NADPH–cytochrome P450 reductase (Cpr) is essential for the function of microsomal cytochrome P450 monooxygenases (P450), including those P450s involved in bile acid (BA) synthesis. Mice with hepatocyte-specific deletion of NADPH–cytochrome P450 reductase (H-Cpr-null) have been engineered to understand the in vivo function of hepatic P450s in the metabolism of xenobiotics and endogenous compounds. However, the impact of hepatic Cpr on BA homeostasis is not clear. The present study revealed that H-Cpr-null mice had a 60% decrease in total BA concentration in liver, whereas the total BA concentration in serum was almost doubled. The decreased level of cholic acid (CA) in both serum and livers of H-Cpr-null mice is likely due to diminished enzyme activity of Cyp8b1 that is essential for CA biosynthesis. Feedback mechanisms responsible for the reduced liver BA concentrations and/or increased serum BA concentrations in H-Cpr-null mice included the following: 1) enhanced alternative BA synthesis pathway, as evidenced by the fact that classic BA synthesis is diminished but chenodeoxycholic acid still increases in both serum and livers of H-Cpr-null mice; 2) inhibition of farnesoid X receptor activation, which increased the mRNA of Cyp7a1 and 8b1; 3) induction of intestinal BA transporters to facilitate BA absorption from the intestine to the circulation; 4) induction of hepatic multidrug resistance–associated protein transporters to increase BA efflux from the liver to blood; and 5) increased generation of secondary BAs. In summary, the present study reveals an important contribution of the alternative BA synthesis pathway and BA transporters in regulating BA concentrations in H-Cpr-null mice.
Cytochrome P450 enzymes (P450s) not only have a major impact on the metabolism of drugs and nutrients, but they also regulate the homeostasis of many endogenous compounds, such as cholesterol and bile acids (BAs). All microsomal P450s receive electrons from a single donor—namely, NADPH–cytochrome P450 reductase (Cpr; EC 188.8.131.52), a housekeeping gene widely expressed in various tissues with the highest level in liver (Black et al., 1979; Black and Coon, 1987; Shen et al., 2002). Whole-body deletion of the Cpr gene causes embryonic lethality in mice, whereas mice with hepatocyte-specific deletion of Cpr (H-Cpr-null) can breed and develop normally, even though their hepatic microsomal P450 activity is ablated (Gu et al., 2003; Henderson et al., 2003; Wu et al., 2003). Therefore, H-Cpr-null mice have been characterized extensively and used for studying the in vivo function of P450s in drug metabolism. It was reported that H-Cpr-null mice have a 90% reduction in bile volume in the gallbladder, accompanied by a 65% reduction of serum cholesterol (Henderson et al., 2003). However, the impact of hepatocyte-specific Cpr depletion on individual BA homeostasis in serum and livers of mice is still not clear.
Bile formation is a major function of the liver. More than 90% of total cholesterol in the body is excreted after conversion into BAs. Detergent properties of BAs not only make them critical for absorption of lipids and fat-soluble vitamins from the intestine, but also contribute to their cytotoxicity during various liver diseases. Recently, BAs also have emerged as signaling molecules by serving as ligands of the farnesoid X receptor (FXR) (Makishima et al., 1999; Parks et al., 1999; Wang et al., 1999), pregnane X receptor (Staudinger et al., 2001), and a G protein–coupled BA receptor, activation of which, in turn, regulates the homeostasis of xenobiotics and endogenous compounds, such as glucose and lipids (Kawamata et al., 2003). Therefore, the synthesis, transport, and metabolism of BAs have to be tightly regulated to ensure that a sufficient amount of cholesterol is metabolized to provide adequate emulsification of lipids in the intestine, and prevent cytotoxic accumulation of BAs in the liver.
BA biosynthesis is initiated by oxidation of cholesterol through two different pathways: the classic (or neutral) pathway and the alternative (or acidic) pathway (Fig. 1). The classic pathway is initiated by microsomal cholesterol 7α-hydroxylase (Cyp7a1), which catalyzes the rate-limiting step in the classic pathway (Ishibashi et al., 1996; Schwarz et al., 1996). The alternative pathway of BA biosynthesis is initiated by sterol 27-hydroxylase (Cyp27a1), which is located in the inner mitochondrial membrane. The rate-limiting step in the alternative pathway is cholesterol delivery to the inner mitochondrial membrane, carried out by the steroidogenic acute regulatory protein (StAR; also named mitochondria cholesterol transporter) (Pandak et al., 2002). The classic pathway of BA synthesis is thought to occur exclusively in the liver, whereas the alternative pathway functions in numerous tissues. Two important microsomal enzymes for bile acid biosynthesis are Cyp7a1 and sterol 12α-hydroxylase (Cyp8b1); the latter is required for the biosynthesis of cholic acid (CA).
In addition to BA biosynthetic enzymes, BA transporters are also critical to maintain BA homeostasis by regulating BA enterohepatic circulation (Fig. 1). Approximately 95% of total BAs in humans and 87% in mice (Dawson et al., 2003) undergo enterohepatic circulation. After BA biosynthesis in the liver, the bile salt export pump (Bsep) transports BAs into bile, whereas multidrug resistance–associated protein (Mrp) 3, 4, and organic solute transporter (Ost) α/β export BAs out of hepatocytes into the blood. Once excreted into bile, BAs either flow directly into the duodenum (rats) or are stored in the gall bladder (humans and mice). In the intestinal lumen, primary BAs [CA, muricholic acid (MCA), and chenodeoxycholic acid (CDCA)] are converted by bacterial enzymes into secondary BAs [deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA)]. The majority of BAs are then taken up into enterocytes by the apical sodium bile-acid transporter (Asbt) in the ileum. Once in the enterocytes, BAs bind to the intestinal bile-acid binding protein (Ibabp) and translocate from the apical to the basolateral membrane of enterocytes. Heterodimer Ostα/β, located in the basolateral membrane of enterocytes, exports BAs from enterocytes into the circulation, where they return to the liver. Sodium-taurocholate cotransporting polypeptide (Ntcp) transports conjugated BAs, and organic anion–transporting polypeptide (Oatp) 1b2 transports unconjugated BAs into hepatocytes from the sinusoidal blood (Csanaky et al., 2011). BAs then return to the liver, and another cycle of enterohepatic circulation of BAs begins.
The classic pathway of BA biosynthesis is considered more important for maintaining normal BA concentrations; the relative importance of the alternative pathway of BA biosynthesis during altered circumstances is not known. A Cyp7a1-null mouse model was engineered (Ishibashi et al., 1996; Schwarz et al., 1996), which eliminated the classic bile-acid biosynthesis pathway. The Cyp7a1-null mice died within 18 days of age, unless supplemented with lipid-soluble vitamins and BAs (Ishibashi et al., 1996). Intriguingly, the Cyp7a1-null mice improved after 3 weeks of age, coincident with expression of the alternative BA biosynthesis pathway involving Cyp7b1, the expression of which is not initiated until 3 weeks of age (Ishibashi et al., 1996). As mentioned earlier, in the H-Cpr-null mouse, the classic pathway of BA biosynthesis is abolished, because of the loss of function of Cpr-dependent microsomal Cyp7a1 and 8b1. In contrast to Cyp7a1-null mice, H-Cpr-null mice breed and develop normally. This suggests the H-Cpr-null mouse model might be a useful model to understand the contribution of the alternative pathway of BA biosynthesis, because mitochondrial Cyp27a1 does not require Cpr to function, and extrahepatic BA biosynthesis should be intact in H-Cpr-null mice.
H-Cpr-null mice have been useful in the pharmaceutical industry for determining the in vivo importance of hepatic P450s in the safety and efficacy of drugs. Although much effort has been made to understand the phenotype of H-Cpr-null mice, BA homeostasis in these mice is still not well characterized. In the present study, individual BA concentrations, BA biosynthetic enzymes, and BA transporters were determined in H-Cpr-null mice to further understand the impact of hepatic Cpr on BA homeostasis.
Materials and Methods
Animals and Tissue Collection.
Breeding pairs of H-Cpr-null mice (Gu et al., 2003), which were backcrossed over 10 generations with C57BL/6 mice, were kindly provided by Dr. Xinxin Ding (Wadsworth Center, State University of New York at Albany, Albany, NY). All mice were bred and maintained on an automatically timed 12-hour dark/light cycle in an American Animal Associations Laboratory Animal Care–accredited facility at the University of Kansas Medical Center (Kansas City, KS), and were allowed water and rodent chow ad libitum (Teklad; Harlan, Indianapolis, IN). At approximately 2 months of age, blood, liver, and small intestine (duodenum, jejunum, and ileum) samples from male H-Cpr-null mice and corresponding male wild-type mice (n = 5/genotype) were collected.
Quantification of Individual Bile Acids in Mouse Serum and Liver.
Blood was allowed to coagulate, and was centrifuged at 10,000g for 15 minutes. The resulting supernatant (serum) was collected for analysis. Total BAs were extracted from mouse serum and liver by a previously described method (Alnouti et al., 2008). Individual BAs (CA, α-MCA, β-MCA, CDCA, UDCA, DCA, and LCA) and their glycine and taurine conjugates were quantified by an ultra-performance liquid chromatography–tandem mass spectrometry method (Alnouti et al., 2008).
Total RNA Isolation.
Total RNA was isolated using RNA Bee reagent (Fisher Scientific Inc., Waltham, MA) per the manufacturer's protocol. The concentration of total RNA in each sample was quantified spectrophotometrically at 260 nm.
Development of Specific Oligonucleotide Probe Sets for Branched DNA Analysis.
Gene sequences of interest were accessed from GenBank, and the target sequences were analyzed by ProbeDesigner Software Version 1.0 (Genospectra, Fremont, CA) as described previously (Hartley and Klaassen, 2000; Cheng et al., 2005). Probes were synthesized by Integrated DNA Technologies (Coralville, IA). Probe sets for mouse Oatps (Cheng et al., 2005), Mrps (Maher et al., 2005), Ntcp, Bsep (Cheng et al., 2007), Cyp7a1 (Dieter et al., 2004), Ostα, Ostβ, Asbt (Cheng and Klaassen, 2006), small heterodimer partner (Shp), Cyp27a1, Cyp39a1, bile acid CoA ligase (BAL), and bile acid CoA:amino acid N-acyltransferase (BAT) (Csanaky et al., 2009) have been reported previously. Probe sequences of mouse Cyp7b1 and StAR are shown in Table 1.
Branched DNA Signal Amplification Assay.
Reagents required for RNA analysis (i.e., lysis buffer, amplifier/label probe buffer, and substrate solution) were supplied in the QuantiGene branched DNA signal amplification assay kit (Panomics Inc., Fremont, CA). Individual total RNA samples from five mice were used to determine the alteration of gene expression in wild-type and H-Cpr-null mice. Data are presented as relative light units per 10 µg of total RNA.
Data were expressed as the mean ± S.E.M. The differences between wild-type and H-Cpr-null mice were analyzed by Student’s t test. Statistical significance was set at P < 0.05.
BA Concentrations in Serum of Adult Male H-Cpr-Null and Wild-Type Mice.
As shown in Fig. 2A, total serum BA concentrations in H-Cpr-null mice were twice those in wild-type (WT) mice. In regard to the individual BAs in the serum, the concentration of unconjugated BAs was approximately 50% lower, and glycine-conjugated BAs were not altered, but taurine-conjugated BAs were doubled in serum of H-Cpr-null mice compared with WT mice (Fig. 2A). The majority of unconjugated BAs, such as α-MCA, β-MCA, CA, and DCA, were decreased in serum of H-Cpr-null mice (Fig. 2B). The concentrations of glycine-conjugated BAs are very low in serum of mice, and were not different between H-Cpr-null and WT mice (Fig. 2C). The serum concentration of taurine-conjugated MCA was decreased, but taurine-conjugated CDCA (T-CDCA), UDCA, DCA (T-DCA), and LCA were increased 18.9-, 11.3-, 10.6-, and 1.6-fold in H-Cpr-null mice, respectively (Fig. 2D).
BA Concentrations in Livers of Adult Male H-Cpr-Null and WT Mice.
As shown in Fig. 3A, total BA concentrations in livers of H-Cpr-null mice were 59.6% lower than in WT mice. The concentrations of unconjugated, glycine-conjugated, and taurine-conjugated BAs were 71.6, 65.1, and 58.5% lower, respectively, in livers of H-Cpr-null mice (Fig. 3A). The concentrations of unconjugated α-MCA, β-MCA, and CA were lower, but CDCA and LCA were higher in livers of H-Cpr-null mice (Fig. 3B). The total concentration of glycine-conjugated CA was lower, but glycine-conjugated DCA and LCA were higher in livers of H-Cpr-null mice (Fig. 3C). The concentrations of taurine-conjugated MCA, CA, and UDCA were markedly lower, but taurine-conjugated CDCA (0.5-fold) and LCA (25-fold) were higher in livers of H-Cpr-null mice (Fig. 3D).
Total Amount of BAs in Livers of H-Cpr-Null and WT Mice.
Hepatomegaly occurs in H-Cpr-null mice (1.2 ± 0.1 g/WT mouse liver; 1.9 ± 0.1 g/H-Cpr-null mouse liver), similar to that previously reported (Gu et al., 2003; Henderson et al., 2003), but the average body weight was not different (23.6 ± 0.3 g/WT mouse; 24.0 ± 0.5 g/H-Cpr-null mouse), leading to a 50% increase in the liver-to-body weight ratio in H-Cpr-null mice (Fig. 4A). Total BA concentrations were 59.6% lower in livers of H-Cpr-null than WT mice (Fig. 4B). In contrast, the total amount of BAs per liver was only 26% lower in H-Cpr-null than WT mice (Fig. 4C).
Percentage of Individual BAs in Serum and Livers of H-Cpr-Null and WT Mice.
The percentage of individual BAs in serum is shown in Fig. 5, top. Serum MCA and CA accounted for 55.4 and 34.7%, respectively, of the total serum BAs in WT mice, but only 26 and 21.2% in H-Cpr-null mice. The percentage of serum CDCA was 9-fold higher in H-Cpr-null than WT mice. UDCA was nearly nondetectable in serum of WT mice, but accounted for 7.7% of total serum BAs in H-Cpr-null mice. DCA contributed 5% to the total BAs in serum of WT mice, but 25.5% in H-Cpr-null mice. Taken together, CDCA, UDCA, and DCA only accounted for 8.5% of total serum BAs in WT mice, but 51.7% in H-Cpr-null mice.
The percentage of individual BAs in liver is shown in the lower panel of Fig. 5. The percentage of MCA in livers was 34% in WT mice, but only 20.7% in H-Cpr-null mice. CA accounts for 51% of the total BAs in livers of WT mice, but only 14% in H-Cpr-null mice. The percentages of CDCA, DCA, and LCA were 6.6-, 3-, and 66-fold higher, respectively, in livers of H-Cpr-null than WT mice. CDCA, DCA, and LCA account for only 13.3% of total liver BAs in WT mice, but 62.2% of that in H-Cpr-null mice.
Concentrations of Primary and Secondary BAs in Serum and Livers of H-Cpr-Null and WT Mice.
BAs can be divided into primary BAs that are synthesized in liver (including CA and CDCA) and secondary BAs (including DCA, LCA, and UDCA) that are converted from primary BAs by intestinal microflora. In livers, CDCA can be further converted to α/β-MCA in rodents (through hepatic 6α/β-hydroxylation). Thus, MCA is also a primary BA.
As shown in Fig. 6, top, the concentrations of primary BAs were similar in serum of WT and H-Cpr-null mice, but secondary BAs were increased 5.9-fold in serum of H-Cpr-null mice. Thus, the percentage of secondary BAs in serum increased from 6.5% in WT mice to 27% in H-Cpr-null mice. Primary BAs account for 93.5% of total serum BAs in WT mice, but 73% in H-Cpr-null mice.
In livers, the concentrations of primary BAs were about 70% lower in H-Cpr-null than WT mice (Fig. 6, bottom). The concentrations of secondary BAs were similar in livers of H-Cpr-null and WT mice. However, the percentage of primary BAs in livers was 91% in WT mice, but 70% in H-Cpr-null mice. In contrast, the percentage of secondary BAs in livers was 9% in WT mice and 30% in H-Cpr-null mice. This suggests an increased microbial metabolism of BAs in the intestine.
BA Biosynthesis Genes in Livers of H-Cpr-Null and WT Mice.
Messenger RNA expression of Cyp7a1 and 8b1, two critical microsomal enzymes for BA biosynthesis, was 3- to 4-fold higher in livers of H-Cpr-null than WT mice. However, these enzymes are nonfunctional in the absence of Cpr. The enzymes in the alternative BA biosynthesis pathway were regulated differently: Cyp27a1 was increased 30%, whereas Cyp39a1 and Cyp7b1 were decreased 63 and 87%, respectively, in livers of H-Cpr-null mice (Fig. 7).
The last step in BA biosynthesis is conjugation of BAs with an amino acid, such as taurine and glycine, by BAL and BAT (Russell, 2003; Thomas et al., 2008). As shown in Fig. 7, mRNA expression of BAL was not altered, but BAT was increased (21%) in livers of H-Cpr-null mice.
Shp, the transcriptional repressor of Cyp7a1 gene expression, decreased 55% in livers of H-Cpr-null mice. However, mRNA of FXR, the BA sensor, was not altered in livers of H-Cpr-null mice (Fig. 7).
Messenger RNA Expression of Transporters in Livers of H-Cpr-Null and WT Mice.
Messenger RNA expression of the two main hepatic uptake transporters, Ntcp and Oatp1b2, was similar in H-Cpr-null and WT mice (Fig. 8). Oatp1a4 mRNA expression was markedly higher, but Oatp1a1 was lower in livers of the H-Cpr-null mice (Fig. 8). Although it was previously thought that Oatp1a1 and 1a4 might be important for the uptake of BAs into livers of rodents, more recent studies have shown that neither Oatp1a1 (Zhang et al., 2012) nor Oatp1a4 (Zhang et al., 2013) is important for BA uptake. Messenger RNA of the StAR that transports cholesterol into mitochondria was 90% higher in livers of H-Cpr-null than WT mice.
Canalicular Bsep mRNA was not altered in livers of H-Cpr-null mice. In contrast, Mrp2, which is located in the canalicular membrane, and Mrp3 and 4 transporters, which are located in the sinusoidal membrane, were higher in livers of H-Cpr-null mice (Fig. 8).
BA Transporters, Fibroblast Growth Factor 15, and FXR in Small Intestines of H-Cpr-Null and WT Mice.
Asbt, Ibabp, and Ostα/β are important in the transfer of BAs from the intestinal lumen into the portal blood, and the fibroblast growth factor 15 (Fgf15) in the ilea is important for regulating BA synthesis. Therefore, the mRNAs of these genes were quantified in the ileum. BAs are primarily taken up into enterocytes by Asbt, and possibly Oatp2b1. As shown in Fig. 9, Oatp2b1 mRNA expression was not different in small intestines of H-Cpr-null and WT mice, but the Asbt mRNA level is higher in the ileum of H-Cpr-null mice. Once in the enterocytes, BAs bind to Ibabp and translocate from the apical to the basolateral region of enterocytes. As shown in Fig. 9, Ibabp is not altered in the ileum of H-Cpr-null mice. mRNA levels of Ostα/β, which is the major BA efflux transporter located in the basolateral membrane of enterocytes (Dawson et al., 2005; Rao et al., 2008), were more than 2.5-fold higher in small intestines of H-Cpr-null than WT mice (Fig. 9). Mrp3, which might play a secondary role in transporting BAs out of enterocytes into the circulation (Inokuchi et al., 2001; Shoji et al., 2004; Thomas et al., 2008), was similar in H-Cpr-null and WT mice (Fig. 9).
Fgf15 plays important roles in controlling BA biosynthesis in liver, as well as gallbladder filling (Inagaki et al., 2005; Choi et al., 2006; Kim et al., 2007). As shown in Fig. 9, Fgf15 mRNA decreased approximately 90% in the ileum of H-Cpr-null mice, compared with WT mice. Messenger RNA expression of the BA sensor FXR was not altered in the intestines of H-Cpr-null mice.
H-Cpr-null mice have a 65% reduction in serum cholesterol and a 90% decrease in gallbladder bile volume (Henderson et al., 2003), leading to a reasonable hypothesis that the amount of BAs would be severely reduced in H-Cpr-null mice due to the loss of function of the Cpr-dependent BA biosynthetic enzymes, in particular Cyp7a1, which is the rate-limiting enzyme in the classic BA biosynthesis pathway. Surprisingly, the total amount of BAs in livers of H-Cpr-null mice is decreased only about 25% compared with WT mice. The considerable presence of CA and its microbiota product DCA suggests that previously synthesized CA in H-Cpr-null mice at an earlier age still remains in the enterohepatic circulation at the time of analysis (approximately 2 months of age). This is not surprising because the enzyme activity of Cpr is not completely diminished in livers of H-Cpr-null mice until 2 months after birth (Gu et al., 2003). Thus, the microsomal Cyp8b1 enzyme, which is essential for the biosynthesis of CA, might remain active until 2 months of age. Interestingly, the concentrations of CDCA and its microbiota product LCA were increased in livers of H-Cpr-null mice. CDCA can be produced by both the classic and alternative BA synthesis pathways. The reason why there is an increase in CDCA is likely due to an increase in the alternative pathway of BA synthesis, as evidenced by an increase in mRNA of Cyp27a1, a mitochondrial enzyme that does not require Cpr to function, as well as a doubling of StAR, which transports steroids into the mitochondria and is the rate-limiting step in the alternative pathway of BA synthesis (Pandak et al., 2002). A prominent increase in the mRNA of both StAR and Cyp27a1 suggests that livers of H-Cpr-null mice tend to compensate for the decreased BA concentrations by enhancing the alternative pathway of BA synthesis. However, the entire alternative BA synthesis pathway is not likely enhanced in livers of H-Cpr-null mice because of the decreased mRNA of Cyp7b1, another enzyme in the alternative pathway of BA synthesis that requires Cpr. Therefore, the increased concentrations of CDCA and its metabolites in livers of H-Cpr-null mice could be due to an enhanced alternative BA synthesis pathway in extrahepatic tissues. Taken together, the severity of impaired BA synthesis in livers of H-Cpr-null mice appears to be compensated by an enhanced alternative pathway of BA synthesis.
Various transcription factors mediate the feedback regulation of BA biosynthesis to ensure the homeostasis of BAs. Cyp7a1 is the rate-limiting enzyme in the classic BA synthesis pathway. Cyp8b1 is the sterol 12α-hydroxylase and thus controls the amount of CA in the BA pool, and represents a critical regulation point for BA diversity in the body. Both Cyp7a1 and 8b1 are negatively regulated by the nuclear receptor FXR, the BA sensor that is important for the downregulation of BA synthesis and the regulation of some BA transporters (Makishima et al., 1999; Parks et al., 1999; Wang et al., 1999). It appears that the induced expression of hepatic Cyp7a1 and 8b1 in H-Cpr-null mice is mainly due to the deactivation of FXR, as reflected by a decrease in mRNAs of Fgf15 in intestines (Fig. 9) and Shp in livers. However, due to lack of Cpr in the livers, these microsomal Cyps are not able to synthesize BAs.
One might suspect that the concentrations of BAs in the serum of the H-Cpr-null mice might resemble that observed in the liver. But surprisingly, the present study shows that liver-specific elimination of Cpr, which is necessary for hepatic BA biosynthesis, did not markedly decrease total BA concentrations in serum. Instead, H-Cpr-null mice have almost twice the serum concentrations of total BAs, mainly due to a 10- to 20-fold increase in T-CDCA and T-DCA. The exact mechanisms by which T-CDCA and T-DCA increase in serum of H-Cpr-null mice are not known. In fact, little is known about the regulation of BA concentrations in serum. Theoretically, concentration and composition of BAs in serum are dependent on many factors, including 1) synthesis of primary BAs in liver, 2) excretion of BAs into bile, 3) expulsion of bile from gallbladder into intestine, 4) conversion of primary BAs to secondary BAs by intestinal flora, 5) reabsorption of BAs from the intestine, 6) transport of BAs into liver, 7) conversion of secondary BAs back to primary BAs in the liver, 8) transport of BAs from liver back into blood, and 9) BA synthesis in extrahepatic tissues. The relative importance of each of these nine processes in determining the composition and concentration of individual BAs in serum is not known.
In H-Cpr-null mice, reduced BA biosynthesis resulted in a decrease in liver BA concentrations, which may send a feedback signal to the intestine to enhance intestinal BA absorption into the circulation. This is reflected by the induction of intestinal BA transporters Asbt and Ostα/β, which function in concert to transport BAs from intestinal lumen to blood. Similarly, it is expected that the reduced hepatic BA concentrations in H-Cpr-null mice may cause the liver to take up more BAs and efflux fewer BAs. However, no alteration was observed in the mRNA expression of the three major hepatic BA transporters, namely, Ntcp, Oatp1b2, and Bsep in H-Cpr-null mice. Recently, Oatp1a1 and 1a4 have been shown to be important for secondary BA metabolism in mice (Zhang et al., 2012, 2013). The decreased Oatp1a1 and increased Oatp1a4 could be due to activation of some transcriptional factors, such as constitutive androstane receptor and pregnane X receptor in the H-Cpr-null mice (Wang et al., 2005; Weng et al., 2005; Cheng and Klaassen, 2006). Further studies are required to elucidate the regulatory mechanisms of Oatp1a1 and 1a4 in livers of H-Cpr-null mice. Mrp2, 3, and 4 play an important role in effluxing both xenobiotics and endogenous compounds, such as BAs, out of the liver. The induction of these Mrps in livers of H-Cpr-null mice would enhance the excretion of toxic compounds, such as BAs, and thus represents a compensatory response to the loss of Cpr-dependent detoxification. BAs in the intestine possess the ability to inhibit the growth of bacteria in the intestine. The reduced hepatic synthesis of BAs in H-Cpr-null mice likely results in an overgrowth of intestinal bacteria, which may contribute to the increase of secondary BAs in the circulation. For instance, despite the reduced hepatic production of CA, its microbiota product DCA is increased in both serum and livers of H-Cpr-null mice. Therefore, it appears that the alterations of BA concentration and composition in serum of H-Cpr-null mice could be attributed to a combined effect from enhanced intestinal BA absorption, hepatic BA excretion, extrahepatic BA synthesis, and increased secondary BA synthesis.
Not much consideration has been given to BA concentrations in serum. Some research has been conducted to determine whether BA concentrations might be useful to differentiate various liver diseases (Bathena et al., 2013). It has generally been thought that an increase in BAs in serum is secondary to high BA concentrations in liver, due to a decrease in uptake of BAs into liver, or an increased retro-transport of BAs from liver to serum to protect the liver from BA-induced toxicity. However, the present study demonstrates that, in the H-Cpr-null mouse model, there is an increase in BAs in serum when there is a decrease in BA concentrations in the liver. Thus, the body might purposely regulate BA concentrations in the serum. In the present study, it appears that the increase in serum BA concentration is primarily due to both an increase in transport of BAs from the intestine to the serum and an increase in retro-transport of BAs from the liver to the serum. If the only purpose of BAs was to aid in the absorption of fats and fat-soluble proteins, there would be no reason for regulating serum concentrations of BAs. However, it is now known that there is a BA receptor (G protein–coupled bile acid receptor) that regulates triglyceride, cholesterol, glucose, and energy metabolism, and thus the regulation of BA concentrations in the serum might be physiologically important.
To conclude, decreasing the activity of all microsomal P450s in the liver by Cre-Lox targeting of Cpr in hepatocytes of mice exerts differential effects on BA concentrations in liver and serum. Reduced BA concentration in livers of Cpr-null mice is attributed to an impaired classic pathway of BA synthesis, which is likely compensated by enhancing the alternative BA synthesis pathway. The increased serum BA concentrations in H-Cpr-null mice appear to be the result of enhanced intestinal BA absorption, hepatic BA retro-transport into the serum, and increased secondary BA synthesis. Overall, the present study suggests the importance of the alternative BA synthesis pathway in regulating BA concentrations in livers and serum of H-Cpr-null mice.
The authors thank Dr. Xinxin Ding (Wadsworth Center, State University of New York at Albany) for providing H-Cpr-null mice, and Lucy Lei for help with sample processing.
Participated in research design: Cheng, Zhang, Klaassen.
Conducted experiments: Cheng, Zhang.
Performed data analysis: Cheng, Zhang.
Wrote or contributed to the writing of the manuscript: Cheng, Zhang, Klaassen.
- Received May 19, 2014.
- Accepted July 15, 2014.
X.C. and Y.Z. contributed equally to this work.
This work was supported by the National Institutes of Health National Institute of Environmental Health Sciences [Grant R01-ES09649].
- apical sodium-dependent bile acid transporter
- bile acid
- bile acid CoA ligase
- bile acid CoA:amino acid N-acyltransferase
- bile salt export pump
- cholic acid
- chenodeoxycholic acid
- NADPH–cytochrome P450 reductase
- deoxycholic acid
- fibroblast growth factor 15
- farnesoid X receptor
- hepatocyte-specific deletion of NADPH–cytochrome P450 reductase
- intestinal bile-acid binding protein
- lithocholic acid
- muricholic acid
- multidrug resistance–associated protein
- Na+/taurocholate cotransporting polypeptide
- organic anion–transporting polypeptide
- organic solute transporter
- cytochrome P450
- small heterodimer partner
- steroidogenic acute regulatory protein
- taurine-conjugated CDCA
- taurine-conjugated DCA
- ursodeoxycholic acid
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics