Subcellular distribution of hepatic bile acid-conjugating enzymes.
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ABSTRACT:
SUBMITTER: Lim WC
PROVIDER: S-EPMC1163172 | biostudies-other | 1981 Sep
REPOSITORIES: biostudies-other
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Project description:To fulfill their physiological functions, bile acids are conjugated with amino acids. In humans, conjugation is catalyzed by bile acid coenzyme A: amino acid N-acyltransferase (BAAT), an enzyme with a highly conserved catalytic triad in its active site. Interestingly, the conjugated amino acids are highly variable among mammals, with some species conjugating bile acids with both glycine and taurine, whereas others conjugate only taurine. The genetic origin of these bile acid conjugation differences is unknown. Here, we tested whether mutations in BAAT's catalytic triad could explain bile acid conjugation differences. Our comparative analysis of 118 mammals first revealed that the ancestor of placental mammals and marsupials possessed two genes, BAAT and BAATP1, that arose by a tandem duplication. This duplication was followed by numerous gene losses, including BAATP1 in humans. Losses of either BAAT or BAATP1 largely happened in a reciprocal fashion, suggesting that a single conjugating enzyme is generally sufficient for mammals. In intact BAAT and BAATP1 genes, we observed multiple changes in the catalytic triad between Cys and Ser residues. Surprisingly, although mutagenesis experiments with the human enzyme have shown that replacing Cys for Ser greatly diminishes the glycine-conjugating ability, across mammals we found that this residue provides little power in predicting the experimentally measured amino acids that are conjugated with bile acids. This suggests that the mechanism of BAAT's enzymatic function is incompletely understood, despite relying on a classic catalytic triad. More generally, our evolutionary analysis indicates that results of mutagenesis experiments may not easily be extrapolatable to other species.
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Project description:Leflunomide is an immunosuppressive agent marketed as a disease-modifying antirheumatic drug. But it causes severe side effects, including fatal hepatitis and liver failure. In this study we investigated the contributions of hepatic metabolism and transport of leflunomide and its major metabolite teriflunomide to leflunomide induced hepatotoxicity in vitro and in vivo.The metabolism and toxicity of leflunomide and teriflunomide were evaluated in primary rat hepatocytes in vitro. Hepatic cytochrome P450 reductase null (HRN) mice were used to examine the PK profiling and hepatotoxicity of leflunomide in vivo. The expression and function of sodium/bile acid cotransporter (NTCP) were assessed in rat and human hepatocytes and NTCP-transfected HEK293 cells. After Male Sprague-Dawley (SD) rats were administered teriflunomide (1,6, 12 mg · kg(-1) · d(-1), ig) for 4 weeks, their blood samples were analyzed.A nonspecific CYPs inhibitor aminobenzotriazole (ABT, 1 mmol/L) decreased the IC50 value of leflunomide in rat hepatocytes from 409 to 216 ?mol/L, whereas another nonspecific CYPs inhibitor proadifen (SKF, 30 ?mol/L) increased the cellular accumulation of leflunomide to 3.68-fold at 4 h. After oral dosing (15 mg/kg), the plasma exposure (AUC0-t) of leflunomide increased to 3-fold in HRN mice compared with wild type mice. Administration of leflunomide (25 mg·kg(-1) · d(-1)) for 7 d significantly increased serum ALT and AST levels in HRN mice; when the dose was increased to 50 mg·kg(-1) · d(-1), all HRN mice died on d 6. Teriflunomide significantly decreased the expression of NTCP in human hepatocytes, as well as the function of NTCP in rat hepatocytes and NTCP-transfected HEK293 cells. Four-week administration of teriflunomide significantly increased serum total bilirubin and direct bilirubin levels in female rats, but not in male rats.Hepatic CYPs play a critical role in detoxification process of leflunomide, whereas the major metabolite teriflunomide suppresses the expression and function of NTCP, leading to potential cholestasis.
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Project description:An improved ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method was established for the simultaneous analysis of various bile acids (BA) and applied to investigate liver BA content in C57BL/6 mice fed 1% cholic acid (CA), 0.3% deoxycholic acid (DCA), 0.3% chenodeoxycholic acid (CDCA), 0.3% lithocholic acid (LCA), 3% ursodeoxycholic acid (UDCA), or 2% cholestyramine (resin). Results indicate that mice have a remarkable ability to maintain liver BA concentrations. The BA profiles in mouse livers were similar between CA and DCA feedings, as well as between CDCA and LCA feedings. The mRNA expression of Cytochrome P450 7a1 (Cyp7a1) was suppressed by all BA feedings, whereas Cyp7b1 was suppressed only by CA and UDCA feedings. Gender differences in liver BA composition were observed after feeding CA, DCA, CDCA, and LCA, but they were not prominent after feeding UDCA. Sulfation of CA and CDCA was found at the 7-OH position, and it was increased by feeding CA or CDCA more in male than female mice. In contrast, sulfation of LCA and taurolithocholic acid (TLCA) was female-predominant, and it was increased by feeding UDCA and LCA. In summary, the present systematic study on BA metabolism in mice will aid in interpreting BA-mediated gene regulation and hepatotoxicity.
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Project description:Retinoic acid (RA) and bile acids share common roles in regulating lipid homeostasis and insulin sensitivity. In addition, the receptor for RA (retinoid x receptor) is a permissive partner of the receptor for bile acids, farnesoid x receptor (FXR/NR1H4). Thus, RA can activate the FXR-mediated pathway as well. The current study was designed to understand the effect of all-trans RA on bile acid homeostasis. Mice were fed an all-trans RA-supplemented diet and the expression of 46 genes that participate in regulating bile acid homeostasis was studied. The data showed that all-trans RA has a profound effect in regulating genes involved in synthesis and transport of bile acids. All-trans RA treatment reduced the gene expression levels of Cyp7a1, Cyp8b1, and Akr1d1, which are involved in bile acid synthesis. All-trans RA also decreased the hepatic mRNA levels of Lrh-1 (Nr5a2) and Hnf4? (Nr2a1), which positively regulate the gene expression of Cyp7a1 and Cyp8b1. Moreover, all-trans RA induced the gene expression levels of negative regulators of bile acid synthesis including hepatic Fgfr4, Fxr, and Shp (Nr0b2) as well as ileal Fgf15. All-trans RA also decreased the expression of Abcb11 and Slc51b, which have a role in bile acid transport. Consistently, all-trans RA reduced hepatic bile acid levels and the ratio of CA/CDCA, as demonstrated by liquid chromatography-mass spectrometry. The data suggest that all-trans RA-induced SHP may contribute to the inhibition of CYP7A1 and CYP8B1, which in turn reduces bile acid synthesis and affects lipid absorption in the gastrointestinal tract.
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Project description:The ubiquitin-mediated degradation system is responsible for controlling various tumor-promoting processes, including DNA repair, cell cycle arrest, cell proliferation, apoptosis, angiogenesis, migration and invasion, metastasis, and drug resistance. The conjugation of ubiquitin to a target protein is mediated sequentially by the E1 (activating)‒E2 (conjugating)‒E3 (ligating) enzyme cascade. Thus, E2 enzymes act as the central players in the ubiquitination system, modulating various pathophysiological processes in the tumor microenvironment. In this review, we summarize the types and functions of E2s in various types of cancer and discuss the possibility of E2s as targets of anticancer therapeutic strategies.
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Project description:Rationale: An improved understanding of thyroid hormone (TH) action on cholesterol metabolism will facilitate the identification of novel therapeutic targets for hypercholesterolemia. TH-regulated microRNAs (miRNAs) have been implicated in TH-controlled biological processes; however, whether and how TH-regulated miRNAs mediate the cholesterol-lowering effect of TH remains unclear. Our aim was to identify TH-regulated microRNAs that have cholesterol-lowering effects and explore the underlying mechanism. Method: Microarray and RNA-seq were performed to identify TH-regulated microRNAs and the genes regulated by mmu-miR-378-3p (miR-378) in the liver of mice, respectively. Recombinant adenoviruses encoding miR-378, Mafg, and shRNA for Mafg, antagomiR-378, liver-specific miR-378 transgenic mice, and miR-378 knockout mice were employed to investigate the roles of hepatic miR-378 and MAFG in cholesterol and bile acid homeostasis. The levels of bile salt species were determined by using UFLC-Triple-time of flight/MS. Results: Here, we show that hepatic miR-378 is positively regulated by TH. Transient overexpression of miR-378 in the liver of mice reduces serum cholesterol levels, accompanied with an increase in the expression of key enzymes in primary bile acid synthetic pathways and corresponding increases in biliary and fecal bile acid levels. Consistently, liver-specific miR-378 transgenic mice with moderate overexpression of hepatic miR-378 display decreased serum cholesterol levels and resistance to diet-induced hypercholesterolemia, while mice lacking miR-378 exhibit defects in bile acid and cholesterol homeostasis. Mechanistically, hepatic miR-378 regulates the expression of key enzymes in both classic and alternative bile acid synthetic pathways through MAFG, a transcriptional repressor, thereby modulating bile acid and cholesterol metabolism. Conclusions: TH-responsive hepatic miR-378 is capable of modulating serum cholesterol levels by regulating both the classic and alternative BA synthetic pathways. Our study not only identifies a previously undescribed role of hepatic miR-378 but also provides new cholesterol-lowering approaches.
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Project description:Bile acids are important for absorbing nutrients. Most mammals produce cholic and chenodeoxycholic bile acids. Here, we investigated genes in the bile acid synthesis pathway in four mammals that deviate from the usual mammalian bile composition. First, we show that naked-mole rats, elephants, and manatees repeatedly inactivated CYP8B1, an enzyme uniquely required for cholic acid synthesis, which explains the absence of cholic acid in these species. Second, no gene-inactivating mutations were found in any pathway gene in the rhinoceros, a species that lacks bile acids, indicating an evolutionarily recent change in its bile composition. Third, elephants and/or manatees that also lack bile acids altogether have lost additional nonessential enzymes (SLC27A5, ACOX2). Apart from uncovering genomic differences explaining deviations in bile composition, our analysis of bile acid enzymes in bile acid-lacking species suggests that essentiality prevents gene loss, while loss of pleiotropic genes is permitted if their other functions are compensated by functionally related proteins.
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Project description:It is well established that, besides facilitating lipid absorption, bile acids act as signaling molecules that modulate glucose and lipid metabolism. Bile acid metabolism, in turn, is controlled by several nutrient-sensitive transcription factors. Altered intrahepatic glucose signaling in type 2 diabetes associates with perturbed bile acid synthesis. We aimed to characterize the regulatory role of the primary intracellular metabolite of glucose, glucose-6-phosphate (G6P), on bile acid metabolism. Hepatic gene expression patterns and bile acid composition were analyzed in mice that accumulate G6P in the liver, that is, liver-specific glucose-6-phosphatase knockout (L-G6pc-/- ) mice, and mice treated with a pharmacological inhibitor of the G6P transporter. Hepatic G6P accumulation induces sterol 12α-hydroxylase (Cyp8b1) expression, which is mediated by the major glucose-sensitive transcription factor, carbohydrate response element-binding protein (ChREBP). Activation of the G6P-ChREBP-CYP8B1 axis increases the relative abundance of cholic-acid-derived bile acids and induces physiologically relevant shifts in bile composition. The G6P-ChREBP-dependent change in bile acid hydrophobicity associates with elevated plasma campesterol/cholesterol ratio and reduced fecal neutral sterol loss, compatible with enhanced intestinal cholesterol absorption. Conclusion: We report that G6P, the primary intracellular metabolite of glucose, controls hepatic bile acid synthesis. Our work identifies hepatic G6P-ChREBP-CYP8B1 signaling as a regulatory axis in control of bile acid and cholesterol metabolism.
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