Dietary cholesterol stimulates CYP7A1 in rats because farnesoid X receptor is not activated

Guorong Xu,1,2 Lu-xing Pan,2 Hai Li,2 Quan Shang,2 Akira Honda,3 Sarah Shefer,2 Jaya Bollineni,2 Yasushi Matsuzaki,3 G. Stephen Tint,1,2 and Gerald Salen1,2

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, 305–8575 Tsukuba City, Japan

Submitted 10 September 2003 ; accepted in final form 13 December 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cholesterol feeding upregulates CYP7A1 in rats but downregulates CYP7A1 in rabbits. To clarify the mechanism responsible for the upregulation of CYP7A1 in cholesterol-fed rats, the effects of dietary cholesterol (Ch) and cholic acid (CA) on the activation of the nuclear receptors, liver X-receptor (LXR-{alpha}) and farsenoid X-receptor (FXR), which positively and negatively regulate CYP7A1, were investigated in rats. Studies were carried out in four groups (n = 12/group) of male Sprague-Dawley rats fed regular chow (control), 2% Ch, 2% Ch + 1% CA, and 1% CA alone for 1 wk. Changes in mRNA expression of short heterodimer partner (SHP) and bile salt export pump (BSEP), target genes for FXR, were determined to indicate FXR activation, whereas the expression of ABCA1 and lipoprotein lipase (LPL), target genes for LXR-{alpha}, reflected activation. CYP7A1 mRNA and activity increased twofold and 70%, respectively, in rats fed Ch alone when the bile acid pool size was stable but decreased 43 and 49%, respectively, after CA was added to the Ch diet, which expanded the bile acid pool 3.4-fold. SHP and BSEP mRNA levels did not change after feeding Ch but increased 88 and 37% in rats fed Ch + CA. This indicated that FXR was activated by the expanded bile acid pool. When Ch or Ch + CA were fed, hepatic concentrations of oxysterols, ligands for LXR-{alpha} increased to activate LXR-{alpha}, as evidenced by increased mRNA levels of ABCA1 and LPL. Feeding CA alone enlarged the bile acid pool threefold and increased the expression of both SHP and BSEP. These results suggest that LXR-{alpha} was activated in rats fed both Ch or Ch + CA, whereas CYP7A1 mRNA and activity were induced only in Ch-fed rats where the bile acid pool was not enlarged such that FXR was not activated. In rats fed Ch + CA, the bile acid pool expanded, which activated FXR to offset the stimulatory effects of LXR-{alpha} on CYP7A1.

bile acid pool; liver X receptor-{alpha}; oxysterol; short heterodimer partner; ABCA1; bile salt export pump; lipoprotein lipase


THE HEPATIC MICROSOMAL enzyme CYP7A1 (cholesterol 7{alpha}-hydroxylase) catalyzes the first and rate-controlling reaction in the classic bile acid synthesis pathway. Feeding cholesterol to New Zealand White (NZW) rabbits for 10 days is associated with suppressed CYP7A1 activity and mRNA levels (32). Furthermore, in cholesterol-fed NZW rabbits, the bile acid pool size increased about twofold, which was responsible for the inhibition of CYP7A1 (33). This opinion that the expanded bile acid pool caused inhibition of CYP7A1 was further supported by the observation in rabbits fed 2% cholesterol for only 1 day. In those rabbits, the bile acid pool size was not enlarged, and CYP7A1 was stimulated rather than suppressed (34). Opposite to rabbits, cholesterol feeding to rats stimulated CYP7A1 and classic bile acid synthesis (15, 22, 25). The different response of CYP7A1 to dietary cholesterol (Ch) between rabbits and rats relates to the enterohepatic bile acid pool size that is increased in rabbits but stable in rats after cholesterol feeding (35). Recent studies (20, 23, 29) showed that CYP7A1 transcription is negatively regulated by the nuclear receptor farnesoid X receptor (FXR) and that bile acids such as lithocholic acid, chenodeoxycholic acid (CDCA), and deoxycholic acid (DCA) are ligands that activate FXR. Although studies in CV-1 cells did not show cholic acid (CA) had strong affinity to activate FXR (20, 23, 29), it has been demonstrated in mice that CA is a powerful activating ligand for FXR in vivo (17). These findings support the idea that, in rabbits, expansion of the bile acid pool that contains 85% DCA and 15% CA is responsible for inhibition of CYP7A1 by providing additional activating ligands to activate FXR, the negative regulator of CYP7A1. Further studies suggest that FXR does not directly bind to the CYP7A1 promoter region (4) but regulates CYP7A1 transcription indirectly by activating short heterodimer partner (SHP), which interacts with LRH-1, a transcription factor for CYP7A1 (9, 19). Liver X receptor (LXR-{alpha}), another nuclear receptor, has been identified as a positive regulator of CYP7A1 transcription (3, 24). The ligands that activate LXR-{alpha} are oxysterols (13, 14, 16), which increase after cholesterol feeding. Recently, we showed that, in rabbits fed cholesterol for 10 days, the inhibitory effects of activated FXR secondary to the enlarged bile acid pool overrode the stimulatory effects of activated LXR-{alpha} to downregulate CYP7A1 (30). However, in other experiments, Gupta et al. (10) suggested that LXR-{alpha} was the dominant regulator of CYP7A1 transcription in rats.

To understand the mechanism why CYP7A1 is stimulated in rats fed cholesterol, this study investigated the role of activation of LXR-{alpha} 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.


    MATERIALS AND METHODS
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 MATERIALS AND METHODS
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Animal Experimental Design

Male Sprague-Dawley rats (n = 48) weighing 250–300 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-{alpha} and LXR-{alpha} 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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Fig. 1. The rat model with bile fistula. A small piece of light and soft aluminum was fixed on the waist of the animal by elastic bandage such that the plastic bottle used to collect bile could be attached continuously. 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.

 


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Fig. 2. Measurement of the functional enterohepatic bile acid (BA) pool size in rats. After bile drainage, the biliary BA outputs declined to reach a low plateau in ~10 h, which represents the basal hepatic BA synthesis rate. Under steady-state conditions, the BA synthesis rate is equal to fecal BA outputs that are not reabsorbed and returned to the liver. The functional BA pool size is calculated as the total "wash out" BA pool subtracted by the contribution from basal BA synthesis. The wash out BA pool is the total BA recovered from the bile collected from the beginning until the point when the low plateau has been reached.

 

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-{alpha} (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.


    RESULTS
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 MATERIALS AND METHODS
 RESULTS
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Plasma cholesterol levels increased 31% [P = not significant (NS)] from 54 ± 7 to 71 ± 14 mg/dl after 1 wk of 2% cholesterol (Ch) feeding. When 1% CA was added to the Ch diet (Ch + CA), plasma cholesterol levels rose 4.6-fold to 250 ± 48 mg/dl (P < 0.001) compared with baseline and increased 3.5-fold (P < 0.001) compared with feeding only Ch. Interestingly, after feeding CA alone, plasma cholesterol increased 20% (65 ± 5 mg/dl, P = NS).

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 {beta}-, {alpha}-, and {omega}-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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Fig. 3. The BA pool sizes in rats fed regular chow (control), 2% cholesterol (Ch), 2% CA + 1% cholic acid (Ch + CA), and 1% CA. The bile acid pool size enlarged 3.4-fold (P < 0.01) and 3-fold (P < 0.01), respectively, in rats fed Ch + CA and CA.

 


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Fig. 4. BA composition in the BA pool after feeding regular chow (control), 2% Ch, 2% Ch + 1% CA, and 1% CA. CA, chenodeoxycholic acid (CDC), and deoxycholic acid (DC) are the major activating ligands for farnesoid X receptor (FXR) in rat bile. The hatched portion is the proportion of nonactivating ligands in the BA pool, which are mainly muricholic acids with a small portion of ursodeoxycholic acid and 7-keto-deoxycholic acid.

 

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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Fig. 5. CYP7A1 activity (open bars) and mRNA levels (hatched bars) in rats fed regular chow (control), 2% Ch, 2% Ch + 1% CA, and 1% CA. Feeding Ch alone increased CYP7A1 activity (P < 0.01) and mRNA (P < 0.01) compared with controls. In rats fed Ch + CA, both CYP7A1 activity and mRNA decreased significantly (P < 0.001 and P < 0.01, respectively) compared with rats fed Ch alone. In rats fed CA alone, CYP7A1 activity and mRNA decreased below the baseline level (P < 0.01 and P < 0.05, respectively).

 


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Fig. 6. Northern blot analysis for hepatic mRNA expression of FXR and FXR target genes short heterodimer partner (SHP) and bile salt export pump (BSEP) in rats fed regular chow (control), 2% Ch, 2% Ch + 1% CA, and 1% CA. Cyclophilin is used as an internal standard.

 

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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Table 1. Hepatic oxysterol concentrations

 

The mRNA levels of ABCA1, an LXR-{alpha} target gene reflecting LXR-{alpha} 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-{alpha}, 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-{alpha} 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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Fig. 7. Northern bloting analysis for hepatic mRNA expression of liver X receptor (LXR)-{alpha} and LXR-{alpha} target genes ABCA1 and lipoprotein lipase (LPL) in rats fed regular chow (control), 2% Ch, 2% Ch + 1% CA, and 1% CA. Cyclophilin is used as an internal standard.

 


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The results of this investigation showed that, unlike rabbits, feeding cholesterol to rats did not expand the bile acid (ligand) pool for FXR but substantially increased LXR-{alpha} ligands, oxysterols in the liver. Therefore, FXR was not activated, as evidenced by unchanged mRNA levels of FXR target genes, SHP and BSEP, whereas LXR-{alpha} was activated with a significant increase in mRNA levels of the LXR-{alpha} target gene ABCA1. As a result, CYP7A1 was upregulated by activated LXR-{alpha} in Ch-fed rats.

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 {beta}-, {alpha}-, and {omega}-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-{alpha} 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-{alpha} 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-{alpha} 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-{alpha} was also simultaneously activated, as evidenced by a further increase in mRNA levels of LXR-{alpha} 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-{alpha} was the dominant regulator of CYP7A1, since 1) in Ch-fed rats, CYP7A1 was upregulated by LXR-{alpha}, 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-{alpha}. 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-{alpha} and FXR were activated compared with rats fed Ch alone where only LXR-{alpha} was activated, demonstrated the inhibitory effects of activated FXR on CYP7A1 in the presence of activated LXR-{alpha} in rats. Therefore, upregulation of CYP7A1 in Ch-fed rats is not because rat CYP7A1 is only sensitive to activated LXR-{alpha} 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-{alpha}. 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-{alpha} were simultaneously activated in the rats fed CA + Ch. Thus in rats, when both LXR-{alpha} and FXR are activated, the stimulatory effects of LXR-{alpha} are offset by the inhibitory effects of FXR on CYP7A1.


    ACKNOWLEDGMENTS
 
GRANTS

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.


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. Xu, Gastrointestinal Lab (15A), Veterans Affairs Medical Center, 385 Tremont Ave., East Orange, NJ 07018-1095 (E-mail: xugu{at}umdnj.edu).

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ananthanarayanan M, Balasubramanian N, Makishima M, Mangelsdorf D, and Suchy FJ. Human bile salt export pump promoter is transactivated by the farnesoid X receptor/bile acid receptor. J Biol Chem 276: 28857-28865, 2001.[Abstract/Free Full Text]
  2. Biesecker LG, Gottschalk LR, and Emerson SG. Identification of four murine cDNAs encoding putative protein kinases from primitive embryonic stem cells differentiated in vitro. Proc Natl Acad Sci USA 90: 7044-7048, 1993.[Abstract]
  3. Chiang JYL, Kimmel R, and Stroup D. Regulation of cholesterol 7{alpha}-hydroxylase gene (CYP7A1) transcription by the liver orphan receptor (LXR{alpha}). Gene 262: 257-265, 2001.[CrossRef][ISI][Medline]
  4. Chiang JYL, Kimmel R, Weinberger C, and Stroup D. Farnesoid X receptor responds to bile acids and represses cholesterol 7{alpha}-hydroxylase gene (CYP7A1) transcription. J Biol Chem 275: 10918-10924, 2000.[Abstract/Free Full Text]
  5. Chomozynski R and Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987.[CrossRef][ISI][Medline]
  6. Costet P, Luo Y, Wang N, and Tall AR. Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor. J Biol Chem 275: 28240-28245, 2000.[Abstract/Free Full Text]
  7. Dzeletovic S, Breuer O, Lund E, and Diczfalusy U. Detemination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry. Anal Biochem 225: 73-80, 1995.[CrossRef][ISI][Medline]
  8. Eriksson S. Biliary excretion of bile acids and cholesterol in bile fistula rats: bile acids and steroids. Proc Soc Exp Biol (NY) 94: 578-582, 1957.
  9. Goodwin B, Jones SA, Price RR, Watson MA, McKee DD, Moore LB, Galardi C, Wilson JG, Lewis MC, Roth ME, Maloney PR, Willson TR, and Kliewer SA. A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell 6: 517-526, 2000.[ISI][Medline]
  10. Gupta S, Pandak WMWM, and Hylemon PB. LXR{alpha} is the dominant regulator of CYP7A transcription. Biochem Biophys Res Commun 292: 338-343, 2002.
  11. Honda A, Salen G, Shefer S, Batta AK, Honda M, Xu G, Tint GS, Matsuzaki Y, Shoda J, and Tanaka N. Bile acid synthesis in the Smith-Lemli-Opitz syndrome: effects of dehydrocholesterols on cholesterol 7alpha-hydroxylase and 27-hydroxylase activities in rat liver. J Lipid Res 40: 1520-1528, 1999.[Abstract/Free Full Text]
  12. Honda A, Shoda J, Tanaka N, Matsuzaki Y, Osuga T, Shigematsu N, Tohma M, and Miyazaki H. Simultaneous assay of the activities of two key enzymes in cholesterol metabolism by gas chromatography-mass spectrometry. J Chromatogr A 565: 53-66, 1991.[CrossRef][ISI]
  13. Janowski BA, Groganm MJ, Jones SA, Wisely GB, Kliewer SA, Corey EJ, and Mangelsdorf DJ. Structural requirements of ligands for the oxysterol liver X receptors LXR{alpha} and LXR{beta}. Proc Natl Acad Sci USA 96: 266-271, 1999.[Abstract/Free Full Text]
  14. Janowski BA, Willy PJ, Devi TR, Falck JR, and Mangelsdorf DJ. An oxysterol signaling pathway mediated by the nuclear receptor LXR{alpha}. Nature 383: 728-731, 1996.[CrossRef][ISI][Medline]
  15. Jelinek DF, Andersson S, Slaughter CA, and Russell DW. Cloning and regulation of cholesterol 7{alpha}-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis. J Biol Chem 265: 8190-8197, 1990.[Abstract/Free Full Text]
  16. Lehmann JM, Kliewer SA, Moore LB, Smith-Oliver TA, Su J-L, Sundseth SS, Winegar DA, Blanchard DE, Spencer TA, and Wilson TM. Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway. J Biol Chem 272: 3137-3140, 1997.[Abstract/Free Full Text]
  17. Li-Hawkins J, Gåfvels M, Olin M, Lund E, Andersson U, Schuster G, Björkhem I, Russell DW, and Eggertsen G. Cholic acid mediates negative feedback regulation of bile acid synthesis in mice. J Clin Invest 110: 1191-1200, 2002.[Abstract/Free Full Text]
  18. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275, 1951.[Free Full Text]
  19. Lu TT, Makishimam M, Repa JJ, Schoonjans K, Kerr TA, Auwerx J, and M DJangelsdorf. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol Cell 6: 507-515, 2000.[ISI][Medline]
  20. Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, Luk A, Hull MV, Lustig KD, Mangelsdorf DJ, and Shan B. Identification of a nuclear receptor for bile acids. Science 284: 1362-1365, 1999.[Abstract/Free Full Text]
  21. Mok HYI, Perry PM, and Dowling RH. The control of bile acid pool size: Effect of jejunal resection and phenobarbitone on bile acid metabolism in the rat. Gut 15: 247-253, 1974.[ISI][Medline]
  22. Moundras C, Behr SR, Rémésy C, and Demigné C. Fecal losses of sterols and bile acids induced by feeding rats guar gum are due to greater pool size and liver bile acid secretion. J Nutr 127: 1068-1076, 1997.[Abstract/Free Full Text]
  23. Pandak WM, Li YC, Chiang JYL, Studer EJ, Gurley EC, Heuman DM, Vlahcevic ZR, and Hylemon PB. Regulation of cholesterol 7{alpha}-hydroxylase mRNA and transcriptional activity by taurocholate and cholesterol in the chronic biliary diverted rat. J Biol Chem 266: 3416-3421, 1992.[ISI]
  24. Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG, Kliewer SA, Stimmel JB, Willson TM, Zavacki AM, Moore DD, and Lehmann JM. Bile acids: natural ligands for an orphan nuclear receptor. Science 284: 1365-1368, 1999.[Abstract/Free Full Text]
  25. Peet DJ, Truly SD, Ma W, Janowski BA, Lobaccaro J-MA, Hammer RE, and Mangelsdorf DJ. Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR-{alpha}. Cell 93: 693-704, 1998.[ISI][Medline]
  26. Shefer S, Nguyen LB, Salen G, Ness GC, Chowdhary IR, Lerner S, Batta AK, and Tint GS. Differing effects of cholesterol and taurocholate on steady state hepatic HMG-CoA reductase and cholesterol 7{alpha}-hydroxylase activities and mRNA levels in the rat. J Lipid Res 33: 1193-1200, 1992.[Abstract]
  27. Shefer S, Salen G, and Batta AK. Methods of assay. In: Cholesterol 7{alpha}-Hydroxylase (7{alpha}-Monooxygenase), edited by Fears R and Sabine JR. Boca Raton, FL: CRC, 1986, p. 43-49.
  28. Smit MJ, Kuipers F, Vonk RJ, Temmerman AM, Jäckle S, and Windler EET. Effects of Dietary cholesterol on bile formation and hepatic processing of chylomicron remnant cholesterol in the rat. Hepatology 17: 445-454, 1993.[ISI][Medline]
  29. Thomas PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77: 5201-5205, 1980.[Abstract]
  30. Wang H, Chen J, Hollister K, Sowers LC, and Forman BM. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol Cell 3: 543-553, 1999.[ISI][Medline]
  31. Xu G, Li H, Pan L, Shang Q, Honda A, Ananthanarayanan M, Erickson SK, Shneider BL, Shefer S, Bollineni J, Forman BM, Matsuzaki Y, Suchy FJ, GS Tint, and Salen G. Farnesoid X receptor (FXR)-mediated down-regulation of CYP7A1 dominates LXR{alpha} in long term cholesterol fed NZW rabbits. J Lipid Res 44: 1956-1962, 2003.[Abstract/Free Full Text]
  32. Xu G, Pan L, Li H, Forman BM, Erickson SK, Shefer S, Bolloneni J, Batta A, Christie J, Wang T, Yang S, Tsai R, Lai L, Shimada K, Tint GS, and Salen G. Regulation of the farnesoid X receptor (FXR) by bile acid flux in rabbits. J Biol Chem 277: 50491-50496, 2002.[Abstract/Free Full Text]
  33. Xu G, Salen G, Shefer S, Ness GC, Nguyen LB, Parker TS, Chen TS, Zhao Z, Donnelly TM, and Tint GS. Unexpected inhibition of cholesterol 7{alpha}-hydroxylase by cholesterol in New Zealand White and Watanabe Heritable Hyperlipidemic rabbits. J Clin Invest 95: 1497-1504, 1995.[ISI][Medline]
  34. Xu G, Salen G, Shefer S, Tint GS, Kren BT, Nguyen LB, Steer CJ, Chen TS, Salen L, and Greenblatt D. Increased bile acid pool inhibits cholesterol 7{alpha}-hydroxylase in cholesterol-fed rabbits. Gastroenterology 113: 1958-1965, 1997.[ISI][Medline]
  35. Xu G, Salen G, Shefer S, Tint GS, Nguyen LB, Chen TS, and Greenblatt D. Increasing dietary cholesterol induces different regulation of classic and alternative bile acid synthesis. J Clin Invest 103: 89-95, 1999.[Abstract/Free Full Text]
  36. Xu G, Shneider BL, Shefer S, Nguyen LB, Batta AK, Tint GS, Arrese M, Thevananther S, Ma L, Stengelin S, Kramer W, Greenblatt D, Pcolinsky M, and Salen G. Ileal bile acid transport regulates bile acid pool, synthesis and plasma cholesterol levels differently in cholesterol-fed rats and rabbits. J Lipid Res 41: 298-304, 2000.[Abstract/Free Full Text]
  37. Zhang Y, Repa JJ, Gautheir K, and Mangelsdorf DJ. Regulation of lipoprotein lipase by the oxysterol receptors, LXR{alpha} and LXR{beta}. J Biol Chem 276: 43018-43024, 2001.[Abstract/Free Full Text]




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