1Medical Service, Veterans Affairs Medical Center, East Orange 07018; 2Department of Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark 07103; and 3Gastrointestinal Division, University of Tsukuba School of Medicine, 3058575 Tsukuba City, Japan
Submitted 10 September 2003 ; accepted in final form 13 December 2003
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ABSTRACT |
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bile acid pool; liver X receptor-; oxysterol; short heterodimer partner; ABCA1; bile salt export pump; lipoprotein lipase
To understand the mechanism why CYP7A1 is stimulated in rats fed cholesterol, this study investigated the role of activation of LXR- and FXR in the regulation of CYP7A1 in rats fed cholesterol and CA. Our results suggest that upregulation of CYP7A1 in rats fed cholesterol is not because rat CYP7A1 is not sensitive to bile acids or activated FXR, but rather because FXR was not activated.
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MATERIALS AND METHODS |
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Male Sprague-Dawley rats (n = 48) weighing 250300 g (Charles River) were used in this study. The rats were divided into the following four groups: controls fed regular rat chow (n = 12); rats fed regular chow containing 2% cholesterol (Purina Mills, St. Louis, MO) for 7 days (n = 12); rats fed regular chow containing 2% cholesterol plus 1% CA for 7 days (n = 12); and rats fed regular chow containing 1% CA for 7 days (n = 12). After completion of the treatments, bile fistulas were constructed in 6/12 rats from each group. After surgery, 10 ml of lactated Ringer solution with 5% glucose was given every 4 h by subcutaneous injection. A small piece of light and soft aluminum was fixed on to the waist of the animal by elastic bandage such that the plastic bottle used to collect bile could be attached continuously (Fig. 1). With this device, the rats were allowed to move around freely after the surgery but could not reach the bottle for bile collection, which was behind the metal sheet. The bile drainage was continued for 18 h to "wash out" the previous bile acid pool. In this study, we measured the functional entrohepatic bile acid pool size, which represents the portion of bile acids that are absorbed and circulate through the liver to activate FXR but not those bile acids that remain in the intestine and are lost in the feces. The functional bile acid pool size was calculated as the amount of total recovered bile acids that was collected by continuous bile drainage until the "low point" on the washout curve (bile acid concentration x volume of bile) corrected by subtracting the contribution resulting from basal bile acid synthesis (basal synthesis rate x h; Fig. 2). The remaining rats in each group (6/12) were killed to collect blood and liver specimens. Blood samples were used for measurements of plasma cholesterol levels. The liver tissues were immediately frozen for measurements of mRNA levels of FXR and FXR target genes, SHP (9, 19) and bile salt export pump (BSEP; see Ref. 1), LXR- and LXR-
target genes ABCA1 (6) and lipoprotein lipase (LPL; see Ref. 36), concentrations of oxysterols (24S,25-epoxycholesterol, 24S-hydroxycholesterol, 22R-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol), and CYP7A1 mRNA levels and activity.
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The animal protocol was approved by the Institutional Animal Care and Use Committee at the Veterans Affairs Medical Center (East Orange, NJ) and the Institutional Animal Care and Use Committee at the University of Medicine and Dentistry, New Jersey Medical School (Newark, NJ).
Biochemical Analyses
Northern blotting analyses. PROBE PREPARATION. The template of cDNAs was synthesized by RT-PCR using a Creator Smart cDNA Library Construction kit (BD Biosciences). The rat cDNA probes were primed from cDNA by PCR as previously described (31). Primers used for FXR were 5'-GGC GGG AAG AAT AAA AGG GGA TGA/5'-AGG AGG GTC TGC TGG TCT ACA, for SHP were 5'-CAA GCC ACC CCA CCA TTC TCT AC/5'-AGC CTC GGC CAC CTC AAA G, for BSEP were 5'-AAC AGT GGC CGC TTT TGG TG/5'-TCG TTT CCC CTG GCT TTA TGA C, for ABCA1 were 5'-CTG ATT GCC CGG CGG AGT AGA AAG/5'-CCC CGA CCA AGC AAG GAG TGTT, for LPL were 5'-ATC GGG CCC AGC AAC ATT ATC C/5'-TGC CTT GCT GGG GTT TTC TTC A, for LXR- (base on human sequences) 5'-CCC AGC TCA GCC CGG AAC AAC T/5'-GCA AGG CAA ACT CGG CAT CAT T, and for cyclophilin 5'-ATT TGC AGA CAA AGT TCC AA/5'-TGA TCT TCT TGC TGG TCT TG. Rat cDNA probe for CYP7A1 was a gift from Dr. John Chiang.
RNA ISOLATION. Total RNA was isolated from frozen rat liver tissues using the single-step RNA isolation method with TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA), as described by Chomozynski and Sacchi (5). Poly(A)+ RNA was isolated from 2 mg total RNA by oligo(dT) cellulose using the FastTrack 2.0 mRNA isolation kit (Invitrogen Life Technologies) described by Biesecker et al. (2).
HYBRIDIZATION. Northern blot hybridization was performed as previously described by Thomas (28). Briefly, 10 µg poly(A)+ RNA were electrophoresed on a formaldehyde-agarose (1.0%) gel and transferred to a nylon membrane (Nytran supercharge nylon transfer membrane; Schleicher & Schuell). The membrane was baked for 2 h at 80°C and hybridized to a [32P]DNA probe for 16 h at 42°C. The membrane was washed at 55°C in 0.2x SSC and 0.1% SDS for 30 min. Relative expression levels were quantified using a Phosphor-Imager (Molecular Dynamics) and standardized against cyclophilin controls.
Oxysterol analysis. Measurement of hepatic oxysterol concentrations was based on the method of Dzeletovic et al. (7) with some modifications. Liver specimen (100 mg wet wt) was homogenized in 2.5 ml distilled water. 27-[2H7]hydroxycholesterol (32 ng, as internal recovery standard), 1 ml of 0.5 N ethanolic KOH, and 5 µg butylated hydroxytoluene were added to 100 µl of the homogenate, and alkaline hydrolysis was allowed to proceed at 37°C for 1 h (11). After the addition of 0.4 ml distilled water and extraction two times with 2 ml of n-hexane, the extract was evaporated to dryness under nitrogen. The residue was dissolved in 1 ml toluene, and oxysterols were purified by a Bond Elut SI cartridge (7). After removal of the solvent under a gentle stream of nitrogen, the oxysterols were converted to trimethylsilyl ether derivatives with 100 µl of trimethylsilyl chloride (TMSI-H) (GL Sciences, Tokyo, Japan) for 15 min at 55°C. High-resolution gas chromatography-mass spectrometry with selected ion monitoring was performed as described previously (12). An Ultra Performance capillary column (25 m x 0.32 mm ID) coated with methylsilicone (Agilent Technologies, Wilmington, DE) was used at a flow rate of helium carrier gas of 1.0 ml/min. The column oven was programmed to change from 100 to 270°C at 30°C/min, after a 1-min delay from the start time. The multiple ion detector was focused on mass-to-charge ratio (m/z) 173.1360 for 22R-hydroxycholesterol, m/z 343.3000 for 24S,25-epoxycholesterol, m/z 413.3239 for 24S-hydroxycholesterol, m/z 456.3787 for 25-hydroxycholesterol and 27-hydroxycholesterol, and m/z 463.4226 for 27-[2H7]hydroxycholesterol.
Assays for activities of CYP7A1. Hepatic microsomes were prepared by differential ultracentrifugation (26). Protein was determined according to Lowry et al. (18). CYP7A1 activity was measured in hepatic microsomes by the isotope incorporation method of Shefer et al. (26).
Assay for bile acids. Bile acid concentration and composition were analyzed by capillary gas-liquid chromatography, as previously described (34).
Statistical method. Data are shown as means ± SD and were compared statistically by the Bonferroni Multiple Comparison Test. BMDP Statistical Software (BMDP Statistical Software, Los Angeles, CA) was used for statistical evaluations.
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RESULTS |
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The functional enterohepatic bile acid pool size did not change after feeding Ch for 1 wk (51 ± 14 vs. 61 ± 16 mg) but enlarged 3.4- and 3-fold, respectively, in rats treated with Ch + CA (175 ± 30 mg, P < 0.01) and CA alone (152 ± 38 mg, P < 0.01; Fig. 3). Bile acid composition in the pool is shown in Fig. 4. DCA, CA, and CDCA, which are activating ligands for FXR, comprised 78 ± 4% of the total bile acids in controls, but the proportion of these activating ligands (bile acids) decreased 31% (54 ± 5%, P < 0.001) in the group fed only Ch. After feeding Ch + CA and CA alone, the proportion of DCA + CA + CDCA in the bile acid pool increased 15% (90 ± 3%, P < 0.01) and 19% (93 ± 3%, P < 0.001), respectively, compared with untreated controls (Fig. 4). The remainder of the bile acid pool was composed chiefly of -,
-, and
-muricholic acids, which contain an additional hydroxyl group at position C-6 and increased after Ch feeding. The muricholic acids are not considered effective activating ligands for FXR (20, 23, 29).
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CYP7A1 activity rose 70% from 25 ± 4 to 43 ± 8 pmol·mg-1·min-1 (P < 0.01), and mRNA increased 95% from 0.83 ± 0.29 to 1.62 ± 0.04 units (P < 0.01) in rats fed only Ch. Adding CA to the Ch diet decreased elevated CYP7A1 activity 51% (21 ± 3 pmol·mg-1·min-1, P < 0.001) and mRNA 43% (0.92 ± 0.10 units, P < 0.01; Figs. 5 and 6). In rats fed CA alone, CYP7A1 activity and mRNA decreased 56% (11 ± 2 pmol·mg-1·min-1, P < 0.01) and 60% (0.33 ± 0.05 units, P < 0.05), respectively, below untreated baseline.
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The mRNA levels of SHP, an FXR target gene reflecting FXR activation, did not change (1.57 ± 0.30 vs. 1.61 ± 0.24 units) in rats fed Ch alone but increased 88% to 2.95 ± 0.42 units (P < 0.01) in rats fed Ch + CA and 2.5-fold to 3.95 ± 0.31 units (P < 0.001) in rats fed CA alone. Similarly, the mRNA abundance of BSEP, another FXR target gene, did not change (4.01 ± 0.49 vs. 3.47 ± 0.22 units) in rats fed Ch alone but increased 37% to 5.63 ± 0.45 units (P < 0.05) and 46% to 5.97 ± 0.86 units (P < 0.05), respectively, in rats fed CA + Ch or CA alone (Fig. 6). However, FXR mRNA levels did not change after any treatment (Fig. 6).
Hepatic concentrations of oxysterols, 24S,25-epoxycholesterol, 24S-hydroxycholesterol, 22R-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol, are reported in Table 1. The data show that, after feeding Ch or Ch + CA, hepatic concentrations of the oxysterols increased and the increase was more in Ch + CA than in feeding Ch alone.
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The mRNA levels of ABCA1, an LXR- target gene reflecting LXR-
activation, increased 72% from 0.90 ± 0.19 to 1.55 ± 0.17 units (P < 0.05) after Ch feeding and rose threefold (2.92 ± 0.28 units, P < 0.001) after feeding Ch + CA (Fig. 7). In rats fed only CA, ABCA1 mRNA levels remained stable compared with controls (1.10 ± 0.15 units). LPL mRNA levels, another target gene of LXR-
, did not change in rats fed only Ch (1.15 ± 0.40 vs. 1.22 ± 0.37 units) but increased threefold to 3.83 ± 0.51 units (P < 0.01) after feeding Ch + CA (Fig. 7). In rats fed CA alone, LPL mRNA levels did not change (0.98 ± 0.13 units) compared with controls. LXR-
mRNA levels remained stable in rats fed Ch or CA alone but increased 79% from 1.72 ± 0.19 to 3.08 ± 0.54 units (P < 0.05) in rats fed Ch + CA (Fig. 7).
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DISCUSSION |
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It is important to note that, in Ch-fed rats, not only the bile acid (ligand) pool size did not expand but also the proportion of DCA + CA + CDCA, which are activating ligands for FXR in the bile acid pool, decreased (Fig. 4) while the remaining portion of the pool, which was composed mainly of the 6-hydroxylated bile acids -,
-, and
-muricholic acids, increased in the bile. It should be emphasized that muricholic acids are not considered activating ligands for FXR (20, 23, 29). In our study of rabbits in which the bile acid flux was replaced with hydrophilic ursocholic acid, activation of FXR did not occur, although the bile acid flux through the liver was equivalent to controls (31). We conclude that, in addition to the bile acid pool size, the proportion of activating bile acid ligands circulating in the bile acid pool through the liver is also crucial for activation of FXR to take place to downregulate CYP7A1. Thus, in Ch-fed rats, only LXR-
was activated by increased oxysterols, but FXR was not activated because the bile acid (ligand) pool size did not expand and the proportion of activating ligands (CA, DCA, and CDCA) in the pool declined, whereas muricholic acids (nonactivating ligands) rose. As a result, the stimulatory effects of activated LXR-
upregulated CYP7A1 in rats fed Ch.
Feeding CA alone or adding CA to cholesterol not only resulted in more than a threefold enlargement of the bile acid pool but also significantly increased the proportion of FXR-activating ligands CA, DCA, and CDCA in the pool (Fig. 4). The increased amount of FXR-activating ligands in the bile acid flux returning through the liver activated hepatic FXR, as indicated by increased mRNA levels of SHP and BSEP. Consequently, FXR became activated simultaneously with activated LXR- and reduced the elevated levels of CYP7A1 mRNA (Fig. 6). This observation not only demonstrated that rat CYP7A1 is also sensitive to activated FXR but, most importantly, supported the hypothesis that activation of FXR also plays a key role in the downregulation of CYP7A1 transcription in rats.
After Ch + CA were fed, not only was FXR activated but LXR- was also simultaneously activated, as evidenced by a further increase in mRNA levels of LXR-
target genes, ABCA1 and LPL, which were significantly higher than in rats fed Ch alone. However, CYP7A1 mRNA levels and activity in rats fed Ch + CA declined significantly from the elevated levels in rats fed Ch alone. These results suggest that CYP7A1 responded differently to dietary Ch in rats than rabbits because FXR was not activated in Ch-fed rats.
Recently, a preliminary paper was published by Gupta et al. (10). The authors concluded that LXR- was the dominant regulator of CYP7A1, since 1) in Ch-fed rats, CYP7A1 was upregulated by LXR-
, although at the same time FXR was activated the expression of FXR target gene SHP was increased; and 2) after adding FXR ligand CA to the Ch diet, CYP7A1 mRNA levels were still increased 300% compared with controls. According to our data, we did not find a significant increase of SHP or BSEP mRNA expression in rats fed Ch alone. Thus FXR was not activated in cholesterol-fed rats such that CYP7A1 received no inhibitory signal from FXR but was stimulated by activated LXR-
. To evaluate hepatic FXR activation, it is important to measure the circulating functional enterohepatic bile acid pool size, the FXR ligand pool that returns through the liver and plays a determining role in activation of hepatic FXR. In our study, we found that the functional bile acid pool was not increased in the rats fed Ch alone. Moreover, in those rats, the proportion of FXR-activating ligands in the pool decreased 31%. Thus the mass of circulating FXR-activating ligands through the liver was not increased but rather decreased in rats fed Ch alone. These two findings also supported our conclusion that hepatic FXR was not activated in Ch-fed rats. Furthermore, we found that plasma cholesterol levels increased limitedly (54 ± 7 to 71 ± 14 mg/dl, P = NS) in rats fed only Ch, whereas in rats fed Ch + CA where FXR was activated, the same amount of Ch increased plasma cholesterol levels 4.6-fold (250 ± 48 mg/dl). These results also support our contention that FXR was not activated in rats fed only Ch but also activated in rats fed Ch + CA. The increase of plasma cholesterol level was limited in rats fed Ch alone because FXR was not activated and activated LXR stimulated CYP7A1. Thus hepatic bile acid synthesis increased significantly to use absorbed Ch and to limit the amount of cholesterol that reached the plasma. However, when FXR was activated after adding CA to the same dose of Ch (Ch + CA), CYP7A1 was not upregulated, much less cholesterol would be used to synthesize bile acids, and that much more cholesterol accumulated in the plasma. If CYP7A1 was still upregulated in rats fed Ch + CA, it would be difficult to explain why plasma cholesterol levels increased 4.6-fold. In addition, our data show that CYP7A1 activity was consistent with CYP7A1 mRNA levels. Both activity and mRNA did not increase in rats fed Ch + CA compared with controls and were significantly lower than in rats fed only Ch. It is also important to emphasize that, to evaluate the effect of CA (activation of FXR) in Ch-fed rats, CYP7A1 mRNA levels in rats fed Ch + CA should be compared with the elevated levels in rats fed Ch alone. The significant reduction of elevated CYP7A1 activity and mRNA levels in rats fed Ch + CA, where both LXR-
and FXR were activated compared with rats fed Ch alone where only LXR-
was activated, demonstrated the inhibitory effects of activated FXR on CYP7A1 in the presence of activated LXR-
in rats. Therefore, upregulation of CYP7A1 in Ch-fed rats is not because rat CYP7A1 is only sensitive to activated LXR-
and not to FXR activation but, rather, because FXR was not activated.
An important issue considered in this experiment is how to measure "the functional entrohepatic bile acid pool size" that circulates through the liver because only that pool reaches hepatic FXR and plays a determining role in activation of FXR and regulation of CYP7A1. Other workers have reported increased bile acid pool sizes when cholesterol was fed to rats (21, 27), although CYP7A1 was stimulated. In those experiments, the entire bile acid mass, including the functional bile acid pool that circulated through the liver plus the amount of bile acids that were not absorbed and lost in the feces, was measured as the bile acid pool size. However, in Ch-fed rats, two times as much bile acids are excreted in the feces (35) and no longer circulate through the liver to affect FXR activation. Thus, in the previous reports, the data for the bile acid pool did not reflect the actual amount of bile acids returned through the liver and activated FXR. After bile fistula was established and the animals reached a steady state, the biliary bile acid secretion rate fell to a new low plateau, which represented basal hepatic bile acid synthesis that equaled the amount of bile acids lost in the feces (fecal bile acid outputs). In the present experiment, using the "wash out" method originally reported by Eriksson (8) and later Mok et al. (20a), the bile acid pool in the rats was calculated as the amount of total bile acids recovered from the bile fistula until the low point on the wash out curve (Fig. 2), corrected by subtracting the contribution resulting from basal bile acid synthesis. In this case, only bile acids that circulated through the liver and recovered in the bile were measured as the functional enterohepatic bile acid pool that regulated hepatic FXR and CYP7A1.
In summary, we have demonstrated that, different from the rabbit, feeding cholesterol to the rat did not activate FXR, since the bile acid pool size remained stable and the proportion of FXR-activating ligands in the pool declined. As a result, CYP7A1 was stimulated by activating LXR-. Adding CA to the Ch diet expanded the bile acid (ligand) pool and increased the proportion of activating ligands in the pool to activate FXR. Elevated levels of CYP7A1 declined, although LXR-
were simultaneously activated in the rats fed CA + Ch. Thus in rats, when both LXR-
and FXR are activated, the stimulatory effects of LXR-
are offset by the inhibitory effects of FXR on CYP7A1.
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ACKNOWLEDGMENTS |
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This study was supported by grants from the Department of Veterans Affairs Research Service, Washington, D. C., and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-56830 and DK-26756.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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