These unsupervised investigations revealed that a gene network associated with cholestatic liver disease was the most statistically overrepresented network in pregnant, cholate-fed, and Fxr−/− mice (Fig. 2E). Therefore, global expression analysis demonstrates that pathways and networks regulated under conditions of bile acid overload or genetic cholestasis are also significantly affected by pregnancy. We aimed to determine whether hepatic genes respond to the accumulation of bile acids during pregnancy or are more likely

to be causative for the raised bile acid concentrations. To this end, we performed qRT-PCR assays on genes known to maintain bile acid homeostasis. These assays also served to confirm changes detected by microarrays. As expected, in cholate-fed MG-132 cost mice, the data were consistent with the adaptation of gene expression of bile acid homeostasis genes by Fxr activation. As such, cholate feeding induced hepatic Shp and Bsep expression, whereas Cyp7a1 and Ntcp expression was repressed (Fig. 3). In contrast, Fxr−/− mice showed elevated Cyp7a1 levels and reduced Bsep, Shp, and Mdr1a levels (Fig. 3). Despite the presence of elevated hepatic bile acid concentrations, there was no evidence of Fxr activation in pregnant mice. Although Fxr protein levels were unaltered, the messenger RNA (mRNA) expression of the Fxr target gene Shp was significantly repressed (−2.8-fold; P < 0.01) during pregnancy, whereas its

targets for repression, the bile acid biosynthesis enzymes Cyp7a1 and Epigenetics inhibitor Cyp8b1,8 were up-regulated 1.6-fold (P < 0.05; Fig. 3). Similarly, pregnancy significantly reduced the expression of hepatic import genes [Ntcp, organic anion-transporting polypeptide 2 (Oatp2), and Oatp4] and export genes [Bsep, Mdr1a, and multidrug resistance–associated protein (Mrp3); Fig. 3]. Defective Fgf15 signaling from the intestine could contribute selleck chemicals llc to the observed bile acid phenotype,9 but this does not seem to be the case because the expression of Fgf15 in the terminal ileum was unaffected

by pregnancy (data not shown). Therefore, raised hepatic bile acid concentrations during pregnancy do not result in hepatic or intestinal Fxr activation. Instead, hepatic gene expression during pregnancy is procholestatic and resembles a state of Fxr inactivation because the majority of these genes are directly or indirectly regulated by Fxr. Our data indicate that hepatic bile acids accumulate in pregnant mice as a result of procholestatic gene expression resembling reduced Fxr function. We therefore assessed whether enhanced Fxr target gene transcription is sufficient to prevent further accumulation of hepatic bile acids during pregnancy. Indeed, hepatic bile acids did not further accumulate in pregnant cholate-fed mice (Fig. 4) in which anticholestatic mechanisms (such as the induction of Shp, Bsep, Oatp2, Mrp3, and Mdr1a and the repression of Cyp7a1 and Cyp8b1) were already induced (see Fig. 3).