, Wilson disease, or ischemia showed readily detectable CPS1 that was not observed in several patients with chronic viral hepatitis or in control donors. Notably, CPS1 rapidly decreased to undetectable levels in sera of patients with acetaminophen-related ALF who ultimately recovered, while alanine aminotransferase levels remained elevated. Therefore, CPS1 becomes readily detectable upon hepatocyte apoptotic and necrotic death in culture or in vivo. Its abundance and short serum half-life, compared with alanine aminotransferase, suggest that it may be a useful prognostic biomarker in human and mouse liver injury. Show less