Transcription of the Cyp8b1 gene was tremendously induced upon colesevelam treatment in the wildtype

but not in the knockdown animals (Fig. 3C). These results show that LRH-1 is a critical transcription factor for adequate up-regulation of Cyp7a1 and Cyp8b1 transcription under conditions of bile salt sequestration. In addition, the apparent paradoxical behavior observed for Cyp7a1 transcription in the LRH-1-KD mice suggest that two LRH-1-dependent, but mechanistically different, mechanisms are involved in the transcriptional regulation of Cyp7a1 expression. A previous study in mice deficient for intestinal Lrh-1 showed a reduction of intestinal Fgf15 mRNA expression, suggesting selleck kinase inhibitor that intestinal LRH-1 directly or indirectly regulates Fgf15 expression.31 Colesevelam did not alter intestinal Lrh-1 expression in wildtype mice Buparlisib mouse but did suppress Shp and Fgf15 expression (Fig. 3D), which is consistent with previous findings.33 Intestinal Shp levels were significantly reduced in LRH-1-KD mice on and off colesevelam (Fig. 3D). Interestingly, we also found a tremendous reduction in Fgf15 mRNA levels in Lrh-1-KD mice on and off colesevelam,

indicating that (intestinal) Lrh-1 regulates the expression of the Fgf15 gene. To further support this relationship, we tested whether LRH-1 would increase expression of FGF19, the human ortholog of murine FGF15, in DLD cells. Transduction of DLD cells with increasing amounts of recombinant LRH-1 encoding adenoviral particles (Supporting Fig. 3A,B) caused a dose-dependent increase in FGF19 mRNA expression (Supporting Fig. 3C). These data indicate that LRH-1 indeed induces Fgf15/19 expression.

We tested whether altered Cyp7a1 expression Olopatadine in colesevelam-treated LRH-1-KD animals also had physiological consequences. Knockdown of LRH-1 did not cause significant alterations in bile flow rate and only tended to reduce biliary bile salt output (Fig. 4A,B). Treatment with colesevelam did not affect bile flow, but reduced biliary bile salt output in both WT mice and LRH-1-KD mice (Fig. 4A,B), in agreement with previous studies from our laboratory.33 In agreement with the observed increase in Cyp7a1 expression levels (Fig. 3C), knockdown of LRH-1 caused a modest increase (+10%) of fecal bile salt output (Fig. 4C). As expected, sequestrant treatment led to a massive induction (+272%) of fecal bile salt output in WT mice. Because colesevelam was given for 2 weeks, a new steady state is established in which fecal loss depicts enhanced bile acid synthesis. In LRH-1-KD mice there was no increase in fecal bile acid output after 2 weeks (Fig. 4C), indicating that LRH-1-KD mice cannot up-regulate bile acid synthesis during colesevelam treatment. As Cyp8b1 expression was also dysregulated in LRH-1-KD mice, we expected that LRH-1 knockdown combined with sequestrant would have profound effects on bile salt composition. Supporting Fig.