Samples were extracted with diethyl ether The water phase was ac

Samples were extracted with diethyl ether. The water phase was acidified with 6 M HCl and extracted with diethyl ether. The samples

were washed with water until neutral, evaporated and methylated with trimethyl silyldiazomethane, and derivatized using hexamethyl-disilazane and trimethylchlorosilane in pyridine. Samples were finally analyzed selleck by gas chromatography–mass spectrometry (GC/MS).16 The following standard methods are described in detail in the Supporting Methods: cell culture, RNA isolation, quantitative real-time polymerase chain reaction (PCR), western (protein) immunoblot, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay, construction of Abcg5 reporters, mutagenesis, and transient transfection assay. Results are expressed as mean ± standard error (SE). Statistical analysis was performed by Student t test. P < 0.05 is considered as statistically significant. In this study, we further investigated the effects and mechanisms of CYP7A1 overexpression on hepatic cholesterol homeostasis in Cyp7a1-tg mice. Cyp7a1-tg mice had a ∼2-fold increase of CYP7A1 enzyme activity.1 As a result, bile acid synthesis and bile acid pool increased 2.5-fold (Fig. 1A) and fecal bile acid content increased 2.5-fold (Fig. 1B). A detailed analysis of bile

acid composition in gallbladder bile using a sensitive GC/MS method showed that gallbladder bile acid composition changed from predominantly tauro-conjugated CA (58%) in wild-type mice to chenodeoxycholic acid (CDCA, 54%) in Cyp7a1-tg mice (Fig. 1C). In FK506 Cyp7a1-tg mice, the CA content was drastically

Progesterone decreased to 1.7%, but α-muricholic acid (α-MCA) content increased two-fold to 20% and β-MCA reduced to 7.4% in comparison with wild-type mice. Ursodeoxycholic acid (UDCA) markedly increased from 3.8% in wild-type to 15% in Cyp7a1-tg mice. This altered bile acid composition can be explained by bile acid inhibition of CYP8B1 and CA synthesis in Cyp7a1-tg mice.5 This may lead to significantly higher CDCA production. In mouse livers, excess CDCA is converted to MCAs by Cyp3a11-mediated 6-hydroxylation and epimerization of a hydroxyl group from the 7α-position to the 7β-position, or to UDCA by epimerization of a 7α-hydroxyl group to the 7β-position. CDCA is more hydrophobic than CA, and MCA and UDCA are highly hydrophilic. Thus, gallbladder bile in Cyp7a1-tg mice is more hydrophobic than that in wild-type mice. Interestingly, despite increased cholesterol catabolism in the liver, Cyp7a1-tg mice still had approximately 2.5-fold higher biliary and fecal cholesterol content than wild-type mice (Fig. 2A,B). Hepatic total cholesterol levels were unaltered (Fig. 2C), but plasma cholesterol was decreased in Cyp7a1-tg mice (Fig. 2D). Biliary cholesterol and bile acid secretion rates were two-fold and four-fold higher, respectively, in Cyp7a1-tg mice than that in wild-type mice (Fig. 3A,B).

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