Expression of the Pregnane X Receptor in Mice Antagonizes the Cholic Acid–Mediated Changes in Plasma Lipoprotein Profile
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动脉硬化血栓血管生物学 2005年第10期
From INSERM U498 (D.M., L.L., A.A., P.G.), Faculté de Médecine, Dijon, France; Pharmaceutical Sciences (C.B.-C., L.L., J.D.S., E.G.S., M.A.), St. Jude Children’s Research Hospital, Memphis, Tenn.
Correspondence to Dr Mahfoud Assem, Department of Pharmaceutical Sciences, St Jude Children’s Research Hospital, 332 N. Lauderdale Ave, Memphis, TN 38105-2794. E-mail Mahfoud.Assem@stjude.org
Abstract
Objective— Modification of lipoprotein metabolism by bile acids has been mainly explained by activation of the farnesyl X receptor (FXR). The aim of the present study was to determine the relative contribution of the pregnane X receptor (PXR), another bile acid–activated nuclear receptor to changes in plasma lipoprotein profile.
Methods and Results— Wild-type mice, Pxr-deficient mice, and Pxr-null mice expressing human PXR (Pxr-null SXR-Tg mice) were fed a cholic acid–containing diet, and consequences on plasma lipoprotein profiles and target gene expression were assessed. Cholic acid produced significant decreases in high-density lipoprotein (HDL) cholesterol, plasma apolipoprotein (apo)A-I and hepatic apoA-I mRNA in wild-type mice. Interestingly, the effect of cholic acid was significantly more pronounced in Pxr-deficient mice, indicating that PXR contributes to the weakening of the effect of bile acids on lipoprotein metabolism. Reciprocally, changes in HDL/apoA-I profiles were abolished in Pxr-null SXR-Tg mice in which PXR-responsive genes, particularly those involved in bile acid detoxification were readily activated after cholic acid treatment.
Conclusion— PXR expression in mice antagonizes the cholic acid–mediated downregulation of plasma HDL cholesterol and apoA-I, and magnification of PXR/SXR-mediated changes may constitute a new mean to counteract the effects of bile acids on plasma lipoproteins.
To determine the contribution of PXR to the effect of bile acids on plasma lipoproteins, wild-type mice, PXR-deficient mice, and Pxr-null mice expressing human PXR were fed a cholic acid–containing diet. Mouse and human PXR were found to antagonize the cholic acid-mediated decreases in plasma HDL cholesterol and apoA-I.
Key Words: apolipoprotein A-I ? bile acids ? farnesyl X receptor ? high-density lipoproteins ? pregnane X receptor ? steroid and xenobiotic receptor
Introduction
High-density lipoproteins (HDL) and their major protein component, apolipoprotein A-I (apoA-I), are protective factors against the development of atherosclerosis.1–4 These observations raised a considerable interest in the development of new therapeutic approaches that aim at increasing plasma HDL cholesterol and apoA-I levels. ApoA-I gene expression is under the control of multiple factors, including several nuclear receptors. ApoA-I gene expression is negatively regulated by the apolipoprotein regulatory protein-I (ARP-1) (NR2F2) and the farnesyl X receptor (FXR) (NR1H4),5–7 whereas it is positively regulated by the hepatocyte nuclear factor-4 (HNF-4) (NR2A1), the peroxisome proliferator receptor alpha (PPAR) (NR1C1), and the liver receptor homolog-1 (LRH-1) (NR5A2).8–10
See page 2016
Although several ligands of pregnane X receptor (PXR) (NR1I2) were reported to increase HDL cholesterol as well as apoA-I and apoA-I mRNA levels in rodents and humans,11–15 the relative contribution of PXR to the regulation of plasma HDL levels in vivo remains to be ascertained. Interestingly, besides its role as a xenobiotic sensor,16–17 PXR responds to bile acids that also constitute well-recognized FXR ligands.18–22 Because PXR and FXR have opposite effects on apoA-I expression, it can be hypothesized that the final effect of bile acids on lipoprotein profile may actually reflect antagonistic contributions of PXR and FXR activations. To address the latter point in a comprehensive way, wild-type (WT) mice, PXR-deficient mice (Pxr-null mice),23 and mice expressing human PXR but not mouse PXR (Pxr-null SXR-Tg mice)23 were fed a 1% cholic acid (CA)-containing diet in the present study. We report here that PXR expression in mice antagonizes the CA-mediated downregulation of plasma HDL cholesterol and apoA-I. Interestingly, the negative effect of CA on plasma HDL profile could be completely reversed by elevated expression of human PXR in Pxr-null SXR-Tg mice, indicating that a magnification of PXR/SXR-mediated changes may constitute a new mean to counteract the negative effects of bile acids on lipoprotein profile.
Materials and Methods
Animals
WT mice, age-matched mice deficient for murine Pxr (Pxr-null),23 and age-matched mice expressing human PXR (Pxr-null SXR-Tg) under the control of the albumin promoter23 were used in the present study. The Pxr-null and Pxr-null SXR-Tg mice were generously provided by Dr Ronald Evans. WT, Pxr-null, SXR-Tg mice and Pxr-null mice were all of mixed genetic background (129/C57BL6). Mice were housed in a pathogen-free animal facility under a standard 12-hour light/12-hour dark cycle. All diets were prepared by Bio-serv (Frenchtown, NJ) and were based on a standard AIN-93G rodent diet. The CA diet was identical to the control diet but supplemented with 1% (w/w) CA. Male mice were 8 to 12 weeks of age at the start of the study. Five to 6 animals per group were used in the present study. All protocols and procedures were approved by the NCI Division of Basic Sciences Animal Care and Use Committee and are in accordance with the National Institutes of Health guidelines.
Plasma and Liver Tissue Sampling
After 5 days of feeding the indicated diet, animals were anesthetized with isoflurane and blood samples were collected by intra-cardiac puncture in heparin-containing tubes that were centrifuged at 5000 rpm for 10 minutes. Plasmas were harvested and stored at –80°C. Livers were excised, weighed, immediately snap-frozen in liquid nitrogen (LN2), and stored at –80°C before mRNA isolation and biochemical analysis.
Hepatic Lipid Analysis
Qualitative analysis of hepatic bile acids was performed by capillary gas-liquid chromatography as described previously.24,25 Quantitative determination of total hepatic bile acids was performed by an enzymatic assay (Colorimetric Total Bile Acid Assay Kit; Bio Quant, San Diego, Calif) on hepatic alcoholic extract obtained as previously described.26 Total cholesterol and triglycerides were determined as described previously.25,27
Plasma Lipid and Apolipoprotein Analysis
All assays were performed on Victor2 1420 Multilabel Counter (Perkin Elmer life Science, Boston, Mass). Cholesterol, phospholipids, and triglycerides were determined by enzymatic methods as described previously.25 HDL and non-HDL cholesterol plasma fractions were determined as cholesterol concentration in d>1.07 and d<1.07 plasma fractions respectively. Mouse apoA-I concentration was determined by a nonimmunologic method. Briefly, plasma samples (0.25 μL) were submitted to electrophoresis on 5% to 15% polyacrylamide gradient gel (Invitrogen, Carlsbad, Calif). Gel were subsequently stained by Coomassie brilliant blue and scanned on a GS-800 densitometer (Biorad, Hercules, Calif). The amount of apoA-I per sample was determined quantitatively by comparison with apoA-I standards that were performed in parallel with the samples.
Native Polyacrylamide Gradient Gel Electrophoresis
Total lipoproteins were separated by ultra centrifugation as the d<1.21 g/mL plasma fraction with one 5.5 hour, 100 000-rpm spin in a TLA100 rotor in a TLX ultracentrifuge (Beckman, Krefeld, Germany). Lipoproteins were then applied to a 1.5% to 25% polyacrylamide gradient gel (Spiragel 1.5 to 25.0; Spiral, Couternon, France), and electrophoresis was conducted as recommended by the manufacturer. Gels were subsequently Coomassie stained and the distribution profiles of lipoprotein were determined by comparison with protein standards (HMW kit; Amersham Bioscience, Uppsala, Sweden) using a GS 800 densitometer.
RNA Isolation and Polymerase Chain Reaction Methods
Total RNA was extracted using Trizol reagent (Life technologies, Carlsbad, Calif). Specific mRNAs were analyzed by quantitative real-time reverse-transcription polymerase chain reaction using the ABI Prism 7900HT (Applied Biosystems, Foster City, Calif). Briefly, 5 μg of RNA were reverse-transcribed into cDNA using MuMLV retrotranscriptase and oligo dT (Life technologies); 50 ng of the cDNA mixture were used. Specific cDNAs were amplified using specific primers (see supplemental data at http://atvb.ahajournals.org).Values were normalized to Gapdh levels. Relative mRNA levels were evaluated using Ct method.
Western Blot
The preparation of liver homogenates and Western blot analysis were performed as described elsewhere.19,28 Anti-Bsep, Mrp4, and cyp 3a11 antibodies were described previously.28–30 Rabbit anti-Mrp2 was provided by Dr Bruno Stieger, Zurich, Switzerland.
Statistical Analysis
Mann–Whitney U test was used to determine the significance between the data means.
Results
Cholic Acid Feeding Reduces Cholesterol and ApoA-I Levels in WT and Pxr-Null Mice, but not in Pxr-Null SXR-Tg Mice
In agreement with previous studies,31 total cholesterol, esterified cholesterol, phospholipid, and HDL cholesterol were significantly lower in WT mice fed a 1% cholic acid diet than in WT mice fed the standard chow (Table 1). Nondenaturing polyacrylamide gradient gel electrophoresis confirmed that the HDL fraction was mostly and selectively affected by the 1% CA treatment (Figure 1), which was accompanied by a significant reduction in plasma apoA-I concentration (0.56 g/L in WT mice fed 1% CA versus 1.18 g/L in WT mice fed the control diet; P<0.05) (Figure 1).
TABLE 1. Effect of 1% CA Diet on Plasma Lipid Parameters
Figure 1. Plasma ApoA-I concentration and lipoprotein profile in WT, Pxr-null, and Pxr-null SXR-Tg mice fed either control or a 1% CA diet. A, Total plasma lipoproteins were submitted to electrophoresis on 1.5% to 25% polyacrylamide gradient gels that were stained for proteins as described in Materials and Methods. B, Plasma apoA-I concentrations were determined as described in Materials and Methods. (*) indicates a significant difference from homologous mice fed the control diet and Pxr-null SXR-Tg mice fed the 1% CA diet (P<0.05 in both cases; Mann–Whitney test).
Whereas lipoprotein phenotypes of Pxr-null mice and WT mice were similar under standard diet, CA induced significantly greater reductions in total cholesterol, esterified cholesterol, phospholipids, triglycerides, and HDL cholesterol in Pxr-null mice after treatment (Table 1). The CA-mediated decreases in plasma apoA-I concentration were of similar magnitude in Pxr-null and WT mice (Figure 1). Strikingly, the CA-mediated changes in lipid concentration and HDL distribution no longer appeared in Pxr-null SXR-Tg mice, with the exception of a reduction in plasma phospholipids (Table 1 and Figure 1).
The Accumulation of Bile Acids in the Liver Is Higher in Pxr-Null Mice Than in WT and Pxr-Null SXR-Tg Mice Under CA Diet
In all mouse lines, levels of cholic, deoxycholic, and chenodeoxycholic acids were increased by 5- to 10-fold by the CA diet, whereas the concentrations of 6-OH bile acids (ie, muricholic acids) remained unchanged (Figure 2). Interestingly, the increase in total bile acids, CA, and deoxycholic acid were significantly higher in the liver of Pxr-null mice than in the liver of WT and Pxr-null SXR-Tg mice (Figure 2). As shown in Table 2, hepatic triglyceride levels remain unchanged in all mouse lines whether they received the control chow or the CA-enriched diet. Hepatic triglyceride concentration was significantly higher in Pxr-null-SXR-Tg mice as compared with WT or Pxr-null mice under control and CA diets. Interestingly, the CA diet induced a significant increase in hepatic cholesterol concentration in WT and Pxr-null mice but not in Pxr-null SXR-Tg mice.
Figure 2. Hepatic bile acid content in WT, Pxr-null and Pxr-null SXR-Tg mice fed a 1% CA diet. Hepatic bile acid content was determined as described in Materials and Methods. *Significant difference from WT and Pxr-null SXR-Tg mice fed the 1% CA diet (P<0.05; Mann–Whitney test).
TABLE 2. Effect of 1% CA Diet on Hepatic Lipid Concentrations
CA Feeding Activates FXR Target Genes in WT, Pxr-Null, and Pxr-Null SXR-Tg Mice
Expression of Cyp7a1 and Cyp8b1, ie, 2 genes indirectly regulated by FXR,32 were dramatically reduced after CA treatment in all cases, indicating an efficient suppression of bile acid synthesis (Figure 3A). The expression of small heterodimeric partner (Shp) and bile salt export pump (Bsep), ie, 2 direct FXR targets,32,33 was significantly increased after cholic treatment. Bsep mRNA levels were increased by 7-fold in the liver of CA-treated Pxr-null SXR-Tg mice, but only by 2-fold in CA-treated WT and Pxr-null liver (P<0.05) (Figure 3A). Induction of Bsep mRNA levels was accompanied by a proportional increase in Bsep protein (data not shown).
Figure 3. Relative changes in hepatic mRNA levels of FXR target genes (A) and PXR target genes (B) in WT, Pxr-null, and Pxr-null SXR-Tg mice fed a 1% CA diet. Total RNA was extracted from the liver and real time quantitative polymerase chain reaction was performed. Data were standardized for Gapdh mRNA, and mRNA levels in WT mice receiving the standard diet was set at 1.00. (a) indicates a significant difference from homologous mice fed the control diet; (b) indicates a significant difference from WT mice fed the 1% CA diet (P<0.05; Mann–Whitney test).
CA Feeding Activates PXR Target Genes Only in Pxr-Null SXR-Tg Mice
As shown in Figure 3B, PXR target genes were minimally affected by the 1% CA diet in WT and Pxr-null mice. In contrast, mRNA levels of Cyp3a11, Mrp2, and Mrp4 were markedly increased (4- to 6-fold increases) in Pxr-null SXR-Tg mice receiving the 1% CA diet as compared with control diet (Figure 3B). Activation patterns were confirmed by hepatic protein analysis (data not shown).
CA Feeding Decreases Hepatic ApoA-I mRNA Levels in WT and Pxr-Null Mice, but not in Pxr-Null SXR-Tg Mice
As shown in Figure 4A, no significant difference in hepatic apoA-I mRNA levels was observed between WT, Pxr-null, and Pxr-null SXR-Tg mice when fed the standard diet. In WT mice, and as previously reported,31 the 1% CA diet led to a significant 40% reduction in hepatic apoA-I mRNA levels as compared with WT mice fed the control diet. Similar reduction in hepatic apoA-I mRNA levels was also observed in Pxr-null mice under the CA diet. In contrast, in Pxr-null SXR-Tg mice, hepatic apoA-I mRNA levels remained constant under the CA-containing diet (Figure 4A), as were plasma apoA-I concentrations (Figure 1).
Figure 4. Abundance of hepatic ApoA-I mRNA (A) and Sr-b1, Abcg1, and Abca1 (B) in WT, Pxr-null, and Pxr-null SXR-Tg mice fed either control or 1% CA diet. Total RNA was extracted from the liver and real-time quantitative polymerase chain reaction was performed and analyzed as described in Figure 3 legend. (a) indicates a significant difference from homologous mice fed the control diet; (b) indicates a significant difference from WT mice fed the 1% CA diet (P<0.05; Mann–Whitney test).
CA Feeding Reduces Hepatic Abca1 mRNA Levels in Pxr-Null Mice, but not in WT and Pxr-Null SXR-Tg Mice
As shown in Figure 4B, the mRNA levels of Sr-b1 and Abcg1, 2 genes involved in HDL metabolism, were significantly increased by 2- to 3-fold in the 3 mouse lines by the CA diet. In addition, a significant reduction of Abca1 mRNA level was observed in Pxr-null mice receiving the CA diet but not in WT mice and Pxr-null SXR-Tg mice.
Discussion
Bile acids modulate the atherogenicity of the lipoprotein profile.34 In particular, they act as negative regulators of hepatic apoA-I transcription;7 this downregulation has been explained either in terms of a direct effect of activated FXR at the apoA-I gene promoter,7 or in terms of an indirect effect of FXR through the SHP/LRH-I pathway.9 Activation of FXR by bile acids also reduces plasma triglyceride concentration by decreasing both hepatic lipogenesis and secretion of very-low-density lipoprotein.35,36 Besides FXR, bile acids are also potent activators of another nuclear receptor, ie, PXR.18–22 Interestingly, several studies have reported that administration of pharmacological PXR agonists is associated with significant increases in both hepatic apoA-I transcription and circulating levels of apoA-I and HDL cholesterol in rats, mice, and humans.11–15, 37–39 The purposes of the present study were: (1) to establish the relative contribution of PXR to the effect of bile acids on plasma lipoprotein profile through the comparison of WT and Pxr-null mice; and (2) to determine whether overexpression of human PXR in Pxr-null SXR-Tg mice was able to counteract the deleterious, FXR-mediated effects of bile acids.
In agreement with previous observations,31 the CA-containing diet significantly reduced hepatic apoA-I expression and HDL cholesterol levels in WT mice. Although the effects of CA were reported to be exacerbated in PXR-deficient mice, in particular with more pronounced effects on total cholesterol and triglyceride levels as compared with WT mice,40 consequences of PXR-deficiency in terms of HDL structure and composition had not been specifically addressed. The present studies demonstrate for the first time that CA treatment of PXR-deficient mice produced more profound effects on plasma HDL cholesterol as compared with WT counterparts. Because exacerbation of the effect of CA on HDL cholesterol in PXR-deficient mice was not accompanied by further decrease in apoA-I gene expression in the present study, we thought further for differential expressions of specific genes that are known to affect lipidation (ie, ABCA1 and ABCG1)41–42 or catabolism (ie, SR-BI)43 of HDL. While induction of SR-BI by bile acids in WT mice confirmed previous studies,44 it is reported for the first time that bile acids positively regulate hepatic ABCG1 mRNA levels. Because hepatic overexpression of either Sr-b1 or Abcg1 are known to induce a reduction of HDL cholesterol levels in mice, caused by an increase in hepatic HDL catabolism,41,45 it is possible that these two proteins contribute in addition to apoA-I to the bile acid-mediated reduction of HDL. More importantly, and in contrast to Sr-b1 and Abcg1 mRNA levels that were induced by the CA treatment in the 3 mouse lines studied, Abca1 mRNA levels were shown in the present study to be significantly reduced by CA in Pxr-null mice only. Recent observations have demonstrated that hepatic ABCA1 is a key factor contributing to lipidation and formation of nascent HDL 42; therefore, downregulation of Abca1 expression arises as a possible mechanism for the greater effect of the CA diet on HDL cholesterol levels in Pxr-null mice as compared with WT mice.
In contrast to observations made with WT and PXR-deficient mice, CA-mediated changes in HDL/apoA-I profiles were completely abolished in mice expressing human PXR (Pxr-null SXR-Tg mice). Moreover, typical PXR-responsive genes were readily activated after CA treatment in this latter mouse line only. Different mechanisms that are not mutually exclusive may explain the positive effect of PXR. First, a direct transactivating effect of PXR on the apoA-I promoter is possible, and it is consistent with the positive effect of phenobarbital on apoA-I transcription as already reported.38–39 Alternatively, it is possible that PXR interferes with some FXR-mediated pathways by inducing qualitative and quantitative changes in hepatic bile acid composition.46 This hypothesis is supported by significant changes in hepatic bile acid composition in Pxr-null mice treated with CA and is susceptible to explain the exacerbation of the effect of the CA diet on lipoprotein profile in Pxr-null mice. However, no qualitative or quantitative differences in hepatic bile acid profiles were observed between Pxr-null SXR-Tg and WT mice fed the CA-enriched diet. Finally, PXR might interfere directly with FXR by targeting their common coactivator peroxisome proliferator-activated receptor- coactivator-1 (Pgc-1) 47 as proposed recently to explain the inhibition of HNF4 signaling pathway by PXR. However, and in contrast to apoA-I, the expression of specific targets genes that are highly responsive to FXR (ie, SHP or CYP7A1) was not affected by PXR in the present study. Finally, although greater induction of PXR responsive genes in Pxr-null SXR-Tg mice under CA diet might be related in part to the highly active albumin promoter,23 it is well-known that mouse and human PXR exhibit marked differences with regard to their ligand specificity48 and lithoCA, the most potent PXR activator among bile acids, is more efficient in activating human PXR than mouse Pxr in vitro.19
In conclusion, our results indicate that FXR and PXR exert distinct, but complementary roles in HDL metabolism, as they actually do in the protection against bile acid toxicity.19,40,49 Additional investigations will be necessary to confirm the pathophysiological relevance of our study. Because of the peculiar sensitivity of SXR to bile acids, the existence of a compensation by PXR of the FXR-mediated changes in lipoprotein metabolism deserves further attention in humans. Overall, magnification of the beneficial effects of PXR in Pxr-null SXRTg mice under CA treatment suggest that beside reduction of bile acid–mediated liver toxicity, PXR agonist may also be useful to counteract the deleterious effects of bile acids on plasma lipoproteins.
Acknowledgments
This work was supported by National Institutes of Health Research grants CA77545, GM60904, ES058571, GM60346, P30 CA21745, the Cancer Center support grant, the American Lebanese Syrian Associated Charities (ALSAC), Conseil Régional de Bourgogne, Fondation de France, Université de Bourgogne, and INSERM U498.
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Correspondence to Dr Mahfoud Assem, Department of Pharmaceutical Sciences, St Jude Children’s Research Hospital, 332 N. Lauderdale Ave, Memphis, TN 38105-2794. E-mail Mahfoud.Assem@stjude.org
Abstract
Objective— Modification of lipoprotein metabolism by bile acids has been mainly explained by activation of the farnesyl X receptor (FXR). The aim of the present study was to determine the relative contribution of the pregnane X receptor (PXR), another bile acid–activated nuclear receptor to changes in plasma lipoprotein profile.
Methods and Results— Wild-type mice, Pxr-deficient mice, and Pxr-null mice expressing human PXR (Pxr-null SXR-Tg mice) were fed a cholic acid–containing diet, and consequences on plasma lipoprotein profiles and target gene expression were assessed. Cholic acid produced significant decreases in high-density lipoprotein (HDL) cholesterol, plasma apolipoprotein (apo)A-I and hepatic apoA-I mRNA in wild-type mice. Interestingly, the effect of cholic acid was significantly more pronounced in Pxr-deficient mice, indicating that PXR contributes to the weakening of the effect of bile acids on lipoprotein metabolism. Reciprocally, changes in HDL/apoA-I profiles were abolished in Pxr-null SXR-Tg mice in which PXR-responsive genes, particularly those involved in bile acid detoxification were readily activated after cholic acid treatment.
Conclusion— PXR expression in mice antagonizes the cholic acid–mediated downregulation of plasma HDL cholesterol and apoA-I, and magnification of PXR/SXR-mediated changes may constitute a new mean to counteract the effects of bile acids on plasma lipoproteins.
To determine the contribution of PXR to the effect of bile acids on plasma lipoproteins, wild-type mice, PXR-deficient mice, and Pxr-null mice expressing human PXR were fed a cholic acid–containing diet. Mouse and human PXR were found to antagonize the cholic acid-mediated decreases in plasma HDL cholesterol and apoA-I.
Key Words: apolipoprotein A-I ? bile acids ? farnesyl X receptor ? high-density lipoproteins ? pregnane X receptor ? steroid and xenobiotic receptor
Introduction
High-density lipoproteins (HDL) and their major protein component, apolipoprotein A-I (apoA-I), are protective factors against the development of atherosclerosis.1–4 These observations raised a considerable interest in the development of new therapeutic approaches that aim at increasing plasma HDL cholesterol and apoA-I levels. ApoA-I gene expression is under the control of multiple factors, including several nuclear receptors. ApoA-I gene expression is negatively regulated by the apolipoprotein regulatory protein-I (ARP-1) (NR2F2) and the farnesyl X receptor (FXR) (NR1H4),5–7 whereas it is positively regulated by the hepatocyte nuclear factor-4 (HNF-4) (NR2A1), the peroxisome proliferator receptor alpha (PPAR) (NR1C1), and the liver receptor homolog-1 (LRH-1) (NR5A2).8–10
See page 2016
Although several ligands of pregnane X receptor (PXR) (NR1I2) were reported to increase HDL cholesterol as well as apoA-I and apoA-I mRNA levels in rodents and humans,11–15 the relative contribution of PXR to the regulation of plasma HDL levels in vivo remains to be ascertained. Interestingly, besides its role as a xenobiotic sensor,16–17 PXR responds to bile acids that also constitute well-recognized FXR ligands.18–22 Because PXR and FXR have opposite effects on apoA-I expression, it can be hypothesized that the final effect of bile acids on lipoprotein profile may actually reflect antagonistic contributions of PXR and FXR activations. To address the latter point in a comprehensive way, wild-type (WT) mice, PXR-deficient mice (Pxr-null mice),23 and mice expressing human PXR but not mouse PXR (Pxr-null SXR-Tg mice)23 were fed a 1% cholic acid (CA)-containing diet in the present study. We report here that PXR expression in mice antagonizes the CA-mediated downregulation of plasma HDL cholesterol and apoA-I. Interestingly, the negative effect of CA on plasma HDL profile could be completely reversed by elevated expression of human PXR in Pxr-null SXR-Tg mice, indicating that a magnification of PXR/SXR-mediated changes may constitute a new mean to counteract the negative effects of bile acids on lipoprotein profile.
Materials and Methods
Animals
WT mice, age-matched mice deficient for murine Pxr (Pxr-null),23 and age-matched mice expressing human PXR (Pxr-null SXR-Tg) under the control of the albumin promoter23 were used in the present study. The Pxr-null and Pxr-null SXR-Tg mice were generously provided by Dr Ronald Evans. WT, Pxr-null, SXR-Tg mice and Pxr-null mice were all of mixed genetic background (129/C57BL6). Mice were housed in a pathogen-free animal facility under a standard 12-hour light/12-hour dark cycle. All diets were prepared by Bio-serv (Frenchtown, NJ) and were based on a standard AIN-93G rodent diet. The CA diet was identical to the control diet but supplemented with 1% (w/w) CA. Male mice were 8 to 12 weeks of age at the start of the study. Five to 6 animals per group were used in the present study. All protocols and procedures were approved by the NCI Division of Basic Sciences Animal Care and Use Committee and are in accordance with the National Institutes of Health guidelines.
Plasma and Liver Tissue Sampling
After 5 days of feeding the indicated diet, animals were anesthetized with isoflurane and blood samples were collected by intra-cardiac puncture in heparin-containing tubes that were centrifuged at 5000 rpm for 10 minutes. Plasmas were harvested and stored at –80°C. Livers were excised, weighed, immediately snap-frozen in liquid nitrogen (LN2), and stored at –80°C before mRNA isolation and biochemical analysis.
Hepatic Lipid Analysis
Qualitative analysis of hepatic bile acids was performed by capillary gas-liquid chromatography as described previously.24,25 Quantitative determination of total hepatic bile acids was performed by an enzymatic assay (Colorimetric Total Bile Acid Assay Kit; Bio Quant, San Diego, Calif) on hepatic alcoholic extract obtained as previously described.26 Total cholesterol and triglycerides were determined as described previously.25,27
Plasma Lipid and Apolipoprotein Analysis
All assays were performed on Victor2 1420 Multilabel Counter (Perkin Elmer life Science, Boston, Mass). Cholesterol, phospholipids, and triglycerides were determined by enzymatic methods as described previously.25 HDL and non-HDL cholesterol plasma fractions were determined as cholesterol concentration in d>1.07 and d<1.07 plasma fractions respectively. Mouse apoA-I concentration was determined by a nonimmunologic method. Briefly, plasma samples (0.25 μL) were submitted to electrophoresis on 5% to 15% polyacrylamide gradient gel (Invitrogen, Carlsbad, Calif). Gel were subsequently stained by Coomassie brilliant blue and scanned on a GS-800 densitometer (Biorad, Hercules, Calif). The amount of apoA-I per sample was determined quantitatively by comparison with apoA-I standards that were performed in parallel with the samples.
Native Polyacrylamide Gradient Gel Electrophoresis
Total lipoproteins were separated by ultra centrifugation as the d<1.21 g/mL plasma fraction with one 5.5 hour, 100 000-rpm spin in a TLA100 rotor in a TLX ultracentrifuge (Beckman, Krefeld, Germany). Lipoproteins were then applied to a 1.5% to 25% polyacrylamide gradient gel (Spiragel 1.5 to 25.0; Spiral, Couternon, France), and electrophoresis was conducted as recommended by the manufacturer. Gels were subsequently Coomassie stained and the distribution profiles of lipoprotein were determined by comparison with protein standards (HMW kit; Amersham Bioscience, Uppsala, Sweden) using a GS 800 densitometer.
RNA Isolation and Polymerase Chain Reaction Methods
Total RNA was extracted using Trizol reagent (Life technologies, Carlsbad, Calif). Specific mRNAs were analyzed by quantitative real-time reverse-transcription polymerase chain reaction using the ABI Prism 7900HT (Applied Biosystems, Foster City, Calif). Briefly, 5 μg of RNA were reverse-transcribed into cDNA using MuMLV retrotranscriptase and oligo dT (Life technologies); 50 ng of the cDNA mixture were used. Specific cDNAs were amplified using specific primers (see supplemental data at http://atvb.ahajournals.org).Values were normalized to Gapdh levels. Relative mRNA levels were evaluated using Ct method.
Western Blot
The preparation of liver homogenates and Western blot analysis were performed as described elsewhere.19,28 Anti-Bsep, Mrp4, and cyp 3a11 antibodies were described previously.28–30 Rabbit anti-Mrp2 was provided by Dr Bruno Stieger, Zurich, Switzerland.
Statistical Analysis
Mann–Whitney U test was used to determine the significance between the data means.
Results
Cholic Acid Feeding Reduces Cholesterol and ApoA-I Levels in WT and Pxr-Null Mice, but not in Pxr-Null SXR-Tg Mice
In agreement with previous studies,31 total cholesterol, esterified cholesterol, phospholipid, and HDL cholesterol were significantly lower in WT mice fed a 1% cholic acid diet than in WT mice fed the standard chow (Table 1). Nondenaturing polyacrylamide gradient gel electrophoresis confirmed that the HDL fraction was mostly and selectively affected by the 1% CA treatment (Figure 1), which was accompanied by a significant reduction in plasma apoA-I concentration (0.56 g/L in WT mice fed 1% CA versus 1.18 g/L in WT mice fed the control diet; P<0.05) (Figure 1).
TABLE 1. Effect of 1% CA Diet on Plasma Lipid Parameters
Figure 1. Plasma ApoA-I concentration and lipoprotein profile in WT, Pxr-null, and Pxr-null SXR-Tg mice fed either control or a 1% CA diet. A, Total plasma lipoproteins were submitted to electrophoresis on 1.5% to 25% polyacrylamide gradient gels that were stained for proteins as described in Materials and Methods. B, Plasma apoA-I concentrations were determined as described in Materials and Methods. (*) indicates a significant difference from homologous mice fed the control diet and Pxr-null SXR-Tg mice fed the 1% CA diet (P<0.05 in both cases; Mann–Whitney test).
Whereas lipoprotein phenotypes of Pxr-null mice and WT mice were similar under standard diet, CA induced significantly greater reductions in total cholesterol, esterified cholesterol, phospholipids, triglycerides, and HDL cholesterol in Pxr-null mice after treatment (Table 1). The CA-mediated decreases in plasma apoA-I concentration were of similar magnitude in Pxr-null and WT mice (Figure 1). Strikingly, the CA-mediated changes in lipid concentration and HDL distribution no longer appeared in Pxr-null SXR-Tg mice, with the exception of a reduction in plasma phospholipids (Table 1 and Figure 1).
The Accumulation of Bile Acids in the Liver Is Higher in Pxr-Null Mice Than in WT and Pxr-Null SXR-Tg Mice Under CA Diet
In all mouse lines, levels of cholic, deoxycholic, and chenodeoxycholic acids were increased by 5- to 10-fold by the CA diet, whereas the concentrations of 6-OH bile acids (ie, muricholic acids) remained unchanged (Figure 2). Interestingly, the increase in total bile acids, CA, and deoxycholic acid were significantly higher in the liver of Pxr-null mice than in the liver of WT and Pxr-null SXR-Tg mice (Figure 2). As shown in Table 2, hepatic triglyceride levels remain unchanged in all mouse lines whether they received the control chow or the CA-enriched diet. Hepatic triglyceride concentration was significantly higher in Pxr-null-SXR-Tg mice as compared with WT or Pxr-null mice under control and CA diets. Interestingly, the CA diet induced a significant increase in hepatic cholesterol concentration in WT and Pxr-null mice but not in Pxr-null SXR-Tg mice.
Figure 2. Hepatic bile acid content in WT, Pxr-null and Pxr-null SXR-Tg mice fed a 1% CA diet. Hepatic bile acid content was determined as described in Materials and Methods. *Significant difference from WT and Pxr-null SXR-Tg mice fed the 1% CA diet (P<0.05; Mann–Whitney test).
TABLE 2. Effect of 1% CA Diet on Hepatic Lipid Concentrations
CA Feeding Activates FXR Target Genes in WT, Pxr-Null, and Pxr-Null SXR-Tg Mice
Expression of Cyp7a1 and Cyp8b1, ie, 2 genes indirectly regulated by FXR,32 were dramatically reduced after CA treatment in all cases, indicating an efficient suppression of bile acid synthesis (Figure 3A). The expression of small heterodimeric partner (Shp) and bile salt export pump (Bsep), ie, 2 direct FXR targets,32,33 was significantly increased after cholic treatment. Bsep mRNA levels were increased by 7-fold in the liver of CA-treated Pxr-null SXR-Tg mice, but only by 2-fold in CA-treated WT and Pxr-null liver (P<0.05) (Figure 3A). Induction of Bsep mRNA levels was accompanied by a proportional increase in Bsep protein (data not shown).
Figure 3. Relative changes in hepatic mRNA levels of FXR target genes (A) and PXR target genes (B) in WT, Pxr-null, and Pxr-null SXR-Tg mice fed a 1% CA diet. Total RNA was extracted from the liver and real time quantitative polymerase chain reaction was performed. Data were standardized for Gapdh mRNA, and mRNA levels in WT mice receiving the standard diet was set at 1.00. (a) indicates a significant difference from homologous mice fed the control diet; (b) indicates a significant difference from WT mice fed the 1% CA diet (P<0.05; Mann–Whitney test).
CA Feeding Activates PXR Target Genes Only in Pxr-Null SXR-Tg Mice
As shown in Figure 3B, PXR target genes were minimally affected by the 1% CA diet in WT and Pxr-null mice. In contrast, mRNA levels of Cyp3a11, Mrp2, and Mrp4 were markedly increased (4- to 6-fold increases) in Pxr-null SXR-Tg mice receiving the 1% CA diet as compared with control diet (Figure 3B). Activation patterns were confirmed by hepatic protein analysis (data not shown).
CA Feeding Decreases Hepatic ApoA-I mRNA Levels in WT and Pxr-Null Mice, but not in Pxr-Null SXR-Tg Mice
As shown in Figure 4A, no significant difference in hepatic apoA-I mRNA levels was observed between WT, Pxr-null, and Pxr-null SXR-Tg mice when fed the standard diet. In WT mice, and as previously reported,31 the 1% CA diet led to a significant 40% reduction in hepatic apoA-I mRNA levels as compared with WT mice fed the control diet. Similar reduction in hepatic apoA-I mRNA levels was also observed in Pxr-null mice under the CA diet. In contrast, in Pxr-null SXR-Tg mice, hepatic apoA-I mRNA levels remained constant under the CA-containing diet (Figure 4A), as were plasma apoA-I concentrations (Figure 1).
Figure 4. Abundance of hepatic ApoA-I mRNA (A) and Sr-b1, Abcg1, and Abca1 (B) in WT, Pxr-null, and Pxr-null SXR-Tg mice fed either control or 1% CA diet. Total RNA was extracted from the liver and real-time quantitative polymerase chain reaction was performed and analyzed as described in Figure 3 legend. (a) indicates a significant difference from homologous mice fed the control diet; (b) indicates a significant difference from WT mice fed the 1% CA diet (P<0.05; Mann–Whitney test).
CA Feeding Reduces Hepatic Abca1 mRNA Levels in Pxr-Null Mice, but not in WT and Pxr-Null SXR-Tg Mice
As shown in Figure 4B, the mRNA levels of Sr-b1 and Abcg1, 2 genes involved in HDL metabolism, were significantly increased by 2- to 3-fold in the 3 mouse lines by the CA diet. In addition, a significant reduction of Abca1 mRNA level was observed in Pxr-null mice receiving the CA diet but not in WT mice and Pxr-null SXR-Tg mice.
Discussion
Bile acids modulate the atherogenicity of the lipoprotein profile.34 In particular, they act as negative regulators of hepatic apoA-I transcription;7 this downregulation has been explained either in terms of a direct effect of activated FXR at the apoA-I gene promoter,7 or in terms of an indirect effect of FXR through the SHP/LRH-I pathway.9 Activation of FXR by bile acids also reduces plasma triglyceride concentration by decreasing both hepatic lipogenesis and secretion of very-low-density lipoprotein.35,36 Besides FXR, bile acids are also potent activators of another nuclear receptor, ie, PXR.18–22 Interestingly, several studies have reported that administration of pharmacological PXR agonists is associated with significant increases in both hepatic apoA-I transcription and circulating levels of apoA-I and HDL cholesterol in rats, mice, and humans.11–15, 37–39 The purposes of the present study were: (1) to establish the relative contribution of PXR to the effect of bile acids on plasma lipoprotein profile through the comparison of WT and Pxr-null mice; and (2) to determine whether overexpression of human PXR in Pxr-null SXR-Tg mice was able to counteract the deleterious, FXR-mediated effects of bile acids.
In agreement with previous observations,31 the CA-containing diet significantly reduced hepatic apoA-I expression and HDL cholesterol levels in WT mice. Although the effects of CA were reported to be exacerbated in PXR-deficient mice, in particular with more pronounced effects on total cholesterol and triglyceride levels as compared with WT mice,40 consequences of PXR-deficiency in terms of HDL structure and composition had not been specifically addressed. The present studies demonstrate for the first time that CA treatment of PXR-deficient mice produced more profound effects on plasma HDL cholesterol as compared with WT counterparts. Because exacerbation of the effect of CA on HDL cholesterol in PXR-deficient mice was not accompanied by further decrease in apoA-I gene expression in the present study, we thought further for differential expressions of specific genes that are known to affect lipidation (ie, ABCA1 and ABCG1)41–42 or catabolism (ie, SR-BI)43 of HDL. While induction of SR-BI by bile acids in WT mice confirmed previous studies,44 it is reported for the first time that bile acids positively regulate hepatic ABCG1 mRNA levels. Because hepatic overexpression of either Sr-b1 or Abcg1 are known to induce a reduction of HDL cholesterol levels in mice, caused by an increase in hepatic HDL catabolism,41,45 it is possible that these two proteins contribute in addition to apoA-I to the bile acid-mediated reduction of HDL. More importantly, and in contrast to Sr-b1 and Abcg1 mRNA levels that were induced by the CA treatment in the 3 mouse lines studied, Abca1 mRNA levels were shown in the present study to be significantly reduced by CA in Pxr-null mice only. Recent observations have demonstrated that hepatic ABCA1 is a key factor contributing to lipidation and formation of nascent HDL 42; therefore, downregulation of Abca1 expression arises as a possible mechanism for the greater effect of the CA diet on HDL cholesterol levels in Pxr-null mice as compared with WT mice.
In contrast to observations made with WT and PXR-deficient mice, CA-mediated changes in HDL/apoA-I profiles were completely abolished in mice expressing human PXR (Pxr-null SXR-Tg mice). Moreover, typical PXR-responsive genes were readily activated after CA treatment in this latter mouse line only. Different mechanisms that are not mutually exclusive may explain the positive effect of PXR. First, a direct transactivating effect of PXR on the apoA-I promoter is possible, and it is consistent with the positive effect of phenobarbital on apoA-I transcription as already reported.38–39 Alternatively, it is possible that PXR interferes with some FXR-mediated pathways by inducing qualitative and quantitative changes in hepatic bile acid composition.46 This hypothesis is supported by significant changes in hepatic bile acid composition in Pxr-null mice treated with CA and is susceptible to explain the exacerbation of the effect of the CA diet on lipoprotein profile in Pxr-null mice. However, no qualitative or quantitative differences in hepatic bile acid profiles were observed between Pxr-null SXR-Tg and WT mice fed the CA-enriched diet. Finally, PXR might interfere directly with FXR by targeting their common coactivator peroxisome proliferator-activated receptor- coactivator-1 (Pgc-1) 47 as proposed recently to explain the inhibition of HNF4 signaling pathway by PXR. However, and in contrast to apoA-I, the expression of specific targets genes that are highly responsive to FXR (ie, SHP or CYP7A1) was not affected by PXR in the present study. Finally, although greater induction of PXR responsive genes in Pxr-null SXR-Tg mice under CA diet might be related in part to the highly active albumin promoter,23 it is well-known that mouse and human PXR exhibit marked differences with regard to their ligand specificity48 and lithoCA, the most potent PXR activator among bile acids, is more efficient in activating human PXR than mouse Pxr in vitro.19
In conclusion, our results indicate that FXR and PXR exert distinct, but complementary roles in HDL metabolism, as they actually do in the protection against bile acid toxicity.19,40,49 Additional investigations will be necessary to confirm the pathophysiological relevance of our study. Because of the peculiar sensitivity of SXR to bile acids, the existence of a compensation by PXR of the FXR-mediated changes in lipoprotein metabolism deserves further attention in humans. Overall, magnification of the beneficial effects of PXR in Pxr-null SXRTg mice under CA treatment suggest that beside reduction of bile acid–mediated liver toxicity, PXR agonist may also be useful to counteract the deleterious effects of bile acids on plasma lipoproteins.
Acknowledgments
This work was supported by National Institutes of Health Research grants CA77545, GM60904, ES058571, GM60346, P30 CA21745, the Cancer Center support grant, the American Lebanese Syrian Associated Charities (ALSAC), Conseil Régional de Bourgogne, Fondation de France, Université de Bourgogne, and INSERM U498.
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