Characterization of organic anion transporting polypeptide 1a1 (Oatp1a1) in the bile acid homeostasis of mice.

摘要

Organic anion transporting polypeptides (human: OATPs; all other species: Oatps; gene symbol: SLCO/Slco) are sodium-independent transport systems that mediate the transmembrane transport of a wide range of amphipathic endogenous and exogenous organic compounds. In mice, Oatp1a1, 1a4, and 1b2 are thought to account for the bulk of Na-independent bile acid (BA) uptake into liver during normal physiological conditions. The overall goal of this dissertation has focused on characterization of the in vivo role of mouse Oatp1a1 in BA homeostasis by using Oatp1a1-null mice. To achieve this overall goal, three specific aims were examined in the present dissertation.;In the first specific aim, a simple and sensitive UPLC-MS/MS method was established and validated for the simultaneous analysis of various BAs, 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). The purpose of this study was to understand the BA metabolic pathways in mice by using this newly developed BA-quantification method, and thus to provide tools and knowledge for the future study in Oatp1a1-null mice. Gender differences in liver BA composition were observed after feeding CA, DCA, CDCA, and LCA, but were not prominent after feeding UDCA. Sulfation of CA and CDCA was found at the 7-OH position, and 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 increased by feeding UDCA and LCA. The metabolic pathways of each BA in vivo are proposed, and can be used to interpret BA-mediated gene regulation and hepatotoxicity.;In the second specific aim, the hypothesis that Oatp1a1 is important in transporting unconjugated BAs was evaluated. The purpose of this study was to determine whether knockout of Oatp1a1 will alter BA metabolism in mice. To address this aim, the concentrations of individual BAs in serum, liver, and bile were compared between WT and Oatp1a1-null mice. The gender-divergent expression of Oatp1a1 was considered in the efforts to identify the endogenous BA substrates for Oatp1a1. In addition, DCA feeding and pharmacokinetic studies were conducted in WT and Oatp1a1-null mice to investigate the role of Oatp1a1 in the disposition of DCA. Data from this study show a critical role of Oatp1a1 in DCA metabolism of mice. Oatp1a1 in mouse liver does not appear to transport DCA, because knockout of Oatp1a1 does not prevent hepatic uptake and hepatotoxicity of DCA. Instead, knockout of Oatp1a1 increases the intestinal permeability and thus increases intestinal absorption of DCA. In addition, Knockout of Oatp1a1 markedly alters the composition and amount of intestinal bacteria. The alterations of intestinal bacteria in Oatp1a1-null mice result in marked changes of BA composition in the intestinal contents and feces, but have no effect on the total fecal BA excretion, due to the same billiary input of BAs in WT and Oatp1a1-null mice.;In the third specific aim, the hypothesis that knockout of Oatp1a1 decreases liver toxicity in mice during extrahepatic cholestasis was evaluated. The purpose of this study was to determine the in vivo role of Oatp1a1 in mice after bile duct ligation (BDL) by using Oatp1a1-null mice. Knockout of Oatp1a1 increased liver toxicity in mice after BDL, which may be due to the increase of secondary BAs in livers of mice. Knockout of Oatp1a1 resulted in an impaired cytoprotective response in mice during BDL-induced cholestasis. In addition, antibiotic treatment potentiated liver toxicity in Oatp1a1-null mice after BDL by increasing the intestinal absorption of BAs. Thus, Oatp1a1 plays a unique and essential protective role in the adaptive response to obstructive cholestasis liver injury.;Altogether, this dissertation demonstrates that: (1) A simple and sensitive UPLC-MS/MS method was established for the simultaneous analysis of various BAs and was applied to investigate the BA metabolism in mice fed CA, CDCA, DCA, LCA, UDCA, or resin; (2) Oatp1a1 does not mediate the hepatic uptake of DCA, but plays a critical role in the intestinal metabolism of DCA; (3) Knockout of Oatp1a1 increases intestinal bacteria and thus alters the urinary metabolomics in mice; and (4) Knockout of Oatp1a1 increases liver toxicity in mice after BDL, which may be due to the increase of secondary BAs in both serum and livers of mice.