The model is based on an shRNA sequence targeting Lrh-1 (NR5A2) cloned behind a doxycycline-responsive promoter. The construct is targeted at the Rosa26 locus along with the enhanced tet-repressor (Fig. 1A). The resulting C57BL/6J mice were bred to be heterozygous for the knockdown cassette and WT littermates lacking the targeting construct were used as controls. Lrh-1 gene knockdown was induced by doxycycline administration by way of the food for 5 weeks. As shown in Fig.

1B, http://www.selleckchem.com/products/PLX-4032.html hepatic Lrh-1 mRNA levels were reduced by ≈90%-95%, whereas the reduction of Lrh-1 expression in small intestine was ≈60%-70% in male and female mice (Fig. 1B). The expression of Shp, a well-established Lrh-1 target gene,22, 23 was robustly reduced in liver (Fig. 1B). In contrast, levels of steroidogenic factor-1, the closest paralog of LRH-1, in the ovaries were unaltered upon expression of the shRNA (data not shown), indicating that knockdown is specific for Lrh-1. There were no overt abnormalities noticed in either group. Plasma aspartate aminotransferase and alanine aminotransferase activities were unchanged (Fig. 1C), implying that knockdown of hepatic Lrh-1 has no detrimental effect on hepatocyte

cell integrity. As our Maraviroc model is fundamentally different from two previously reported ones,30, 31 we first analyzed a number of general metabolic parameters. As shown in Supporting Table 1, plasma cholesterol and triglyceride levels were unaltered and plasma lipoprotein profiles were found to be unchanged between wildtype and knockdown animals (data not shown). Two previous reports showed that bile salt composition rather than synthesis rate was altered in liver-specific

Lrh-1 knockout mice.30, 31 Consistent with this, hepatic Cyp7a1 mRNA levels remained unaltered or were even slightly induced, whereas those of Cyp8b1 were reduced. We also found that knockdown of LRH-1 resulted in a significant reduction of Cyp8b1 mRNA levels (Fig. 2A). Surprisingly, hepatic MCE Cyp7a1 mRNA levels were increased upon LRH-1 knockdown (Fig. 2A). Several genes implicated in hepatic bile salt transport (e.g., Ntcp, Abcb11/Bsep, and Abcb4/Mdr2) were all mildly reduced upon LRH-1 knockdown (Fig. 2A), in agreement with previous findings.31 We next tested whether the physicochemical properties of the neutral sterol fraction as well as the bile salt pool were affected upon LRH-1 knockdown. LRH-1 knockdown did not significantly alter amounts or relative abundances of each of the major neutral sterols in feces (Supporting Fig. 1A-C). In agreement with induced Cyp7a1 levels, the total amount of fecal bile salts secreted per day, reflecting hepatic synthesis, was slightly increased (males +57%, females +59%) (Fig. 2B). The primary bile salts cholate (CA) and chenodeoxycholate (CDCA) are the direct products of de novo bile salt synthesis. Modifications of these bile salts in liver and intestine give rise to differentially structured primary and secondary bile salts, respectively.