https://orcid.org/0000-0003-0222-5476
Abstract
Accumulation of bile acids (BAs) in hepatocytes has a role in liver disease and also in drug-induced liver injury. The constitutive androstane receptor (CAR) has been shown to protect against BA-induced liver injury. The polymorphism of CAR has recently been shown to modify the pharmacokinetics and pharmacodynamics of various drugs. Thus, it was hypothesized that polymorphism of CAR may also influence BA homeostasis. Using CAR-null and WT mice, this study modeled the potential consequences of CAR polymorphism on BA homeostasis. Our previous study showed that chemical activation of CAR decreases the total BA concentrations in livers of mice. Surprisingly the absence of CAR also decreased the BA concentrations in livers of mice, but to a lesser extent than in CAR-activated mice. Neither CAR activation nor elimination of CAR altered the biliary excretion of total BAs, but CAR activation increased the proportion of 6-OH BAs (TMCA), whereas the lack of CAR increased the excretion of taurocholic acid, TCDCA, and TDCA. Serum BA concentrations did not parallel the decrease in BA concentrations in the liver in either the mice after CAR activation or mice lacking CAR. Gene expression of BA synthesis, transporter and regulator genes were mainly similar in livers of CAR-null and WT mice. In summary, CAR activation decreases primarily the 12-OH BA concentrations in liver, whereas lack of CAR decreases the concentrations of 6-OH BAs in liver. In bile, CAR activation increases the biliary excretion of 6-OH BAs, whereas absence of CAR increases the biliary excretion of 12-OH BAs and TCDCA.
The primary functions of BA production are to eliminate water-insoluble cholesterol, generate bile flow, and to assist digestion by emulsification and formation of micelles with fatty acids, monoglycerides, and fat-soluble vitamins (Monte et al., 2009). This beneficial function of BAs is mainly attributable to the amphipathic nature of BAs, with each BA having a hydrophilic and hydrophobic “face” (Hofmann and Hagey, 2014). A gradient of hydrophobicity among the predominant BA species has been described as UDCA<CA<CDCA<DCA<LCA, and the hydrophobicity of each BA is considered to be related to the potential for causing hepatotoxicity (Billington et al., 1980; Heuman, 1989; Palmer, 1972).
Thus, accumulation of bile acids (BAs) in hepatocytes is known to play a significant role in certain forms of liver disease and is also a contributing factor in drug-induced liver injury (Bhamidimarri and Schiff, 2013; Trauner et al., 1998). The reason for this is because of the hydrophobic detergent-like properties of BAs, which can induce oxidative stress, disrupt phospholipid bilayer membranes, stimulate an inflammatory response, and ultimately cause hepatocyte death (Danielsson et al., 1963; Lindstedt, 1957; Perez and Briz, 2009). Therefore, understanding the regulation of the synthesis, disposition, and pharmacodynamics of BAs are vital to prevent toxicity of endogenous and exogenous compounds.
Synthesis of BAs from cholesterol is the principal route of cholesterol elimination, and occurs in the liver through a series of hydroxylation, reduction, and oxidation reactions (Russell, 2003). The “classical” pathway of BA synthesis includes cholesterol 7α-hydroxylase Cytochrome P450 7a1 (Cyp7a1), the rate-limiting enzyme of BA synthesis. To a lesser extent, the alternative pathway of BA synthesis (through Cyp27a1 and Cyp7b1) also contributes to the total BA synthesis. The sterol 12α-hydroxylase (Cyp8b1) is required for synthesis of the major 12α-OH BA, cholic acid (CA) (Chiang, 2003). BAs synthetized by the liver are referred to as “primary” BAs, and in mice, the primary BAs are CA, chenodeoxycholic acid (CDCA), α-muricholic acid (αMCA), and βMCA. In mouse liver, BAs undergo conjugation primarily with taurine before biliary excretion. In intestine, secondary BAs are formed via deconjugation, dehydroxylation, epimerization, and oxidation reactions by bacteria to form “secondary” BAs (Russell, 2003).
Farnesoid X receptor (FXR) is a BA receptor that plays a central role in regulation of BA synthesis, and individual BAs differ in terms of capacity to activate FXR (CDCA>DCA>LCA>CA) (Parks et al., 1999). Activation of FXR in liver by BAs promotes transcription of small heterodimer partner (Shp), which represses transcription of Cyp7a1 by inhibiting the binding of Liver receptor homolog 1 (Lrh1) to the Cyp7a1 promoter (Goodwin et al., 2000). In enterocytes of the ileum, activation of FXR by BAs induces Fibroblast growth factor 15 (Fgf15) (FGF19 in humans). Following synthesis in the ileum, Fgf15 is released into the blood and circulates to the liver where it activates the FGF Receptor 4 (Fgfr4) to signal negative feedback regulation of Cyp7a1 in liver (Holt et al., 2003; Inagaki et al., 2005; Lu et al., 2000; Song et al., 2009; Xie et al., 1999). It has been shown that intestinal FXR activation appears to be more important in the regulation of the overall BA synthesis than hepatic FXR activation (Kong et al., 2012).
Adaptation of the liver is essential for effective biliary elimination of exogenous and endogenous compounds. Various nuclear receptors and transcription factors (e.g., AhR, PXR, CAR, PPARα, Nrf2) work as chemosensors and orchestrate the drug metabolism genes and transporters to increase the elimination of toxic compounds (Aleksunes and Klaassen, 2012). There is accumulating evidence that these chemosensors have effects on BA homeostasis as part of the adaptive responses to xenobiotics (Csanaky et al., 2018; Fader et al., 2017; Lickteig et al., 2016; Zhang et al., 2017,, 2018a).
One of the main xenobiotic sensors is constitutive androstane receptor (CAR; NR1I3), which regulates genes of ADME (Cui and Klaassen, 2016; Qatanani and Moore, 2005). CAR has been explored for the purpose of protecting against BA-induced liver injury (Halilbasic et al., 2013). We recently showed that CAR activation by the ligand 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) decreases total BAs in livers of mice, by decreasing the 12α-hydroxylated BA taurocholic acid (TCA), as well as increasing the biliary excretion of muricholic acids (Lickteig et al., 2016).
To gain a better understanding of the role of CAR in BA metabolism, gene expression of BA regulators, synthesis enzymes, and transporters have been explored previously in livers of CAR knockout mice. However, a relationship between mRNAs of BA synthesis enzymes, transporters in liver and transporters in ileum of CAR knockout mice has yet to be explored. In the previous studies, CAR knockout mice were treated with lithocholic acid (LCA), generally considered the most potent BA that causes liver injury (Beilke et al., 2009; Uppal et al., 2005; Zhang et al., 2004). Although these studies showed that CAR-null mice are more sensitive to LCA-induced hepatotoxicity, the effect of CAR deletion on the profile of individual BAs in blood, liver, or biliary excretion of BAs was not described.
Recently it has been shown that the polymorphism of CAR can modify the pharmacokinetics and pharmacodynamics of drugs, such as efavirenz, atazanavir, tacrolimus, artemisinin, and irinotecan (Chen et al., 2014; Mbatchi et al., 2018; Swart et al., 2012; Zang et al., 2015). Based on these findings it can be hypothesized that various polymorphisms of CAR may also influence BA homeostasis.
Given these deficiencies in our understanding of the potential role of genetic variation of CAR in BA homeostasis in the mouse model, the objectives of the present study were (1) to compare the individual BAs in liver and serum, as well as (2) to determine the biliary excretion of individual BAs in male and female CAR-null and WT-mice. In addition, the effect of CAR deletion on major pathways of BA regulation, synthesis, excretion, and uptake was also determined to gain insight into the pathways responsible for altered BA profile in the absence of CAR.
MATERIALS AND METHODS
Chemicals
Standards and internal standards for quantification of BAs were purchased from Steraloids, Inc (Newport, Rhode Island) and Sigma-Aldrich (St. Louis, Missouri). Unless otherwise noted, all other chemicals were purchased from Sigma-Aldrich.
Animals
Breeder pairs from the CAR-null mouse line on the C57BL/6 background, engineered by Tularik, Inc (South San Francisco, California) were obtained from the laboratory of Dr Ivan Rusyn and have been described previously (Ueda et al., 2002). Male and female C57BL/6 wild-type (WT) mice were obtained from Charles River Laboratories, Inc (Wilmington, Massachusetts). All mice underwent environment acclimation for at least one week and were housed in a standard temperature-, light- (12:12 h light:dark cycle), and humidity-controlled facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Mice had access to chow (Teklad Rodent Diet No. 8604, Harlan Teklad, Madison, Wisconsin) and water ad libitum. This study was approved by the Institutional Animal Care and Use Committee at the University of Kansas Medical Center. To provide a similar weight of animals for surgery, all male mice were 12–15 weeks of age, whereas females were 16–19 weeks of age (5–8 mice per group).
Collection of livers, blood, and ilea
Mice were anesthetized (pentobarbital, 50 mg/kg, IP) and blood was collected from the retro-orbital sinus. Blood samples were processed to obtain serum using Microtainer separating tubes (BD Biosciences, San Jose, California). Livers and ilea were removed, rinsed briefly with saline, and flash frozen (liquid nitrogen). All samples were collected between 8 am and 12 pm and stored at −80°C until analysis.
Bile collection surgery
Mice were anesthetized (ketamine, 100 mg/kg, IP/midazolam, 5 mg/kg, IP), and the common bile duct was cannulated with a 30-gauge needle attached to PE-10 tubing. Bile was collected for 40 min per mouse in 0.6-ml tubes on ice. Each volume of bile was determined gravimetrically, using 1.0 for specific gravity. All samples were collected between 8 am and 12 pm and stored at −80°C until analysis.
RNA extraction and messenger RNA quantification
Total RNA of livers and ilea was extracted using RNA-Bee reagent (Tel-Test, Inc, Friendswood, Texas) and dissolved in 0.1% diethyl pyrocarbonate. RNA concentrations were quantified spectrophotometrically at a wavelength of 260 nm. RNA integrity and quality were confirmed by agarose gel electrophoresis to visualize intact 18S and 28S bands. Messenger RNA of genes was determined using QuantiGene Plex 2.0 Assay (Affymetrix/Panomics, Inc, Fremont, California). Gene-specific probe sets were developed by Affymetrix/Panomics, Inc (sets Nos 21330 and 21383; Affymetrix/Panomics has been acquired by ThermoFisher Scientific; https://www.thermofisher.com/us/en/home/life-science/gene-expression-analysis-genotyping/quantigene-rna-assays/quantigene-plex-assay.html, Accessed June 25, 2019.). Samples were analyzed using a Bio-Plex 200 System Array reader with Luminex 100 xMAP, and data acquired using Bio-Plex Data Manager version 5.0 (Bio-Rad, Hercules, California).
Real-time RT-PCR was used to analyze Abca1, Abcg5, Abcg8, Atp8b1, Bcrp, β-klotho, Ent1, I-Babp, Mate1, Mdr1, Mrp1, Mrp4, Mrp6, Oatp1a4, Oatp1b2, Oat2, Oct1, Ostα, and Ostβ. These were normalized to ribosomal protein L13A (Rpl13a) of the same sample, as described earlier (Cui et al., 2012; Lickteig et al., 2016; Rockwell et al., 2012). Relative mRNA levels were calculated with WT controls set at 100%.
Ultraperformance liquid chromatography-tandem mass spectrometry analysis of BAs in mouse liver, bile, and serum
Bile acids were extracted and quantified using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), per previously published methods (Alnouti et al., 2008; Zhang and Klaassen, 2010).
Calculations of primary, secondary, hydroxylated, and 12α-hydroxylated BAs
Concentrations of BA groups, including primary (1°), secondary (2°), 6-hydroxylated (6-OH), and 12α-hydroxylated (12α-OH) were calculated by the sum concentration of the group members (Lickteig et al., 2016).
Statistical analysis
Individual values were log-transformed to obtain normal distributions. Student’s t tests were performed, and statistical significance was based on a p value of 0.05. Asterisks (*) denote differences between WT and CAR-null male or female mice.
RESULTS
Effects of CAR Deletion on Bile Acid Concentrations and Composition in Serum of Mice
The absence of CAR in male and female mice did not result in changes in the concentration of total (Σ) BAs in serum (Figure 1, top panels). However, in male CAR-null mice, taurine-conjugated BAs (TBAs) were lower (−60%), which was mainly attributable to decreases in TωMCA (−89%) and the tendency of decreases in TCA (Figure 1, middle left). Although unconjugated-BA (UBA) concentrations as a group did not significantly increase in serum of male CAR-null mice, several individual BAs increased, including DCA (+117%), CDCA (+118%), αMCA (+350%), ωMCA (+115%), and HDCA (+531%) (Figure 1, bottom left). In serum of female CAR-null mice, the only significant change was a 49% lower concentration of DCA (Figure 1, bottom right).
Effect of the absence of CAR on bile acid (BA) concentrations in sera of male and female mice. Individual BAs in sera of male and female, wild-type (WT), and CAR-null mice were quantified by ultraperformance liquid chromatography-tandem mass spectrometry (n ≥ 5 mice per group). Data represent means ± SEM. Asterisks (*) denote statistical differences from respective male or female WT mice (p < .05). Abbreviations: 1° BAs, primary bile acids; 2° BAs, secondary bile acids; 6-OH, 6-hydroxylated; 12α-OH, 12α-hydroxylated; BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; F, females; HDCA, hyodeoxycholic acid; LCA, lithocholic acid; M, males; MCA, muricholic acid; Σ-BAs, total bile acids; T-BAs, taurine-conjugated bile acids; U-BAs, unconjugated bile acids; UDCA, ursodeoxycholic acid; WT, wild-type. Color image is available in the online version of the article.
The relative proportion of each BA in the serum of male and female WT and CAR-null mice is shown in Figure 2. In each graph of WT mice, the most abundant BA is shown at the top, with the remaining BAs listed in order of most- to least-abundant (clockwise). For the graphs of the CAR-null mice, the clockwise order of BAs is identical to the order for WT mice of the same sex. The percent of each BA is shown for those individual BAs with composition of ≥ 5%, as well as for those that were statistically different. TCA (29%) was the most abundant BA in WT male mice, whereas the unconjugated counterpart CA was the most abundant in the CAR-null male mice (25%) (Figure 2). Consistent with the decrease in TωMCA concentrations, the relative proportion of TωMCA in serum also decreased from 17% to 1.9%, as did TDCA (from 3.9% to 1.3%) in the male CAR-null mice. The proportions of numerous unconjugated BAs were higher in the serum of male CAR-null mice than in CAR-WT mice, including βMCA (from 6.3% to 17%), DCA (from 5.2% to 12%), ωMCA (from 4.8% to 11%), αMCA (from 0.8% to 3.9%), CDCA (from 0.7% to 1.6%), and HDCA (from 0.2% to 1.6%) (Figure 2). The increased proportions of individual UBAs in the CAR-null male mice resulted in an overall increase in the unconjugated proportion of BAs in the serum (from 39% to 74%), whereas the proportion of taurine-conjugated BAs decreased from 61% to 26% (Supplementary Table 1).
Effect of the absence of CAR on serum composition of individual bile acids (BAs) in male and female mice. Individual BAs in sera of male and female, wild-type (WT), and CAR-null mice were quantified by ultraperformance liquid chromatography-tandem mass spectrometry (n ≥ 5 mice per group). Each section in pie charts was calculated to represent the mean proportion of an individual BA relative to the total BA concentration. Asterisks (*) denote statistical differences from respective male or female WT mice (p < .05). Abbreviations: 1° BAs, primary bile acids; 2° BAs, secondary bile acids; 6-OH, 6-hydroxylated; 12α-OH, 12α-hydroxylated; BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; F, females; HDCA, hyodeoxycholic acid; LCA, lithocholic acid; M, males; MCA, muricholic acid; Σ-BAs, total bile acids; T-BAs, taurine-conjugated bile acids; U-BAs, unconjugated bile acids; UDCA, ursodeoxycholic acid; WT, wild-type. Color image is available in the online version of the article.
The absence of CAR resulted in very few changes in the relative proportion of BAs in the serum of female CAR-null mice. The only significant alteration was an increase in CA (from 3.0% to 8.2%) (Figure 2). In summary, the effect of the absence of CAR on serum BA composition was different between males and females. In male CAR-null mice, the ratio of unconjugated to conjugated BAs shifted to an increase in unconjugated BAs. However, in serum of female CAR-null mice, BA composition was largely unchanged from the CAR-WT mice.
Effects of CAR Deletion on Bile Acid Concentrations and Composition in Livers of Mice
In contrast to serum, the effect of the absence of CAR was similar in livers of male and female mice. In livers of male CAR-null mice, concentrations of each of the major categories of BAs tended to be lower, although the decreases were statistically significant only for 6-OH BAs (−38%) (Figure 3, top left). The decrease in 6-OH BAs was attributable to decreased TωMCA (−49%) and βMCA (−44%), as well as Tα + βMCA tended to be decreased (Figure 3, middle and bottom left). The major BA TCA also tended to be decreased (−29%) in liver by the absence of CAR in male mice, although not statistically significant (Figure 3, middle left).
Effect of the absence of CAR on concentrations of bile acids (BAs) in livers of male and female mice. Livers were collected from male and female, wild-type (WT), and CAR-null mice and individual BAs were quantified by ultraperformance liquid chromatography-tandem mass spectrometry (n ≥ 5 mice per group). Data represent means ± SEM. Asterisks (*) denote statistical differences from respective male or female WT mice (p < .05). The numbers above some bars indicate the p values that were relatively close to statistical significance. Abbreviations: 1° BAs, primary bile acids; 2° BAs, secondary bile acids; 6-OH, 6-hydroxylated; 12α-OH, 12α-hydroxylated; BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; F, females; LCA, lithocholic acid; M, males; MCA, muricholic acid; Σ-BAs, total bile acids; T-BAs, taurine-conjugated bile acids; U-BAs, unconjugated bile acids; UDCA, ursodeoxycholic acid; WT, wild-type. Color image is available in the online version of the article.
In livers of female CAR-null mice, concentrations of each of the major categories of BAs were lower, including total (Σ) BAs (−38%), TBAs (−37%), UBAs (−65%), primary BAs (−35%), secondary BAs (−53%), 6-OH BAs (−34%), and 12α-OH BAs (−43%) (Figure 3, top right). In livers of female CAR-null mice, each of the 12α-OH BAs was decreased, including TCA (−42%), TDCA (−46%), CA (−52%), and DCA (−75%). Likewise, all of the 6-OH BAs analyzed were lower in the female CAR-null mice, including Tα + βMCA (−24%), TωMCA (−54%), αMCA (−43%), βMCA (−49%), and ωMCA (−69%). In addition, LCA (−96%) and UDCA (−61%) were lower in livers of female CAR-null mice.
The fraction of each BA in the livers of male CAR-null mice was very similar to that of male WT mice (Figure 4, Supplementary Table 2). This is consistent with the fact that the concentrations for many of the various individual BAs in the CAR-null mice tended to be lower by a similar extent. Just as the concentrations of TωMCA were lower in livers of CAR-null males than the CAR-WT male mice, the percent composition of BAs that was TωMCA also decreased (from 7.2% to 5.3%) (Figure 4, Supplementary Table 2). The TDCA concentration was similar in male WT and CAR-null mice, but the fraction that was TDCA was higher in livers of male CAR-null mice (from 5.4% to 8.3%).
Effect of the absence of CAR on liver composition of individual bile acids (BAs) in male and female mice. Livers were collected from male and female, wild-type (WT) and CAR-null mice and individual BAs were quantified by ultraperformance liquid chromatography-tandem mass spectrometry (n ≥ 5 mice per group). Each section in pie charts was calculated to represent the mean proportion of an individual BA relative to the total BA concentration. Asterisks (*) denote statistical differences from respective male or female WT mice (p < .05). Abbreviations: 1° BAs, primary bile acids; 2° BAs, secondary bile acids; 6-OH, 6-hydroxylated; 12α-OH, 12α-hydroxylated; BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; F, females; LCA, lithocholic acid; M, males; MCA, muricholic acid; Σ-BAs, total bile acids; T-BAs, taurine-conjugated bile acids; U-BAs, unconjugated bile acids; UDCA, ursodeoxycholic acid; WT, wild-type. Color image is available in the online version of the article.
In livers of CAR-null female mice, TCA was the most abundant BA but was less than in the WT mice (decreased from 53% to 49%) (Figure 4, Supplementary Table 2). The TωMCA fraction of BAs in livers of CAR-null female mice was lower to a similar extent (from 7.9% to 5.9%) as in CAR-null male mice. The two BAs with increased proportion in livers of female CAR-null mice were the non6, 12α-OH BAs, TCDCA (from 2.6% to 5.2%) and Tα + βMCA (from 22% to 27%). Given all of these changes, the balance of 12α-OH and non12α-OH BAs shifted toward increased non12α-OH BAs (from 39% to 44%), whereas the 12α-OH portion was lowered (from 61% to 56%) (Supplementary Table 2).
To summarize the effect of CAR deletion on the BA profile in livers of male and female mice, BA concentrations were generally lowered (by 31% in males, by 38% in females), with a decrease in both 12α-OH BAs (by 29% in males, by 43% in females) and 6-OH BAs (by 29% in males, by 34% in females). It is notable that liver concentrations of BAs derived from CDCA, including TCDCA, TLCA, and TUDCA, were not altered by the absence of CAR in either male or female mice.
Effects of CAR Deletion on Liver Weight and Bile Flow
The absence of CAR in male mice resulted in a 15% decrease in liver weight, expressed as a percent of body weight. In contrast, liver weight was not altered in female CAR-null mice (Figure 5, top panel). Bile flow relative to body weight and liver weight was similar in both male or female WT and CAR-null mice (Figure 5, middle and bottom panels).
Effect of the absence of CAR on liver weight and bile flow in male and female mice. Mice were anesthetized and bile was collected for 40 min from male and female, wild-type (WT), and CAR-null mice (n ≥ 5 mice per group). Liver weight is expressed as a percent of bodyweight (BW) (upper panel). Bile flow rates were normalized to BW (middle panel) and liver weight (lower panel). Data represent means ± SEM. Asterisks (*) denote statistical differences from respective male or female WT mice (p < .05). Abbreviations: F, females; M, males; WT, wild-type. Color image is available in the online version of the article.
Effects of CAR Deletion on Biliary Excretion of Bile Acids
The absence of CAR tended to increase the biliary excretion of BAs in both male and female mice (Figure 6). In male CAR-null mice, the biliary excretion of several BA categories tended to increase, but only the 12α-OH BAs were statistically significant (+113%), (Figure 6, top left), even though the livers of the CAR-null male mice were significantly smaller (Figure 5, top). The increased excretion of 12α-OH BAs in CAR-null male mice was largely attributable to increased excretion of TCA (+110%), as well as TDCA (+290%) and CA (+1272%) (Figure 6, middle and bottom left). Although the biliary excretion of 6-OH BAs was not altered in male CAR-null mice, excretion of TαMCA and αMCA were increased +143% and +405%, respectively. Increased biliary excretion of two additional non12α-OH BAs, TCDCA (+128%) and TLCA (+226%), was also observed in male CAR-null mice (Figure 6, middle left).
Effect of the absence of CAR on biliary excretion of bile acids (BAs) in male and female mice. Bile was collected from male and female, wild-type (WT), and CAR-null mice for 40 min (n ≥ 5 mice per group). Bile acids were quantified by ultraperformance liquid chromatography-tandem mass spectrometry. Data represent means ± SEM. Asterisks (*) denote statistical differences from respective male or female WT mice (p < .05). The numbers above some bars indicate the p values that were relatively close to statistical significance. Abbreviations: 1° BAs, primary bile acids; 2° BAs, secondary bile acids; 6-OH, 6-hydroxylated; 12α-OH, 12α-hydroxylated; BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; F, females; HCA, hyocholic acid; HDCA, hyodeoxycholic acid; LCA, lithocholic acid; M, males; MCA, muricholic acid; Σ-BAs, total bile acids; T-BAs, taurine-conjugated bile acids; U-BAs, unconjugated bile acids; UDCA, ursodeoxycholic acid; WT, wild-type. Color image is available in the online version of the article.
In female CAR-null mice, the trend of increased biliary excretion of the major categories of BAs, total BAs, TBAs, primary BAs, and 12α-OH BAs, was consistent with that of the male CAR-null mice, although none reached statistical significance (Figure 6, top right). Biliary excretion was higher for five individual BAs in female CAR-nulls, namely TCDCA (+109%), TαMCA (+197%), TDCA (+131%), TLCA (+249%), and TUDCA (+249%) (Figure 6, middle right and bottom right). Again, generally consistent with male CAR-null mice, biliary excretion in female CAR-null mice appears to be higher for TCA and CA than in female WT mice, although not statistically significant (Figure 6, middle right and bottom right).
In bile of male mice, TCA was the predominant BA in both WT and CAR-null mice, with TCA being higher in male CAR-null mice than WT mice (66% vs 56%) (Figure 7, Supplementary Table 3). The minor BAs TLCA (from 0.03% to 0.05%) and CA (from 0.01% to 0.08%) were also higher in the bile of male CAR-null than WT mice (Figure 7, Supplementary Table 3). There was also less of the quantitatively major TβMCA (from 32% to 20%), as well as the minor BAs THDCA (from 2.4% to 1.1%) and THCA (from 0.16% to 0.08%) in bile of male CAR-null than male CAR-WT mice. The relative proportion of 12α-OH BAs was higher (from 57% to 68%), whereas the non12α-OH BA proportion was less (from 43% to 32%) in the male CAR-null mice. This shift was largely due to a higher proportion of TCA in bile of male CAR-null mice (Supplementary Table 3). The lower 6-OH proportion (from 39% to 29%) is largely attributable to the lower TβMCA (Supplementary Table 3).
Effect of the absence of CAR on bile composition of individual bile acids (BAs) in male and female mice. Bile was collected from male and female, wild-type (WT), and CAR-null mice for 40 min (n ≥ 5 mice per group). Each section in pie charts was calculated to represent the mean proportion of an individual BA relative to the total BA concentration. Asterisks (*) denote statistical differences from respective male or female WT mice (p < .05). Abbreviations: 1° BAs, primary bile acids; 2° BAs, secondary bile acids; 6-OH, 6-hydroxylated; 12α-OH, 12α-hydroxylated; BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; F, females; HCA, hyocholic acid; HDCA, hyodeoxycholic acid; LCA, lithocholic acid; M, males; MCA, muricholic acid; Σ-BAs, total bile acids; T-BAs, taurine-conjugated bile acids; U-BAs, unconjugated bile acids; UDCA, ursodeoxycholic acid; WT, wild-type. Color image is available in the online version of the article.
In bile of female mice, TCA made up the greatest proportion of BAs in both WT and CAR-null mice (from 63% in both). As in CAR-null male mice, the proportion of TαMCA was higher in CAR-null females than WT females (from 5.4% to 9.2%). The quantitatively minor BAs THCA (from 0.11% to 0.07%) and THDCA (from 1.7% to 0.87%) were also lower in bile of female CAR-null mice.
To summarize the effect of CAR deletion on BA composition in bile of male and female mice, biliary excretion was similarly enhanced in male and female CAR-null mice. However, the enhancing effect on biliary excretion was more pronounced in male CAR-null mice than in female CAR-null mice, especially considering the significantly smaller livers of CAR-null male mice than WT male mice. Consistent with this, BA composition in bile of male CAR-null mice had more changes than in female CAR-null mice. In bile of male CAR-null mice, the increased TCA (110%) was responsible for the increase in the proportion of 12α-OH-BAs.
Effect of CAR Deletion on mRNAs of Bile Acid Synthesis and Regulation
In livers of male mice, the absence of CAR resulted in increased mRNA of Cyp27a1 (+26%), a cholesterol 27-hydroxylase that initiates the alternative pathway of BA synthesis (Figure 8, top left). Also in male mice, the absence of CAR in liver resulted in decreased mRNA of Cyp7b1 (−67%), an oxysterol 7α-hydroxylase that is responsible for the conversion of 25- and 27-hydroxycholesterol to intermediates in BA synthesis (Figure 8, top left).
Effect of the absence of CAR on mRNA of BA synthesis and regulation in livers of male and female mice. Livers were collected from male and female, wild-type (WT), and CAR-null mice (n ≥ 5 mice per group). Total RNA was analyzed by QuantiGene Plex 2.0 Assay, as well as by RT-qPCR. Relative mRNA levels were calculated with vehicle controls set as 100%. Data represent means ± SEM. Asterisks (*) denote statistical differences from respective male or female WT mice (p < .05). Abbreviations: Baat, Bile acid CoA:amino acid N-acyltransferase; Bal, bile acid CoA ligase; Cyp, cytochrome p450; FXR, farnesoid x receptor; F, females; Fgfr4, fibroblast growth factor receptor; Hnf4α, hepatocyte nuclear factor 4α; Lrh-1, liver receptor homolog-1; M, males; Shp, small heterodimer partner; WT, wild-type. Color image is available in the online version of the article.
In livers of CAR-null female mice, mRNA of Cyp7a1, the rate-limiting enzyme of BA synthesis, was increased (+123%) (Figure 8, top right). The absence of CAR in livers of female mice resulted in decreased mRNA of Shp (−66%) and Fgfr4 (−26%) (Figure 8, bottom right).
To summarize, expression of mRNAs of BA synthesis enzymes and regulators are different in male and female mice in the absence of CAR. The changes in male CAR-null mice do not indicate enhanced synthesis of BAs. In contrast, in livers of female CAR-null mice, the upregulation of Cyp7a1 and downregulation of Shp indicate a regulatory effect to increase BA synthesis.
Effect of CAR Deletion on mRNAs of Bile Acid and Xenobiotic Transporters in Livers of Male and Female Mice
In male mice, the absence of CAR resulted in relatively few changes in mRNA of transporters involved in BA transport in liver (Figure 9, top left). In livers of male CAR-null mice, Oatp1a4 was increased 54%; however, this transporter may not contribute to the in vivo hepatic uptake of BAs, but rather, is important for secondary BA metabolism (Zhang et al., 2013). Among mRNAs of BA transporters in liver, the major canalicular BA efflux transporter Bsep was increased 36%, and the sinusoidal BA efflux transporter Ostβ was increased 182% in livers of CAR-null male mice. Ostβ functions as a heterodimer with Ostα to mediate sinusoidal efflux of BAs, and Ostα mRNA did not change. In addition, in livers of male CAR-null mice, mRNA of the canalicular phospholipid flippase, Mdr2, and the sterol half-transporter, Abcg8, were increased by 53% and 87%, respectively.
Effect of the absence of CAR on mRNA of transporters in livers of male and female mice. Livers were collected from male and female, wild-type (WT), and CAR-null mice (n ≥ 5 mice per group). Total RNA was analyzed by QuantiGene Plex 2.0 Assay, as well as by RT-qPCR. Relative mRNA levels were calculated with vehicle controls set as 100%. Data represent means ± SEM. Asterisks (*) denote statistical differences from respective male or female WT mice (p < .05). Abbreviations: Bcrp, breast cancer resistance protein; Bsep, bile salt export pump; Ent, equilibrative nucleoside transporter; F, females; M, males; Mate, multidrug and toxin extrusion transporter; Mdr, multidrug resistance protein; Mrp, multidrug resistance-associated protein; Ntcp, Na(+)-taurocholate cotransporting polypeptide; Oatp, organic anion transporting polypeptide; Oct, organic cation transporter; Ost, organic solute transporter; WT, wild-type. Color image is available in the online version of the article.
In livers of female mice, the absence of CAR resulted in few and modest changes in mRNAs of BA transporters. The sinusoidal efflux transporter Mrp1, which transports glutathione-conjugated drugs and prostaglandins, was decreased (−45%) in livers of female CAR-null mice. Ostα, which is capable of sinusoidal efflux transport of BAs, was increased (+286%), but the mRNA of the Ostα partner Ostβ did not change.
To summarize, the absence of CAR had only a few effects on the abundance of mRNAs of BA and xenobiotic transporters in livers of male and female mice. Notably, the absence of CAR did not produce any changes in the mRNA of liver BA transporters in male and female mice.
Effect of CAR Deletion on mRNAs in Ilea of Male and Female Mice
In ilea of male mice, mRNA of Niemann-Pick c1-like 1 (Npc1l1) decreased (−26%) in the absence of CAR, but was unchanged in ilea of female CAR-null mice (Figure 10, top panels). In ilea of female mice, the oxysterol sensor Liver X receptor (Lxrα) was increased (+43%), whereas it was unchanged in male CAR-null mice (Figure 10, middle panels). In ilea of male CAR-null mice, mRNA of Mrp3 was increased 70%, but was not altered in CAR-null female mice (Figure 10, bottom panels). The absence of CAR in male mice resulted in decreased mRNA of Shp (−79%) and Mrp2 (−45%) in ilea, whereas the absence of CAR in female mice resulted in increased mRNA of Shp (+165%) and Mrp2 (+117%) (Figure 10, middle and bottom panels). In summary, most of the mRNAs of BA regulation and transport analyzed in ilea were unchanged by the absence of CAR in both males and females. However, the absence of CAR in male and female mice had the opposite effects on expression of mRNA of Shp and Mrp2 in ilea.
Effect of the absence of CAR on mRNA of BA regulators and transporters in ilea of male and female mice. Ilea were collected from male and female, wild-type (WT), and CAR-null mice (n ≥ 5 mice per group). Total RNA was analyzed by QuantiGene Plex 2.0 Assay, as well as by RT-qPCR. Relative mRNA levels were calculated with vehicle controls set as 100%. Data represent means ± SEM. Asterisks (*) denote statistical differences from respective male or female WT mice (p < .05). Abbreviations: Abc, ATP-binding cassette; Asbt, apical sodium-dependent bile acid transporter; FXR, farnesoid X receptor; F, females; Fgf, fibroblast growth factor; I-babp, ileal bile acid binding protein; Lxrα, liver x receptor α; M, males; Mrp, multidrug resistance-associated protein; Npc1l1, Niemann-Pick c1-like 1; Ost, organic solute transporter; Σ-BAs, total bile acids; Shp, small heterodimer partner; Tgr5, transmembrane G protein-coupled receptor 5; WT, wild-type. Color image is available in the online version of the article.
DISCUSSION
In clinical practice, the determination of BA concentrations in serum has been shown to be useful in monitoring liver function and BA metabolism (Korman et al., 1974; Skrede et al., 1978). In serum of male and female mice, the absence of CAR resulted in very few changes in relative BA composition (Figs. 1 and 2). However, the hepatic concentrations of BAs tended to be lower whereas the biliary excretion was higher in CAR-null male and female mice compared with WT mice (Figs. 3 and 6). In addition, whereas the relative composition of BAs in liver did not change, the proportion of 12α-OH BAs was higher in the bile of CAR-null mice (Figure 4). Differences in BA composition in liver and biliary excretion of BAs between WT and CAR-null mice, does not correlate with changes in serum. Therefore, BA concentrations in serum are not a useful indicator of either the liver or biliary BA profile in CAR-null mice.
To gain insight into potential mechanisms for decrease in BA concentrations of liver and the increased biliary BA excretion in CAR-null mice, the mRNAs of BA synthesis, regulation and transport in livers were analyzed. Cyp7a1, the rate-limiting enzyme of BA synthesis is increased in livers of female CAR-null mice (Figure 8). Given that BA concentrations in liver decrease in the absence of CAR in female mice, higher expression of Cyp7a1 to increase BA synthesis might be expected as a means to compensate for the lower concentrations of BAs in the liver.
FXR is a BA receptor that plays a central regulatory role in BA homeostasis of the liver and intestine. In liver, it is through FXR that BAs exert negative feedback regulation of BA synthesis. Specifically, activation of hepatic FXR by BA ligands induces transcription of Shp, which in turn represses Lrh1 from promoting Cyp7a1 and Cyp8b1 transcription (Chiang, 2003; Goodwin et al., 2000). Shp inhibits Lrh1-mediated upregulation of Cyp7a1. Small heterodimer partner mRNA is lower in livers of female CAR-null mice than WT mice, which would allow the increase in Cyp7a1 mRNA. In contrast, Cyp7a1 mRNA in male CAR-null mice is not changed, despite the decrease in total BAs in liver, including the 6-OH- and 12α-OH-BAs. Cyp27a1 and Cyp7b1 of the alternative pathway are increased and decreased, respectively, in livers of male CAR-null mice, which also does not clarify a mechanism for the decreases in 6-OH- or 12α-OH-BAs. Given that the BA profile in livers of males and females is similar in the absence of CAR, the contribution of these changes in expression of mRNAs of BA synthesis and regulation in liver is unclear.Previously our laboratory showed that absence of CAR in male mice does not change free and total cholesterol levels in serum but significantly decreases the free and total cholesterol levels in liver (Zhang et al., 2018b). Therefore, it is highly possible that the lower concentrations of BAs and increased expression of BA synthetic enzymes are the consequences of the shortage of cholesterol, the precursor of the BA synthesis.
With regard to the contribution of BA transporters to the decreased BAs in liver and increased biliary excretion of BAs in the absence of CAR, there were few changes in mRNA of transporters in liver (Figure 9). Na(+)-taurocholate cotransporting polypeptide (Ntcp) and Oatp1b2 are the major uptake transporters of conjugated and unconjugated BAs, respectively, into mouse liver (Csanaky et al., 2011; Slijepcevic et al., 2015). Oatp1b2 and Ntcp mRNA levels are not different in the absence of CAR, in either male or female mice, and thus there is no evidence that altered uptake plays a role in the major decreases in liver BA concentrations observed in this study. In livers of male mice, the mRNA of Bsep, the major biliary export transporter of BAs, is increased (36%) in CAR-null mice, which may explain the decrease in total BAs in liver and the increase in biliary excretion of total BAs. Concomitantly with Bsep, the mRNA of the canalicular phospholipid floppase Mdr2 is also increased in CAR-null male mice (Figure 8). Enhanced expression of Mdr2 and a concomitant increase in phospholipids have been shown to be protective against the canalicular damage caused by the enhanced biliary excretion of BAs after partial hepatectomy (Csanaky et al., 2009). Just as liver mass is decreased following partial hepatectomy, livers of CAR-null male mice are also 15% smaller than livers of WT mice.
Despite a similar decrease in total BAs in liver and a tendency for increased biliary excretion of BAs in livers of female CAR-null mice, the liver size and the mRNA of Bsep was unchanged. Although Ostα and Ostβ mRNAs are increased in female and male CAR-null mice, respectively, the functional Ost transporter is an Ostα-Ostβ heterodimer, and thus increased mRNA of one subunit may not result in increased Ostα-Ostβ protein (Dawson et al., 2005). Overall, changes in the expression of BA transporters in absence of CAR do not appear to fully explain the changes of BA profile of liver and bile, especially the differences between male and female mice.
Given the increased biliary excretion of BAs that occurs in male and female CAR-null mice, it is expected that ilea of CAR-null mice are exposed to higher amounts of BAs compared with WT mice. With respect to regulation of BA synthesis, it is known that BA activation of FXR in ilea enterocytes induces the expression of Fgf15 mRNA (Kong et al., 2012). Fgf15 is released from enterocytes into the blood to signal back to the liver to decrease BA synthesis. In both male and female mice, the absence of CAR does not alter mRNA levels of the intestinal FXR target gene Fgf15. This indicates that although BA concentrations are decreased in livers of CAR-null mice, this decrease does not appear to be mediated by the FXR-Fgf15 pathway.
In addition to the aforementioned differences in mRNA levels of Bsep (Figure 9) and Cyp7a1 (Figure 8) in livers of male and female CAR-null mice, mRNAs of Shp and Mrp2 in ilea are also regulated differently in male and female CAR-null mice. Shp is a target gene of FXR in mouse enterocytes. Absence of CAR decreases Shp mRNA in ilea of males, but increases it in females. The same effect of the absence of CAR occurs in ilea for Mrp2. Mrp2 is known to be an efflux transporter of sulfate-conjugated BAs, which are a very minor constituent of the BA pool in mice (Huang et al., 2010). Taken together, these sex differences in mRNA levels of BA synthesis and transport genes in CAR-null mice seem to indicate that the regulatory interactions of CAR are different between male and female mice.
We have previously shown that CAR activation in mice by the synthetic CAR ligand TCPOBOP decreases BA concentrations in livers of mice, primarily by decreasing the concentration of 12α-hydroxylated BAs, especially TCA (Lickteig et al., 2016). Following TCPOBOP treatment, decreased concentrations of TCA in liver were likely due to downregulation of expression of Cyp8b1, which is responsible for the 12α-hydroxylation of BAs. Given the decrease in TCA in liver, biliary excretion of the quantitatively major muricholic acids, TαMCA and TβMCA, was significantly increased. Considering these effects of CAR activation, one might expect the absence of CAR to produce effects that are the opposite of those that resulted from CAR activation. Surprisingly, that is not the effect that occurs in CAR-null mice: First, whereas CAR activation by TCPOBOP appears to be more selective for 12α-OH BAs, the absence of CAR results in a decrease in liver concentrations of most of the major structural categories of BAs. Second, whereas CAR activation with TCPOBOP primarily increased the biliary excretion of 6-OH BAs, the absence of CAR resulted in enhanced biliary excretion of 12α-OH BAs and TαMCA. Third, CAR activation by TCPOBOP increases the liver weight and bile flow in male and female mice. Although, the absence of CAR does decrease the liver weight in male mice, CAR absence does not alter the liver weight in female mice, nor bile flow in male or female mice.
CAR-null mice have been shown to be more sensitive to LCA-induced hepatotoxicity than WT mice (Beilke et al., 2009; Uppal et al., 2005; Zhang et al., 2004). This seems to indicate that CAR contributes to BA homeostasis and has, to a certain degree, a protective role in liver of WT mice, even in the absence of activation by classical CAR activators, such as phenobarbital. Considering this, the decreased concentrations of BAs in livers of CAR-null mice and increased biliary excretion may be a regulated response of the liver that is intended to reduce the risk of liver injury by BAs, should BAs accumulate in the absence of CAR. To our knowledge, there is no evidence indicating altered development of the liver or biliary system of CAR-null mice that can help explain the decreased liver BA concentrations and enhanced biliary excretion. Based on the data presented in this manuscript, investigation into association of CAR polymorphisms and liver diseases having BA-mediated injury may be warranted.
To summarize, the gene expression of BA synthesis and transporters are largely similar in livers of male and female mice in the absence of CAR compared with WT mice. The increased Cyp7a1 mRNA in female CAR-null versus the decreased liver size and increased Bsep and Mdr2 in male CAR-null indicate that the absence of CAR does have some sex-specific effects on gene regulation of BA homeostasis in mice. The absence of CAR from male and female mice results in slightly decreased concentrations of BAs in liver and increased biliary excretion of 12α-OH BAs without altering bile flow. The BA concentrations in the serum minimally change and do not reflect the changes of the concentrations in liver and bile of CAR-null mice.
SUPPLEMENTARY DATA
Supplementary data are available at Toxicological Sciences online.
DECLARATION OF CONFLICTING INTERESTS
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
ACKNOWLEDGMENTS
The authors would like to thank all members of our laboratories for technical assistance with blood and tissue collection.
FUNDING
This work was supported by the National Institute of Environmental Health Sciences (R01 ES009649); National Institute of Diabetes and Digestive and Kidney Diseases (F32 DK092069); and Children's Mercy Startup for ILC.
REFERENCES
Author notes
These authors contributed equally to this study.
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