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Comparative regulation of major enzymes in the bile acid biosynthesis pathway by cholesterol, cholate and taurine in mice and rats
Life Sciences 77 (2005) 746 – 757 www.elsevier.com/locate/lifescie
Comparative regulation of major enzymes in the bile acid biosynthesis pathway by cholesterol, cholate and taurine in mice and rats Wen Chena,c, Kazuhito Surugaa, Naomichi Nishimurab, Toshinao Goudaa, Vinh Nien Lama, Hidehiko Yokogoshia,T a
Laboratory of Nutritional Biochemistry, School of Food and Nutritional Sciences, and COE Program in the 21st Century, The University of Shizuoka, Shizuoka 422-8526, Japan b Department of Human Life and Development, Nayoro City College, Nayoro 096-8641, Japan c Beijing Laboratory of Bioactive Substances and Functional Foods, Beijing Union University, Beijing 100083, China Received 16 January 2004; accepted 23 November 2004
Abstract These enzymes play important roles in the biosynthesis of bile acids. They are cholesterol 7a-hydroxylase (CYP7A1), the rate limiting enzyme in the classic pathway, sterol 12a-hydroxylase (CYP8B1), the key enzyme for synthesis of cholic acid (CA), and sterol 27-hydroxylase (CYP27), the initial enzyme in the alternative pathway. In the present study, the susceptibility of these three enzymes to dietary cholesterol and cholate, and the cholesterol lowering effect of taurine were determined in male C57BL/6 mice and Wistar rats. Both mice and rats were divided into 6 groups: control group (N), high cholesterol diet group (C), high cholesterol and cholate diet group (CB), and their 1% taurine-supplemented groups (NT, CT, CBT, respectively). After animals were fed with the respective diets for one week, the mRNA levels of CYP7A1 increased in the C-group compared with those of the N-group, and decreased in the CB-group compared with those of the C-group in both mice and rats. But the extent of decrease is different between the two species. CYP8B1 was also markedly repressed by cholate in mice, but not in rats. These results are consistent with the changes in serum and liver cholesterol concentrations. Taurine significantly increased CYP7A1 mRNA levels in the CBT-group compared with the CB-group in both animal models, with a subsequent decrease in serum and liver cholesterol levels and increase in fecal bile acid excretion. Up-regulated CYP8B1 was also observed after taurine supplementation in
T Corresponding author. Tel.: +81 54 264 5559; fax: +81 54 263 7079. E-mail address: [email protected] (H. Yokogoshi). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.11.036
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the CBT-group in mice. No increase in CYP7A1 was produced by taurine in the CT-group compared with that of the C-group in mice, although the changes of serum and liver cholesterol and fecal bile acids indicated taurine showed an efficient cholesterol lowering effect. In addition, CYP27 was induced in both C- and CBgroups of rats but not of mice, and no changes were produced by taurine. The overall results suggest that there are differences between mice and rats in susceptibility of the three enzymes to dietary cholesterol and cholate, and taurine induced CYP7A1 to produce its cholesterol-lowering effect only in the presence of cholate in the cholesterol diet. D 2005 Elsevier Inc. All rights reserved. Keywords: Cholesterol; Sodium cholate; Taurine; CYP7A1; CYP8B1; CYP27; Mouse; Rat
Introduction Cholesterol homeostasis is maintained by coordinate regulation of three primary pathways which include de novo synthesis, uptake from plasma, and conversion into bile acids in the liver (Russel and Setchell, 1992). Bile acid biosynthesis from cholesterol occurs via two pathways: one, the classic (also known as neutral) pathway, and second, the alternative (also known as acidic or mitochondrial) pathway (Chiang, 1998; Vlahcevic et al., 1999). In the classic pathway, 7a-hydroxylation of cholesterol is initiated by microsomal CYP7A1, a specific enzyme in the liver, followed by a sequence of 14 enzymatic steps catalyzed by a variety of enzymes located in cytosol, microsomes, mitochondria, and peroxisomes, to yield the primary bile acids cholic acid (CA) and chenodexycholic acid (CDCA). One of the intermediate, 7a-hydroxy-4-cholesten-3-one, is a branch point intermediate which can be converted into the precursors of either CA or CDCA. CA can be formed when this intermediate is hydroxylated at the C-12 position by CYP8B1, the key enzyme for CA formation. In the alternative pathway, bile acid synthesis is initiated by mitochondrial CYP27 to convert cholesterol to 27-hydroxycholesterol, and the main product is believed to be CDCA (Chiang, 1998; Bjorkhem, 1992). The above three enzymes, CYP7A1, CYP8B1 and CYP27 play critical roles in the biosynthesis of bile acids. Within the liver the bile acids are conjugated with either glycine or taurine to form glycoconjugates or tauroconjugates, respectively, before their being secreted into the bile ducts. Research on the cholesterollowering effect of taurine were initiated for this reason (Danielsson, 1963; Tsuji et al., 1979; Sugiyama et al., 1989). Taurine (2-aminoethanesulfonic acid) is a conditionally essential amino acid which is not incorporated into proteins, but rather is found free in the body. Taurine had already been known to play an important role in numerous physiological functions including: antioxidation, detoxification, osmoregulation, membrane stabilization, neuromodulation, modulation of cellular calcium levels and conjugation with bile acids etc. (Kuriyama, 1980; Wright et al., 1986; Thurston et al., 1980; Pasantes et al., 1985; Huxtable, 1992). Several research groups have reported that the cholesterol-lowering effect of taurine was carried out by enhancing CYP7A1 activity and fecal bile acid excretion in hypercholesterolemic rats (Tsuji et al., 1979; Sugiyama et al., 1989; Venkatesan et al., 1993; Gandhi et al., 1992). Our previous publications not only agreed with the above conclusions, but also described the reverse effect of taurine on cholesterol catabolism in different types of hypercholesterolemic rats (Yokogoshi et al., 1999; Yokogoshi and Oda, 2002; Mochizuki et al., 2001).
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Although widely reported that taurine has outstanding cholesterol-lowering effects by improving CYP7A1 activity, there is a paucity of literature on taurine’s effect on the other two enzymes. The major objectives of this study were to compare not only the susceptibility of CYP7A1, CYP8B1 and CYP27 to dietary cholesterol and cholate in mice and rats, but also to compare the effects of taurine on these three enzymes and its cholesterol-lowering effects.
Materials and methods Materials Taurine was obtained from Taisho Pharmaceutical Co. (Ohmiya, Japan). All other chemicals whose sources were not indicated were from Wako Pure Chemical Co. (Osaka, Japan). Animals and diets Ten-week old male C57BL/6 mice and 5-week old Wistar male rats (SLC Inc., Hamamatsu, Japan) were housed in individual cages at 23 F 1 8C with a 12-h light/dark cycle (lights on 0700). In order to accustom them to our experimental conditions, they were fed with a control diet (20% casein) for 3 days. On the 4th day, both mice and rats were divided into 6 groups and designated as: control group (N), high cholesterol diet group (C), high cholesterol and cholate diet group (CB), and their 1% taurine-supplemented groups (NT, CT, CBT, respectively), based on their serum cholesterol levels and body weights. The diet composition in each group is shown in Table 1. Two-24h fecal samples were collected from each animal towards the end of the feeding period, lyophilized, and stored at 208C until assay. After the animals were allowed free access to water and their respective diets for one week, they were anesthetized with diethyl ester and decapitated at 23:00, the blood then collected, and the liver removed for determination of lipid and mRNA levels of CYP7A1, CYP8B1 and CYP27.
Table 1 The composition of diet in each group (g/kg) Group a
Casein Cellulosea Corn oila Choline cloride Vitamin mix AIN-93G a Mineral mix AIN-93G a Sucrosea Corn starcha Cholesterol Sodium cholate Taurine a
N
NT
C
CT
CB
CBT
200 50 50 1.5 10 35 217.8 435.7 – –
200 50 50 1.5 10 35 214.5 429 – – 10
200 50 50 1.5 10 35 214.5 429 10 – –
200 50 50 1.5 10 35 211.1 422.4 10 – 10
200 50 50 1.5 10 35 213.7 427.3 10 2.5 –
200 50 50 1.5 10 35 210.3 420.7 10 2.5 10
Purchased from Oriental Yeast Co. (Japan).
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The study was subject to approval by the Animal Use Committee of University of Shizuoka, and the animals were maintained in accordance with the guidelines for the care and use of laboratory animals, University of Shizuoka. Determination of serum and liver lipids and fecal bile acids Serum total cholesterol and HDL-cholesterol were determined by commercial kits (Cholesterol Ctest kit and HDL-cholesterol-test kit, respectively, Wako Pure Chemical Co., Japan). The concentration of (VLDL + LDL) was calculated as the difference between total cholesterol and the HDL-cholesterol levels. TLiver lipids were extracted by the method of Folch et al. (Folch et al., 1957). The extract was evaporated to dryness under N2 and then resuspended in isopropanol. Cholesterol levels were also measured using the commercial kit (Cholesterol C-test kit, Wako Pure Chemical Co., Japan). TFaeces were collected, lyophilized, ground, and steroids were extracted by ethanol and petroleum ether according to the method of Sheltawy and Losowsky (Sheltawy and Losowsky, 1975). The extract was dried under N2 and then resuspended in methanol. Total bile acids were measured by an enzymatic method which recognizes only the 3a-hydroxyl moiety of the steroid structure. Quantitation of mRNA for CYP7A1, CYP8B1 and CYP27 The CYP7A1 cDNA probe was a generous gift from Dr. Hiroaki Oda (Nagoya University, Japan). For CYP8B1 and CYP27, cDNA probes were prepared as follows: first-strand cDNA was prepared from mouse liver total RNA using QIAGEN OneStep RT-PCR Kit (QIAGEN K.K., Japan), and the cDNA was used as a template in the following PCR reactions with the following primer pairs, 5VTGAATTCTTGAAGGGGATGC-3V (forward) and 5V-CCTTGCTCCCTCAGAAACTG-3V (reverse) for CYP8B1, and 5V-TTCTCAGACACGATCTATGGCTGT-3V (forward) and 5V-CTACTGTCTCTGCAGAAAGCGTA-3V (reverse) for CYP27. The resulting PCR products were subcloned into pGEM-T easy vector (Promega Co.) and then purified on 1% agarose gels. Sequencing of subcloned products was performed to confirm that we had isolated the correct cDNA. All cDNA probes were radiolabeled with 32P-dCTP using Takara Random Primer DNA labelling Kit Ver.2 (Takara Bio INC., Japan). Total RNA was prepared from mouse or rat liver using TRIZOL Reagent (Invitrogen/Life Technologies, Japan), and then mRNA levels of the enzymes were determined by Northern blot. Finally, bands were quantified by Storm 820 Molecular Dynamics (Amersham Pharmacia Biotech.), and the results were corrected with regard to the signal generated from GAPDH mRNA obtained from the same membrane. Statistics The means and SE of 4 mice or rats per group are reported. Statistical analyses were performed by one-way ANOVA followed by the Duncan’s multiple range test. P-values of less than 0.05 were considered to be significant.
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Results Serum and liver cholesterol and fecal bile acid levels in mice Data are shown in Table 2. After having free access to the diets and water for one week, the animals in the six groups showed no significant differences in body weight, food intake, and liver weight. Both serum total cholesterol and VLDL + LDL cholesterol concentrations in the C- and CB-groups were significantly higher than those of the N-group by cholesterol loading in the diets, and both of them were notably decreased by taurine supplementation. HDL cholesterol levels did not change by cholesterol loading in the C-group but decreased remarkably in the CB-group, and it was significantly improved by taurine supplementation in the CBT-group. These data suggest that the decrease in serum cholesterol level by taurine supplementation is mainly due to the decrease in VLDL and LDL cholesterol. The amount of liver total cholesterol was notably enhanced by cholesterol loading in the C-group and further increased by adding cholate to the cholesterol diet in the CB-group. The cholesterol levels of both CT- and CBT-groups were significantly reduced by taurine supplementation. With the exception that the amount of dried feces in the CBT-group was significantly increased compared with that of the N-group, there were no differences among the other five groups. The fecal bile acid levels were markedly enhanced in the C-group compared with that of the N-group, which increased further by cholate loading in the CB-group. The fecal bile acid excretion was further raised by taurine supplementation in both the CT- and CBT-groups compared with the C- and CB-groups, respectively. These data suggest that cholesterol degradation is enhanced by cholesterol loading in the diet, and is further promoted by taurine supplementation. The amounts of serum and liver cholesterol and fecal bile acids in the rats Data are shown in Table 3. Table 2 The effect of taurine on cholesterol metabolism in mice fed high cholesterol diets with or without cholate for 7 days N Final body weight (g) Food intake (g/d) Liver weight (mg/g body weight) Serum lipids (mmol/L) Total cholesterol HDL-cholesterol VLDL + LDL-cholesterol Liver cholesterol (umol/g) Feces weight (g/2d) Fecal total bile acids (umol/g)
NT
CT
CB
CBT
27.00 F 0.63 27.69 F 1.15 28.06 F 0.86 4.11 F 0.03 4.18 F 0.09 4.30 F 0.09 50.42 F 3.27 52.94 F 4.91 52.66 F 0.86
26.65 F 1.08 4.08 F 0.19 55.35 F 2.92
26.00 F 1.29 4.09 F 0.11 54.51 F 4.54
27.42 F 0.67 4.25 F 0.04 58.33 F 2.18
3.45 F 0.14a 2.04 F 0.07b 1.41 F 0.10a 22.23 F 0.32a 0.72 F 0.03a 16.50 F 0.17a
3.55 F 0.31a 1.87 F 0.12b 1.68 F 0.21a 29.01 F 0.52b 0.86 F 0.04ab 31.71 F 1.43c
4.48 F 0.13b 1.39 F 0.09a 3.10 F 0.17c 53.45 F 2.54e 0.85 F 0.07ab 61.60 F 1.85d
3.50 F 0.12a 1.88 F 0.13b 1.62 F 0.14a 39.38 F 1.50d 0.93 F 0.04b 71.18 F 2.08e
3.27 F 0.2a 2.00 F 0.07b 1.27 F 0.15a 23.01 F 1.15a 0.75 F 0.04a 17.38 F 0.45a
C
4.41 F 0.23b 2.14 F 0.18b 2.27 F 0.19b 34.14 F 1.12c 0.86 F 0.04ab 27.32 F 0.67b
Values are mean F SE, n = 4. Values with diferent superscripts within the same line are significantly different at p b 0.05.
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Table 3 The effect of taurine on cholesterol metabolism in rats fed high cholesterol diets with or without cholate for 7 days N Final body weight (g) Food intake (g/d) Liver weight (mg/g body weight) Serum lipids (mmol/L) Total cholesterol HDL-cholesterol VLDL + LDL-cholesterol Liver cholesterol (umol/g) Feces wieght (g/2d) Fecal total bile acids (umol/g)
NT
C
CT
168.7 F 4.0 175.5 F 6.1 173.0 F 6.4 174.4 F 3.8 15.36 F 0.56 16.31 F 0.39 15.93 F 0.84 16.05 F 0.53 3.94 F 0.10a 4.06 F 0.13a 4.27 F 0.12b 4.37 F 0.06b
1.99 F 0.14a 1.96 F 0.11a 1.32 F 0.11b 1.34 F 0.08b 0.67 F 0.10a 0.62 F 0.15a 20.76 F 0.73a 20.91 F 1.47a 2.28 F 0.05a 2.51 F 0.12ab 23.77 F 0.95a 25.65 F 0.35a
2.29 F 0.13a 1.12 F 0.10ab 1.17 F 0.08b 32.09 F 0.73b 2.82 F 0.18b 60.08 F 1.96b
CB 174.1 F 5.0 16.02 F 066 4.66 F 0.09c
CBT 168.1 F 2.1 15.05 F 0.24 4.43 F 0.06bc
2.22 F 0.14a 3.37 F 0.21b 1.94 F 0.12a 1.21 F 0.04b 1.04 F 0.04a 1.15 F 0.10ab ab c 1.00 F 0.14 2.33 F 0.21 0.79 F 0.03a b d 30.39 F 3.43 57.69 F 1.22 41.39 F 1.82c 2.68 F 0.10b 2.98 F 0.16 b 2.87 F 0.08b 64.14 F 1.75b 103.79 F 0.68c 126.02 F 2.32d
Values are mean F SE, n = 4. Values with diferent superscripts within the same line are significantly different at p b 0.05.
After having free access to the diets and water for one week, the animals among the six groups showed no differences in body weight and food intake. Liver weight was increased by a cholesterol loading diet and further raised by adding cholate to the cholesterol diet. Unlike in mice, serum total cholesterol concentrations in rats were significantly higher in the CBgroup but not in the C-group, and it was normalized by taurine supplementation in the CBT-group. HDL cholesterol was notably lower in the CB-group compared with that of the N-group, and no change was observed by taurine supplementation. VLDL + LDL cholesterol level was greatly increased by a cholesterol loading diet, which was further increased by adding cholate to the cholesterol diet and significantly decreased by taurine supplementation in the CBT-group. As in the case with mice, the decrease of serum cholesterol by taurine supplementation in the rat is due mainly to the decrease in VLDL and LDL. The amount of liver total cholesterol was notably raised by cholesterol loading in the C-group, and further increased by adding cholate to the cholesterol diet in the CB-group. In contrast, it was significantly reduced by taurine supplementation in the CBT-group compared with that of the CB-group, but there was no difference between the C- and CT-groups. The dried feces weights in the C- and CB-groups were significantly higher than those of the N-group. The fecal bile acid levels dramatically increased in the C-group compared with that of the N-group and further increased by cholate loading in the CB-group. In contrast to mice, the increase in fecal bile acid excretion was observed in the CBT-group but not the CT-group compared with the CB- and C-groups, respectively. This is in agreement with the results of no difference in the levels of serum and liver cholesterol between the C- and CT-groups. mRNA levels of the three enzymes in mice and rats The mRNA levels of CYP7A1 (Fig. 1) were increased in the C-group compared with those of the Ngroup, but decreased in the CB-group compared with those of the C-group in both mice and rats. This is in agreement with a regulatory mechanism in which CYP7A1 can be induced by cholesterol and repressed by bile acids (Yokogoshi and Oda, 2002; Russel and Setchell, 1992). CYP7A1 mRNA levels
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Fig. 1. Effect of diets on CYP7A1 mRNA level in mice (A) and rats (B). After animals were fed with the respective diets for 7 days, their livers were obtained at 23:00 in the dark cycle for later extraction of total RNA. The relative amounts of CYP7A1 mRNA were quantified relative to GAPDH using Northern blot. N-control diet group, NT-taurine supplemented control diet group, C-cholesterol diet group, CT-taurine supplemented cholesterol diet group, CB-cholesterol and cholate diet group, CBTtaurine supplemented cholesterol and cholate diet group. Bars with different superscript are significantly different at p b 0.05. (A). CYP7A1 mRNA was significantly repressed in the CB-group and notably increased in the CBT-group by taurine supplementation. (B). CYP7A1 mRNA was markedly increased in the C-group and repressed in the CB-group by adding cholate to the cholesterol diet. It was not enhanced by taurine in the CT-group but dramatically improved in the CBT-group compared with that of the C-group and CB-group respectively.
were notably increased by taurine supplementation in the CBT-group compared with those of the CBgroup, but there was no difference between the C- and CT-group in the two animal models. These results suggest that taurine induces CYP7A1 only in the presence of cholate in the cholesterol diet. In mice the CYP7A1 mRNA levels were repressed in the CB-group not only to be less than those of the C-group but also the N-group, whereas in rats they were less than those of the C-group but higher than those of the N-
Fig. 2. Effect of diets on CYP8B1 mRNA level in mice (A) and rats (B). After animals were fed with the respective diets for 7 days, their livers were obtained at 23:00 in the dark cycle for later extraction of total RNA. The relative amount of CYP8B1 mRNA was quantified relative to GAPDH using Northern blot. N-control diet group, NT-taurine supplemented control diet group, C-cholesterol diet group, CT-taurine supplemented cholesterol diet group, CB-cholesterol and cholate diet group, CBTtaurine supplemented cholesterol and cholate diet group. Bars with different superscript are significantly different at p b 0.05. (A). CYP8B1 mRNA was significantly repressed in the CB-group and notably increased in the CBT-group by taurine supplementation. (B). There are no notable differences among the six groups.
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Fig. 3. Effect of diets on CYP27 mRNA level in mice (A) and rats (B). After animals were fed with the respective diets for 7 days, their livers were obtained at 23:00 in the dark cycle for later extraction of total RNA. The relative amount of CYP27 mRNA was quantified relative to GAPDH using Northern blot. N-control diet group, NT-taurine supplemented control diet group, C-cholesterol diet group, CT-taurine supplemented cholesterol diet group, CB-cholesterol and cholate diet group, CBTtaurine supplemented cholesterol and cholate diet group. Bars with different superscript are significantly different at p b 0.05. (A). There are no changes among the six groups. (B). CYP27 mRNA levels tend to increase in the C- and CB-group compared to that of the N-group, but are not significantly increased by taurine.
group. These data suggest that the susceptibility of CYP7A1 to cholesterol and bile acid is very different between the two species. No changes were observed in CYP8B1 mRNA levels (Fig. 2) by cholesterol loading, but they were significantly repressed by adding cholate to the cholesterol diet, and were increased by taurine supplementation in the CBT-group compared with the CB-group in mice. However, no differences were observed in the six groups of rats. As was shown with CYP7A1, the susceptibility of CYP8B1 to bile acid is very different between mice and rats. CYP27 mRNA (Fig. 3) was improved in the C- and CB-groups of rats but not of mice, and it was not significantly up-regulated by taurine supplementation although it seemed much higher in the CBT-group than in the CB-group of rats.
Discussion CYP7A1 is the rate-limiting enzyme in the classic pathway, which is the main pathway of bile acid biosynthesis. It has been widely reported that its gene transcription is regulated by several factors or nuclear receptors to balance the elimination of cholesterol as a response to optimize the physiological status of the body (Janowski et al., 1996; Wang et al., 1999). Although CYP7A1 is probably responsible for regulating the overall rates of bile acids biosynthesis, CYP8B1 also plays an important role in determining the ratio of CA to CDCA to affect the bile composition and its relative hydrophobicity. In addition, the alternative pathway was reported to contribute less than 10% to total bile acid biosynthesis (Vlahcevic et al., 1980; Swell et al., 1980). However, recent studies have demonstrated that it could become a more important contributor under certain pathological conditions, in vitro studies suggesting that it accounted for 50% of bile acid synthesis (Axelson et al., 1989; Stravitz et al., 1996). Therefore, there is little doubt that CYP27 is important in the conversion of cholesterol to bile acids. In the present study, we discussed the susceptibility of the three enzymes described above to cholesterol and bile acids in mice and rats.
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It has been known that the rat is a poor model for developing experimental atherosclerosis since in this species a high cholesterol diet induces CYP7A1 which facilitates disposal of excess cholesterol (Jones et al., 1993; Doerner et al., 1995). In our study, we also observed that a diet high in cholesterol in the C-group induced CYP7A1 mRNA in rats, which agrees with previous reports. Subsequently fecal bile acid excretion was significantly increased and serum cholesterol concentration was maintained at normal levels. It was reported that CYP7A1 activity was not affected by adding cholate to a high cholesterol diet (Vlahcevic et al., 2000). However, we found that CYP7A1 mRNA levels were significantly repressed by diets high in combination of cholesterol and cholate in the CB-group compared with those of the C-group, but still notably higher than those of the N-group. These results suggest that the overall effect of the combination of cholesterol and cholate was to induce CYP7A1 mRNA, and to show that rat is more susceptible to cholesterol than to cholate. It was reported that the activity of CYP27 was increased by a cholesterol diet, while CYP8B1 activity was significantly reduced by adding 1% cholate to a high cholesterol diet in rat (Nguyen et al., 1999; Vlahcevic et al., 2000). However, only the increasing trend in CYP27 mRNA was observed by both the cholesterol diets with or without cholate in the present study; this may be due to our use of a smaller amount of cholate in the diet than reported in other studies. In mice, CYP7A1 mRNA levels tended to increase and CYP8B1 was unchanged by the diet high in cholesterol, but they were markedly decreased by adding cholate to the cholesterol diet compared with those of the C- and N-groups. These data suggest that CYP7A1 and CYP8B1 are more susceptible to cholate than to cholesterol. Serum and liver cholesterol levels were significantly increased in the Cgroup and further enhanced by the combination of cholesterol and cholate in the CB-group. All groups showed that cholesterol accumulated in the liver as CYP7A1 and CYP8B1 were repressed, although the serum cholesterol concentration in the CB-group was maintained at the same level as that of the C-group. No significant change in CYP27 mRNA levels was produced by either cholesterol or cholate. The above data indicate that mice and rats show different responses to cholesterol and cholate. Cholesterol degradation in mice is more susceptible to repression by dietary cholate, whereas in the rat it is less sensitive to cholate than to cholesterol since it is increased even by a diet high in both cholesterol and cholate. These data suggest that there is an apparent difference in the regulation of cholesterol degradation or bile acid biosynthesis between mice and rats. In order to interpret further the cholesterol-lowering effect of taurine, we investigated its effects on mRNA levels of the aforementioned three enzymes in the two animal models. Although it has been well accepted that taurine could induce CYP7A1 to improve bile acid biosynthesis (Tsuji et al., 1979; Sugiyama et al., 1989; Venkatesan et al., 1993; Gandhi et al., 1992), we found that under our experimented conditions taurine only enhanced the mRNA levels of CYP7A1 in both mice and rats fed the CB-diet but not the C-diet. In mice, cholesterol concentrations in serum and liver were reduced, and fecal bile acid excretion was increased by taurine supplementation in the CT-group compared with that of the C-group, in spite of unchanged mRNA levels in any of the enzymes. These results suggest that taurine may enhance protein levels of the enzymes to express its cholesterol-lowering effect. Liver cholesterol concentration was reduced 26% by taurine supplementation in the CBT-group compared with that of CB-group; however only a 14% decrease was observed in the CT-group compared with that of the C-group. This may be attributed to the marked increase in mRNA levels of CYP7A1 and CYP8B1 by taurine supplementation in the CBT-group.
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In rats, taurine did not show an efficient action to reduce liver cholesterol, although liver cholesterol and fecal bile acid excretion tended to decrease and increase, respectively, in the CT-group compared with those of the C-group. But liver cholesterol levels were lowered 28% in the CBT-group compared with those of the CB-group as CYP7A1 was dramatically induced by taurine supplementation. Since CYP7A1 is subject to feedback inhibition by bile acids returning to the liver via the enterohepatic circulation (Russel and Setchell, 1992), the increased excretion of bile acids in the feces results in a decrease in bile acids in the enterohepatic circulation, subsequently activating CYP7A1, and finally accelerating cholesterol degradation. This is well confirmed in the CBT-group of the two animal models in our experiment that the sequence of reducing cholesterol concentration is accompanied by increase of fecal bile acid excretion and up-regulation of CYP7A1 mRNA. But no improvement in CYP7A1 mRNA level was observed in the CT-group of mice although serum and liver cholesterols were significantly decreased and fecal bile acid excretion was notably increased. These results suggest that cholate is required for taurine to induce CYP7A1 mRNA, or alternatively, exogenous cholate may not impair taurine from inducing CYP7A1 mRNA. In contrast, taurine induces CYP7A1 gene transcription only in the presence of cholate in the cholesterol diet, and then it shows its cholesterol-lowering effect. In other words, taurine induces CYP7A1 mRNA when high dietary cholesterol is assimilated because cholate is well known for its activity on cholesterol uptake. Biosynthesis, fecal excretion, and reabsorption from ileum are three important aspects of bile acid homeostasis in the body. Biosynthesis and fecal excretion of bile acids are increased by taurine, whereas reabsorption from the ileum is decreased. Our data may be interpreted as taurine inhibiting the intestinal transport of bile acids in the ileum, which would result in the up-regulation of CYP7A1 in the liver and lowering of liver and serum cholesterol. Ileum-bile acid binding protein (I-BABP) and ileum-bile acid transporter (I-BAT) are reported to be responsible for bile acid reabsorption in the ileum (Grober et al., 1999; Zhang et al., 2002). The nature of these responses is unclear, and further experiments are required to reveal the mechanism of the cholesterol-lowering effect of taurine. The overall results of our experiments suggest two points. 1) There are differences between mice and rats on the susceptibility of CYP7A1 and CYP8B1 to dietary cholesterol and cholate. It seems that they are more sensitive to cholate in mice, but more susceptible to cholesterol in rats. 2) Taurine induces CYP7A1 mRNA only in the presence of cholate in the cholesterol diet, which subsequently exerts its efficient cholesterol lowering effect. Acknowledgment This work was supported by a grant for scientific research from Shizuoka Prefecture, and 21st century COE program from Ministry of Education, Culture, Sports, Science and Technology of Japan. References Axelson, M., Mork, B., Aly, A., Wisen, O., Sjovall, J., 1989. Concentrations of cholestenoic acids in plasma from patients with liver disease. Journal of Lipid Research 30, 1877 – 1882. Bjorkhem, I., 1992. Mechanism of degradation of the steroid side chain in the formation of bile acids. Journal of Lipid Research 33, 455 – 471.
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Chiang, J.Y.L., 1998. Regulation of bile acid synthesis. Frontiers Bioscience 3, 176 – 193. Danielsson, H., 1963. Present states of research on catabolism and excretion of cholesterol. Advanced Lipid Research 1, 335 – 385. Doerner, K.C., Gurley, E.C., Vlahcevic, Z.R., Hylemon, P.B., 1995. Regulation of cholesterol 7a-hydroxylase expression by sterols in primary hepatocyte cultures. Journal of Lipid Research 36, 168 – 177. Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497 – 509. Gandhi, V.M., Cherian, K.M., Mulky, M.J., 1992. Hypolipidemic action of taurine in rats. Indian Journal of Experimental Biology 30 (15), 413 – 417. Grober, J., Zaghini, I., Fujii, H., Jones, S.A., Kliewer, S.A., Willson, T.M., Ono, T., Besnard, P., 1999. Identification of a bile acid-responsive element in the human ileal bile acid-binding protein gene. Involvement of the farnesoid receptor/9-cisretinoic acid receptor heterodimer. Journal of Biological Chemistry 274, 29749 – 29754. Huxtable, R.J., 1992. Physiological actions of taurine. Physiological Review 72, 101 – 163. Janowski, B.A., Willy, P.J., Devi, T.R., Falck, J.R., Mangelsdorf, D.J., 1996. An oxysterol signalling pathway mediated by nuclear receptor LXR alpha. Nature 383, 728 – 731. Jones, M.P., Pandak, W.M., Hylemon, P.B., Chiang, J.Y., Heuman, D.W., Vlahcevic, Z.R., 1993. Cholesterol 7a-hydroxylase: evidence for transcriptional regulation by cholesterol and/or metabolic products of cholesterol in the rat. Journal of Lipid Research 34, 885 – 892. Kuriyama, K., 1980. Taurine as a neuromodulator. Federation Proceeding 39, 2680 – 2684. Mochizuki, H., Oda, H., Yokogoshi, H., 2001. Dietary taurine potentiates polychlorinated biphenyl-induced hypercholesterolemia in rats. Journal of Nutritional Biochemistry 12 (2), 109 – 115. Nguyen, L.B., Xu, G., Shefer, S., Stephen Tint, G., Batta, A., Salen, G., 1999. Comparative regulation of hepatic sterol 27hydroxylase and cholesterol 7a-hydroxylase activities in the rat, guinea pig, and rabbit: Effects of cholesterol and bile acid. Metabolism 48 (12), 1542 – 1548. Pasantes, M.H., Wright, C.E., Gaull, G.E., 1985. Taurine protection of lymphoblastoid cells from iron-ascorbate-induced damage. Biochemical Pharmacology 34, 2205 – 2207. Russel, D.W., Setchell, K.D.R., 1992. Bile acid biosynthesis. Biochemistry 31, 4737 – 4748. Sheltawy, M.J., Losowsky, M.S., 1975. Determination of fecal bile acid by an enzymatic method. Clinica Chimica Acta 64, 127 – 132. Stravitz, R.T., Vlahcevic, Z.R., Russell, T.L., Heizer, M.L., Avadhani, N.G., Hylemon, P.B., 1996. Regulation of sterol 27hydroxylase and an alternative pathway of bile acid biosynthesis in primary cultures of rat hepatocytes. Journal of Steroid Biochemistry and Molecular Biology 57, 337 – 347. Sugiyama, K., Ohishi, A., Ohnuma, Y., Muramatsu, K., 1989. Comparison between the plasma cholesterol-lowering effects of glycine and taurine in fats fed on high cholesterol diets. Agric. Biological. Chemistry 53, 1647 – 1652. Swell, L., Gustafsson, J., Schwartz, C.C., Halloran, G., Danielsson, H., Vlahcevic, Z.R., 1980. An in vivo evaluation of the quantitative significance of several potential pathways to cholic and chenodeoxycholic acid from cholesterol in man. Journal of Lipid Research 21, 455 – 466. Thurston, J.H., Hauhart, R.E., Dirgo, J.A., 1980. Taurine: A role in osmotic regulation of mammalian brain and possible clinical significances. Life Sciences 26, 1561 – 1568. Tsuji, K., Seki, T., Iwao, H., 1979. Cholesterol-lowering effects of taurine and sulfur-containing amino acids in serum and liver of rats. Sulfur-Containing Amino Acids 2, 143 – 145. Venkatesan, N., Rao, P.V., Arumugam, V., 1993. Inhibitory effect of taurine on puromycin aminonucleoside-induced hyperlipidemia in rats. Journal of Clinical Biochemistry and Nutrition 15, 203 – 210. Vlahcevic, Z.R., Eggertsen, G., Bjorkhem, I., Hylemon, P.B., Redford, K., Pandak, W.M., 2000. Regulation of sterol 12ahydroxylase and cholic acid biosynthesis in the rat. Gastroenterology 118, 599 – 607. Vlahcevic, Z.R., Pandak, W.M., Stravitz, R.T., 1999. Regulation of bile acid biosynthesis. Gastroenterological Clinics of North America 28, 1 – 25. Vlahcevic, Z.R., Schwartz, C.C., Gustafsson, J., Halloran, L.G., Danielsson, H., Swell, L., 1980. Biosynthesis of bile acids in man. Multiple pathways to cholic and chenodeoxycholic acid. Journal of Biological Chemistry 255, 2925 – 2933. Wang, H., Chen, J., Hollister, K., Sowers, L.C., Forman, B.M., 1999. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Molecular Cell 3, 543 – 553. Wright, C.E., Tallan, H.H., Lin, Y.Y., 1986. Taurine: biological update. Annual Review of Biochemistry 55, 427 – 453.
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Yokogoshi, H., Mochizuki, H., Nanami, K., Hida, Y., Miyachi, F., Oda, H., 1999. Dietary taurine enhances cholesterol degradation and reduces serum and liver cholesterol concentrations in rats fed a high-cholesterol diet. Journal of Nutrition 129 (9), 1705 – 1712. Yokogoshi, H., Oda, H., 2002. Dietary taurine enhances cholesterol degradation and reduces serum and liver cholesterol concentrations in rats fed a high-cholesterol diet. Amino Acids 23 (4), 433 – 439. Zhang, E.Y., Phelps, M.A., Cheng, C., Ekins, S., Swaan, P.W., 2002. Modeling of active transport systems. Advanced Drug Delivery Reviews 54, 329 – 354.
Comparative regulation of major enzymes in the bile acid biosynthesis pathway by cholesterol, cholate and taurine in mice and rats Wen Chena,c, Kazuhito Surugaa, Naomichi Nishimurab, Toshinao Goudaa, Vinh Nien Lama, Hidehiko Yokogoshia,T a
Laboratory of Nutritional Biochemistry, School of Food and Nutritional Sciences, and COE Program in the 21st Century, The University of Shizuoka, Shizuoka 422-8526, Japan b Department of Human Life and Development, Nayoro City College, Nayoro 096-8641, Japan c Beijing Laboratory of Bioactive Substances and Functional Foods, Beijing Union University, Beijing 100083, China Received 16 January 2004; accepted 23 November 2004
Abstract These enzymes play important roles in the biosynthesis of bile acids. They are cholesterol 7a-hydroxylase (CYP7A1), the rate limiting enzyme in the classic pathway, sterol 12a-hydroxylase (CYP8B1), the key enzyme for synthesis of cholic acid (CA), and sterol 27-hydroxylase (CYP27), the initial enzyme in the alternative pathway. In the present study, the susceptibility of these three enzymes to dietary cholesterol and cholate, and the cholesterol lowering effect of taurine were determined in male C57BL/6 mice and Wistar rats. Both mice and rats were divided into 6 groups: control group (N), high cholesterol diet group (C), high cholesterol and cholate diet group (CB), and their 1% taurine-supplemented groups (NT, CT, CBT, respectively). After animals were fed with the respective diets for one week, the mRNA levels of CYP7A1 increased in the C-group compared with those of the N-group, and decreased in the CB-group compared with those of the C-group in both mice and rats. But the extent of decrease is different between the two species. CYP8B1 was also markedly repressed by cholate in mice, but not in rats. These results are consistent with the changes in serum and liver cholesterol concentrations. Taurine significantly increased CYP7A1 mRNA levels in the CBT-group compared with the CB-group in both animal models, with a subsequent decrease in serum and liver cholesterol levels and increase in fecal bile acid excretion. Up-regulated CYP8B1 was also observed after taurine supplementation in
T Corresponding author. Tel.: +81 54 264 5559; fax: +81 54 263 7079. E-mail address: [email protected] (H. Yokogoshi). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.11.036
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the CBT-group in mice. No increase in CYP7A1 was produced by taurine in the CT-group compared with that of the C-group in mice, although the changes of serum and liver cholesterol and fecal bile acids indicated taurine showed an efficient cholesterol lowering effect. In addition, CYP27 was induced in both C- and CBgroups of rats but not of mice, and no changes were produced by taurine. The overall results suggest that there are differences between mice and rats in susceptibility of the three enzymes to dietary cholesterol and cholate, and taurine induced CYP7A1 to produce its cholesterol-lowering effect only in the presence of cholate in the cholesterol diet. D 2005 Elsevier Inc. All rights reserved. Keywords: Cholesterol; Sodium cholate; Taurine; CYP7A1; CYP8B1; CYP27; Mouse; Rat
Introduction Cholesterol homeostasis is maintained by coordinate regulation of three primary pathways which include de novo synthesis, uptake from plasma, and conversion into bile acids in the liver (Russel and Setchell, 1992). Bile acid biosynthesis from cholesterol occurs via two pathways: one, the classic (also known as neutral) pathway, and second, the alternative (also known as acidic or mitochondrial) pathway (Chiang, 1998; Vlahcevic et al., 1999). In the classic pathway, 7a-hydroxylation of cholesterol is initiated by microsomal CYP7A1, a specific enzyme in the liver, followed by a sequence of 14 enzymatic steps catalyzed by a variety of enzymes located in cytosol, microsomes, mitochondria, and peroxisomes, to yield the primary bile acids cholic acid (CA) and chenodexycholic acid (CDCA). One of the intermediate, 7a-hydroxy-4-cholesten-3-one, is a branch point intermediate which can be converted into the precursors of either CA or CDCA. CA can be formed when this intermediate is hydroxylated at the C-12 position by CYP8B1, the key enzyme for CA formation. In the alternative pathway, bile acid synthesis is initiated by mitochondrial CYP27 to convert cholesterol to 27-hydroxycholesterol, and the main product is believed to be CDCA (Chiang, 1998; Bjorkhem, 1992). The above three enzymes, CYP7A1, CYP8B1 and CYP27 play critical roles in the biosynthesis of bile acids. Within the liver the bile acids are conjugated with either glycine or taurine to form glycoconjugates or tauroconjugates, respectively, before their being secreted into the bile ducts. Research on the cholesterollowering effect of taurine were initiated for this reason (Danielsson, 1963; Tsuji et al., 1979; Sugiyama et al., 1989). Taurine (2-aminoethanesulfonic acid) is a conditionally essential amino acid which is not incorporated into proteins, but rather is found free in the body. Taurine had already been known to play an important role in numerous physiological functions including: antioxidation, detoxification, osmoregulation, membrane stabilization, neuromodulation, modulation of cellular calcium levels and conjugation with bile acids etc. (Kuriyama, 1980; Wright et al., 1986; Thurston et al., 1980; Pasantes et al., 1985; Huxtable, 1992). Several research groups have reported that the cholesterol-lowering effect of taurine was carried out by enhancing CYP7A1 activity and fecal bile acid excretion in hypercholesterolemic rats (Tsuji et al., 1979; Sugiyama et al., 1989; Venkatesan et al., 1993; Gandhi et al., 1992). Our previous publications not only agreed with the above conclusions, but also described the reverse effect of taurine on cholesterol catabolism in different types of hypercholesterolemic rats (Yokogoshi et al., 1999; Yokogoshi and Oda, 2002; Mochizuki et al., 2001).
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Although widely reported that taurine has outstanding cholesterol-lowering effects by improving CYP7A1 activity, there is a paucity of literature on taurine’s effect on the other two enzymes. The major objectives of this study were to compare not only the susceptibility of CYP7A1, CYP8B1 and CYP27 to dietary cholesterol and cholate in mice and rats, but also to compare the effects of taurine on these three enzymes and its cholesterol-lowering effects.
Materials and methods Materials Taurine was obtained from Taisho Pharmaceutical Co. (Ohmiya, Japan). All other chemicals whose sources were not indicated were from Wako Pure Chemical Co. (Osaka, Japan). Animals and diets Ten-week old male C57BL/6 mice and 5-week old Wistar male rats (SLC Inc., Hamamatsu, Japan) were housed in individual cages at 23 F 1 8C with a 12-h light/dark cycle (lights on 0700). In order to accustom them to our experimental conditions, they were fed with a control diet (20% casein) for 3 days. On the 4th day, both mice and rats were divided into 6 groups and designated as: control group (N), high cholesterol diet group (C), high cholesterol and cholate diet group (CB), and their 1% taurine-supplemented groups (NT, CT, CBT, respectively), based on their serum cholesterol levels and body weights. The diet composition in each group is shown in Table 1. Two-24h fecal samples were collected from each animal towards the end of the feeding period, lyophilized, and stored at 208C until assay. After the animals were allowed free access to water and their respective diets for one week, they were anesthetized with diethyl ester and decapitated at 23:00, the blood then collected, and the liver removed for determination of lipid and mRNA levels of CYP7A1, CYP8B1 and CYP27.
Table 1 The composition of diet in each group (g/kg) Group a
Casein Cellulosea Corn oila Choline cloride Vitamin mix AIN-93G a Mineral mix AIN-93G a Sucrosea Corn starcha Cholesterol Sodium cholate Taurine a
N
NT
C
CT
CB
CBT
200 50 50 1.5 10 35 217.8 435.7 – –
200 50 50 1.5 10 35 214.5 429 – – 10
200 50 50 1.5 10 35 214.5 429 10 – –
200 50 50 1.5 10 35 211.1 422.4 10 – 10
200 50 50 1.5 10 35 213.7 427.3 10 2.5 –
200 50 50 1.5 10 35 210.3 420.7 10 2.5 10
Purchased from Oriental Yeast Co. (Japan).
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The study was subject to approval by the Animal Use Committee of University of Shizuoka, and the animals were maintained in accordance with the guidelines for the care and use of laboratory animals, University of Shizuoka. Determination of serum and liver lipids and fecal bile acids Serum total cholesterol and HDL-cholesterol were determined by commercial kits (Cholesterol Ctest kit and HDL-cholesterol-test kit, respectively, Wako Pure Chemical Co., Japan). The concentration of (VLDL + LDL) was calculated as the difference between total cholesterol and the HDL-cholesterol levels. TLiver lipids were extracted by the method of Folch et al. (Folch et al., 1957). The extract was evaporated to dryness under N2 and then resuspended in isopropanol. Cholesterol levels were also measured using the commercial kit (Cholesterol C-test kit, Wako Pure Chemical Co., Japan). TFaeces were collected, lyophilized, ground, and steroids were extracted by ethanol and petroleum ether according to the method of Sheltawy and Losowsky (Sheltawy and Losowsky, 1975). The extract was dried under N2 and then resuspended in methanol. Total bile acids were measured by an enzymatic method which recognizes only the 3a-hydroxyl moiety of the steroid structure. Quantitation of mRNA for CYP7A1, CYP8B1 and CYP27 The CYP7A1 cDNA probe was a generous gift from Dr. Hiroaki Oda (Nagoya University, Japan). For CYP8B1 and CYP27, cDNA probes were prepared as follows: first-strand cDNA was prepared from mouse liver total RNA using QIAGEN OneStep RT-PCR Kit (QIAGEN K.K., Japan), and the cDNA was used as a template in the following PCR reactions with the following primer pairs, 5VTGAATTCTTGAAGGGGATGC-3V (forward) and 5V-CCTTGCTCCCTCAGAAACTG-3V (reverse) for CYP8B1, and 5V-TTCTCAGACACGATCTATGGCTGT-3V (forward) and 5V-CTACTGTCTCTGCAGAAAGCGTA-3V (reverse) for CYP27. The resulting PCR products were subcloned into pGEM-T easy vector (Promega Co.) and then purified on 1% agarose gels. Sequencing of subcloned products was performed to confirm that we had isolated the correct cDNA. All cDNA probes were radiolabeled with 32P-dCTP using Takara Random Primer DNA labelling Kit Ver.2 (Takara Bio INC., Japan). Total RNA was prepared from mouse or rat liver using TRIZOL Reagent (Invitrogen/Life Technologies, Japan), and then mRNA levels of the enzymes were determined by Northern blot. Finally, bands were quantified by Storm 820 Molecular Dynamics (Amersham Pharmacia Biotech.), and the results were corrected with regard to the signal generated from GAPDH mRNA obtained from the same membrane. Statistics The means and SE of 4 mice or rats per group are reported. Statistical analyses were performed by one-way ANOVA followed by the Duncan’s multiple range test. P-values of less than 0.05 were considered to be significant.
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Results Serum and liver cholesterol and fecal bile acid levels in mice Data are shown in Table 2. After having free access to the diets and water for one week, the animals in the six groups showed no significant differences in body weight, food intake, and liver weight. Both serum total cholesterol and VLDL + LDL cholesterol concentrations in the C- and CB-groups were significantly higher than those of the N-group by cholesterol loading in the diets, and both of them were notably decreased by taurine supplementation. HDL cholesterol levels did not change by cholesterol loading in the C-group but decreased remarkably in the CB-group, and it was significantly improved by taurine supplementation in the CBT-group. These data suggest that the decrease in serum cholesterol level by taurine supplementation is mainly due to the decrease in VLDL and LDL cholesterol. The amount of liver total cholesterol was notably enhanced by cholesterol loading in the C-group and further increased by adding cholate to the cholesterol diet in the CB-group. The cholesterol levels of both CT- and CBT-groups were significantly reduced by taurine supplementation. With the exception that the amount of dried feces in the CBT-group was significantly increased compared with that of the N-group, there were no differences among the other five groups. The fecal bile acid levels were markedly enhanced in the C-group compared with that of the N-group, which increased further by cholate loading in the CB-group. The fecal bile acid excretion was further raised by taurine supplementation in both the CT- and CBT-groups compared with the C- and CB-groups, respectively. These data suggest that cholesterol degradation is enhanced by cholesterol loading in the diet, and is further promoted by taurine supplementation. The amounts of serum and liver cholesterol and fecal bile acids in the rats Data are shown in Table 3. Table 2 The effect of taurine on cholesterol metabolism in mice fed high cholesterol diets with or without cholate for 7 days N Final body weight (g) Food intake (g/d) Liver weight (mg/g body weight) Serum lipids (mmol/L) Total cholesterol HDL-cholesterol VLDL + LDL-cholesterol Liver cholesterol (umol/g) Feces weight (g/2d) Fecal total bile acids (umol/g)
NT
CT
CB
CBT
27.00 F 0.63 27.69 F 1.15 28.06 F 0.86 4.11 F 0.03 4.18 F 0.09 4.30 F 0.09 50.42 F 3.27 52.94 F 4.91 52.66 F 0.86
26.65 F 1.08 4.08 F 0.19 55.35 F 2.92
26.00 F 1.29 4.09 F 0.11 54.51 F 4.54
27.42 F 0.67 4.25 F 0.04 58.33 F 2.18
3.45 F 0.14a 2.04 F 0.07b 1.41 F 0.10a 22.23 F 0.32a 0.72 F 0.03a 16.50 F 0.17a
3.55 F 0.31a 1.87 F 0.12b 1.68 F 0.21a 29.01 F 0.52b 0.86 F 0.04ab 31.71 F 1.43c
4.48 F 0.13b 1.39 F 0.09a 3.10 F 0.17c 53.45 F 2.54e 0.85 F 0.07ab 61.60 F 1.85d
3.50 F 0.12a 1.88 F 0.13b 1.62 F 0.14a 39.38 F 1.50d 0.93 F 0.04b 71.18 F 2.08e
3.27 F 0.2a 2.00 F 0.07b 1.27 F 0.15a 23.01 F 1.15a 0.75 F 0.04a 17.38 F 0.45a
C
4.41 F 0.23b 2.14 F 0.18b 2.27 F 0.19b 34.14 F 1.12c 0.86 F 0.04ab 27.32 F 0.67b
Values are mean F SE, n = 4. Values with diferent superscripts within the same line are significantly different at p b 0.05.
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Table 3 The effect of taurine on cholesterol metabolism in rats fed high cholesterol diets with or without cholate for 7 days N Final body weight (g) Food intake (g/d) Liver weight (mg/g body weight) Serum lipids (mmol/L) Total cholesterol HDL-cholesterol VLDL + LDL-cholesterol Liver cholesterol (umol/g) Feces wieght (g/2d) Fecal total bile acids (umol/g)
NT
C
CT
168.7 F 4.0 175.5 F 6.1 173.0 F 6.4 174.4 F 3.8 15.36 F 0.56 16.31 F 0.39 15.93 F 0.84 16.05 F 0.53 3.94 F 0.10a 4.06 F 0.13a 4.27 F 0.12b 4.37 F 0.06b
1.99 F 0.14a 1.96 F 0.11a 1.32 F 0.11b 1.34 F 0.08b 0.67 F 0.10a 0.62 F 0.15a 20.76 F 0.73a 20.91 F 1.47a 2.28 F 0.05a 2.51 F 0.12ab 23.77 F 0.95a 25.65 F 0.35a
2.29 F 0.13a 1.12 F 0.10ab 1.17 F 0.08b 32.09 F 0.73b 2.82 F 0.18b 60.08 F 1.96b
CB 174.1 F 5.0 16.02 F 066 4.66 F 0.09c
CBT 168.1 F 2.1 15.05 F 0.24 4.43 F 0.06bc
2.22 F 0.14a 3.37 F 0.21b 1.94 F 0.12a 1.21 F 0.04b 1.04 F 0.04a 1.15 F 0.10ab ab c 1.00 F 0.14 2.33 F 0.21 0.79 F 0.03a b d 30.39 F 3.43 57.69 F 1.22 41.39 F 1.82c 2.68 F 0.10b 2.98 F 0.16 b 2.87 F 0.08b 64.14 F 1.75b 103.79 F 0.68c 126.02 F 2.32d
Values are mean F SE, n = 4. Values with diferent superscripts within the same line are significantly different at p b 0.05.
After having free access to the diets and water for one week, the animals among the six groups showed no differences in body weight and food intake. Liver weight was increased by a cholesterol loading diet and further raised by adding cholate to the cholesterol diet. Unlike in mice, serum total cholesterol concentrations in rats were significantly higher in the CBgroup but not in the C-group, and it was normalized by taurine supplementation in the CBT-group. HDL cholesterol was notably lower in the CB-group compared with that of the N-group, and no change was observed by taurine supplementation. VLDL + LDL cholesterol level was greatly increased by a cholesterol loading diet, which was further increased by adding cholate to the cholesterol diet and significantly decreased by taurine supplementation in the CBT-group. As in the case with mice, the decrease of serum cholesterol by taurine supplementation in the rat is due mainly to the decrease in VLDL and LDL. The amount of liver total cholesterol was notably raised by cholesterol loading in the C-group, and further increased by adding cholate to the cholesterol diet in the CB-group. In contrast, it was significantly reduced by taurine supplementation in the CBT-group compared with that of the CB-group, but there was no difference between the C- and CT-groups. The dried feces weights in the C- and CB-groups were significantly higher than those of the N-group. The fecal bile acid levels dramatically increased in the C-group compared with that of the N-group and further increased by cholate loading in the CB-group. In contrast to mice, the increase in fecal bile acid excretion was observed in the CBT-group but not the CT-group compared with the CB- and C-groups, respectively. This is in agreement with the results of no difference in the levels of serum and liver cholesterol between the C- and CT-groups. mRNA levels of the three enzymes in mice and rats The mRNA levels of CYP7A1 (Fig. 1) were increased in the C-group compared with those of the Ngroup, but decreased in the CB-group compared with those of the C-group in both mice and rats. This is in agreement with a regulatory mechanism in which CYP7A1 can be induced by cholesterol and repressed by bile acids (Yokogoshi and Oda, 2002; Russel and Setchell, 1992). CYP7A1 mRNA levels
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Fig. 1. Effect of diets on CYP7A1 mRNA level in mice (A) and rats (B). After animals were fed with the respective diets for 7 days, their livers were obtained at 23:00 in the dark cycle for later extraction of total RNA. The relative amounts of CYP7A1 mRNA were quantified relative to GAPDH using Northern blot. N-control diet group, NT-taurine supplemented control diet group, C-cholesterol diet group, CT-taurine supplemented cholesterol diet group, CB-cholesterol and cholate diet group, CBTtaurine supplemented cholesterol and cholate diet group. Bars with different superscript are significantly different at p b 0.05. (A). CYP7A1 mRNA was significantly repressed in the CB-group and notably increased in the CBT-group by taurine supplementation. (B). CYP7A1 mRNA was markedly increased in the C-group and repressed in the CB-group by adding cholate to the cholesterol diet. It was not enhanced by taurine in the CT-group but dramatically improved in the CBT-group compared with that of the C-group and CB-group respectively.
were notably increased by taurine supplementation in the CBT-group compared with those of the CBgroup, but there was no difference between the C- and CT-group in the two animal models. These results suggest that taurine induces CYP7A1 only in the presence of cholate in the cholesterol diet. In mice the CYP7A1 mRNA levels were repressed in the CB-group not only to be less than those of the C-group but also the N-group, whereas in rats they were less than those of the C-group but higher than those of the N-
Fig. 2. Effect of diets on CYP8B1 mRNA level in mice (A) and rats (B). After animals were fed with the respective diets for 7 days, their livers were obtained at 23:00 in the dark cycle for later extraction of total RNA. The relative amount of CYP8B1 mRNA was quantified relative to GAPDH using Northern blot. N-control diet group, NT-taurine supplemented control diet group, C-cholesterol diet group, CT-taurine supplemented cholesterol diet group, CB-cholesterol and cholate diet group, CBTtaurine supplemented cholesterol and cholate diet group. Bars with different superscript are significantly different at p b 0.05. (A). CYP8B1 mRNA was significantly repressed in the CB-group and notably increased in the CBT-group by taurine supplementation. (B). There are no notable differences among the six groups.
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Fig. 3. Effect of diets on CYP27 mRNA level in mice (A) and rats (B). After animals were fed with the respective diets for 7 days, their livers were obtained at 23:00 in the dark cycle for later extraction of total RNA. The relative amount of CYP27 mRNA was quantified relative to GAPDH using Northern blot. N-control diet group, NT-taurine supplemented control diet group, C-cholesterol diet group, CT-taurine supplemented cholesterol diet group, CB-cholesterol and cholate diet group, CBTtaurine supplemented cholesterol and cholate diet group. Bars with different superscript are significantly different at p b 0.05. (A). There are no changes among the six groups. (B). CYP27 mRNA levels tend to increase in the C- and CB-group compared to that of the N-group, but are not significantly increased by taurine.
group. These data suggest that the susceptibility of CYP7A1 to cholesterol and bile acid is very different between the two species. No changes were observed in CYP8B1 mRNA levels (Fig. 2) by cholesterol loading, but they were significantly repressed by adding cholate to the cholesterol diet, and were increased by taurine supplementation in the CBT-group compared with the CB-group in mice. However, no differences were observed in the six groups of rats. As was shown with CYP7A1, the susceptibility of CYP8B1 to bile acid is very different between mice and rats. CYP27 mRNA (Fig. 3) was improved in the C- and CB-groups of rats but not of mice, and it was not significantly up-regulated by taurine supplementation although it seemed much higher in the CBT-group than in the CB-group of rats.
Discussion CYP7A1 is the rate-limiting enzyme in the classic pathway, which is the main pathway of bile acid biosynthesis. It has been widely reported that its gene transcription is regulated by several factors or nuclear receptors to balance the elimination of cholesterol as a response to optimize the physiological status of the body (Janowski et al., 1996; Wang et al., 1999). Although CYP7A1 is probably responsible for regulating the overall rates of bile acids biosynthesis, CYP8B1 also plays an important role in determining the ratio of CA to CDCA to affect the bile composition and its relative hydrophobicity. In addition, the alternative pathway was reported to contribute less than 10% to total bile acid biosynthesis (Vlahcevic et al., 1980; Swell et al., 1980). However, recent studies have demonstrated that it could become a more important contributor under certain pathological conditions, in vitro studies suggesting that it accounted for 50% of bile acid synthesis (Axelson et al., 1989; Stravitz et al., 1996). Therefore, there is little doubt that CYP27 is important in the conversion of cholesterol to bile acids. In the present study, we discussed the susceptibility of the three enzymes described above to cholesterol and bile acids in mice and rats.
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It has been known that the rat is a poor model for developing experimental atherosclerosis since in this species a high cholesterol diet induces CYP7A1 which facilitates disposal of excess cholesterol (Jones et al., 1993; Doerner et al., 1995). In our study, we also observed that a diet high in cholesterol in the C-group induced CYP7A1 mRNA in rats, which agrees with previous reports. Subsequently fecal bile acid excretion was significantly increased and serum cholesterol concentration was maintained at normal levels. It was reported that CYP7A1 activity was not affected by adding cholate to a high cholesterol diet (Vlahcevic et al., 2000). However, we found that CYP7A1 mRNA levels were significantly repressed by diets high in combination of cholesterol and cholate in the CB-group compared with those of the C-group, but still notably higher than those of the N-group. These results suggest that the overall effect of the combination of cholesterol and cholate was to induce CYP7A1 mRNA, and to show that rat is more susceptible to cholesterol than to cholate. It was reported that the activity of CYP27 was increased by a cholesterol diet, while CYP8B1 activity was significantly reduced by adding 1% cholate to a high cholesterol diet in rat (Nguyen et al., 1999; Vlahcevic et al., 2000). However, only the increasing trend in CYP27 mRNA was observed by both the cholesterol diets with or without cholate in the present study; this may be due to our use of a smaller amount of cholate in the diet than reported in other studies. In mice, CYP7A1 mRNA levels tended to increase and CYP8B1 was unchanged by the diet high in cholesterol, but they were markedly decreased by adding cholate to the cholesterol diet compared with those of the C- and N-groups. These data suggest that CYP7A1 and CYP8B1 are more susceptible to cholate than to cholesterol. Serum and liver cholesterol levels were significantly increased in the Cgroup and further enhanced by the combination of cholesterol and cholate in the CB-group. All groups showed that cholesterol accumulated in the liver as CYP7A1 and CYP8B1 were repressed, although the serum cholesterol concentration in the CB-group was maintained at the same level as that of the C-group. No significant change in CYP27 mRNA levels was produced by either cholesterol or cholate. The above data indicate that mice and rats show different responses to cholesterol and cholate. Cholesterol degradation in mice is more susceptible to repression by dietary cholate, whereas in the rat it is less sensitive to cholate than to cholesterol since it is increased even by a diet high in both cholesterol and cholate. These data suggest that there is an apparent difference in the regulation of cholesterol degradation or bile acid biosynthesis between mice and rats. In order to interpret further the cholesterol-lowering effect of taurine, we investigated its effects on mRNA levels of the aforementioned three enzymes in the two animal models. Although it has been well accepted that taurine could induce CYP7A1 to improve bile acid biosynthesis (Tsuji et al., 1979; Sugiyama et al., 1989; Venkatesan et al., 1993; Gandhi et al., 1992), we found that under our experimented conditions taurine only enhanced the mRNA levels of CYP7A1 in both mice and rats fed the CB-diet but not the C-diet. In mice, cholesterol concentrations in serum and liver were reduced, and fecal bile acid excretion was increased by taurine supplementation in the CT-group compared with that of the C-group, in spite of unchanged mRNA levels in any of the enzymes. These results suggest that taurine may enhance protein levels of the enzymes to express its cholesterol-lowering effect. Liver cholesterol concentration was reduced 26% by taurine supplementation in the CBT-group compared with that of CB-group; however only a 14% decrease was observed in the CT-group compared with that of the C-group. This may be attributed to the marked increase in mRNA levels of CYP7A1 and CYP8B1 by taurine supplementation in the CBT-group.
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In rats, taurine did not show an efficient action to reduce liver cholesterol, although liver cholesterol and fecal bile acid excretion tended to decrease and increase, respectively, in the CT-group compared with those of the C-group. But liver cholesterol levels were lowered 28% in the CBT-group compared with those of the CB-group as CYP7A1 was dramatically induced by taurine supplementation. Since CYP7A1 is subject to feedback inhibition by bile acids returning to the liver via the enterohepatic circulation (Russel and Setchell, 1992), the increased excretion of bile acids in the feces results in a decrease in bile acids in the enterohepatic circulation, subsequently activating CYP7A1, and finally accelerating cholesterol degradation. This is well confirmed in the CBT-group of the two animal models in our experiment that the sequence of reducing cholesterol concentration is accompanied by increase of fecal bile acid excretion and up-regulation of CYP7A1 mRNA. But no improvement in CYP7A1 mRNA level was observed in the CT-group of mice although serum and liver cholesterols were significantly decreased and fecal bile acid excretion was notably increased. These results suggest that cholate is required for taurine to induce CYP7A1 mRNA, or alternatively, exogenous cholate may not impair taurine from inducing CYP7A1 mRNA. In contrast, taurine induces CYP7A1 gene transcription only in the presence of cholate in the cholesterol diet, and then it shows its cholesterol-lowering effect. In other words, taurine induces CYP7A1 mRNA when high dietary cholesterol is assimilated because cholate is well known for its activity on cholesterol uptake. Biosynthesis, fecal excretion, and reabsorption from ileum are three important aspects of bile acid homeostasis in the body. Biosynthesis and fecal excretion of bile acids are increased by taurine, whereas reabsorption from the ileum is decreased. Our data may be interpreted as taurine inhibiting the intestinal transport of bile acids in the ileum, which would result in the up-regulation of CYP7A1 in the liver and lowering of liver and serum cholesterol. Ileum-bile acid binding protein (I-BABP) and ileum-bile acid transporter (I-BAT) are reported to be responsible for bile acid reabsorption in the ileum (Grober et al., 1999; Zhang et al., 2002). The nature of these responses is unclear, and further experiments are required to reveal the mechanism of the cholesterol-lowering effect of taurine. The overall results of our experiments suggest two points. 1) There are differences between mice and rats on the susceptibility of CYP7A1 and CYP8B1 to dietary cholesterol and cholate. It seems that they are more sensitive to cholate in mice, but more susceptible to cholesterol in rats. 2) Taurine induces CYP7A1 mRNA only in the presence of cholate in the cholesterol diet, which subsequently exerts its efficient cholesterol lowering effect. Acknowledgment This work was supported by a grant for scientific research from Shizuoka Prefecture, and 21st century COE program from Ministry of Education, Culture, Sports, Science and Technology of Japan. References Axelson, M., Mork, B., Aly, A., Wisen, O., Sjovall, J., 1989. Concentrations of cholestenoic acids in plasma from patients with liver disease. Journal of Lipid Research 30, 1877 – 1882. Bjorkhem, I., 1992. Mechanism of degradation of the steroid side chain in the formation of bile acids. Journal of Lipid Research 33, 455 – 471.
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Chiang, J.Y.L., 1998. Regulation of bile acid synthesis. Frontiers Bioscience 3, 176 – 193. Danielsson, H., 1963. Present states of research on catabolism and excretion of cholesterol. Advanced Lipid Research 1, 335 – 385. Doerner, K.C., Gurley, E.C., Vlahcevic, Z.R., Hylemon, P.B., 1995. Regulation of cholesterol 7a-hydroxylase expression by sterols in primary hepatocyte cultures. Journal of Lipid Research 36, 168 – 177. Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497 – 509. Gandhi, V.M., Cherian, K.M., Mulky, M.J., 1992. Hypolipidemic action of taurine in rats. Indian Journal of Experimental Biology 30 (15), 413 – 417. Grober, J., Zaghini, I., Fujii, H., Jones, S.A., Kliewer, S.A., Willson, T.M., Ono, T., Besnard, P., 1999. Identification of a bile acid-responsive element in the human ileal bile acid-binding protein gene. Involvement of the farnesoid receptor/9-cisretinoic acid receptor heterodimer. Journal of Biological Chemistry 274, 29749 – 29754. Huxtable, R.J., 1992. Physiological actions of taurine. Physiological Review 72, 101 – 163. Janowski, B.A., Willy, P.J., Devi, T.R., Falck, J.R., Mangelsdorf, D.J., 1996. An oxysterol signalling pathway mediated by nuclear receptor LXR alpha. Nature 383, 728 – 731. Jones, M.P., Pandak, W.M., Hylemon, P.B., Chiang, J.Y., Heuman, D.W., Vlahcevic, Z.R., 1993. Cholesterol 7a-hydroxylase: evidence for transcriptional regulation by cholesterol and/or metabolic products of cholesterol in the rat. Journal of Lipid Research 34, 885 – 892. Kuriyama, K., 1980. Taurine as a neuromodulator. Federation Proceeding 39, 2680 – 2684. Mochizuki, H., Oda, H., Yokogoshi, H., 2001. Dietary taurine potentiates polychlorinated biphenyl-induced hypercholesterolemia in rats. Journal of Nutritional Biochemistry 12 (2), 109 – 115. Nguyen, L.B., Xu, G., Shefer, S., Stephen Tint, G., Batta, A., Salen, G., 1999. Comparative regulation of hepatic sterol 27hydroxylase and cholesterol 7a-hydroxylase activities in the rat, guinea pig, and rabbit: Effects of cholesterol and bile acid. Metabolism 48 (12), 1542 – 1548. Pasantes, M.H., Wright, C.E., Gaull, G.E., 1985. Taurine protection of lymphoblastoid cells from iron-ascorbate-induced damage. Biochemical Pharmacology 34, 2205 – 2207. Russel, D.W., Setchell, K.D.R., 1992. Bile acid biosynthesis. Biochemistry 31, 4737 – 4748. Sheltawy, M.J., Losowsky, M.S., 1975. Determination of fecal bile acid by an enzymatic method. Clinica Chimica Acta 64, 127 – 132. Stravitz, R.T., Vlahcevic, Z.R., Russell, T.L., Heizer, M.L., Avadhani, N.G., Hylemon, P.B., 1996. Regulation of sterol 27hydroxylase and an alternative pathway of bile acid biosynthesis in primary cultures of rat hepatocytes. Journal of Steroid Biochemistry and Molecular Biology 57, 337 – 347. Sugiyama, K., Ohishi, A., Ohnuma, Y., Muramatsu, K., 1989. Comparison between the plasma cholesterol-lowering effects of glycine and taurine in fats fed on high cholesterol diets. Agric. Biological. Chemistry 53, 1647 – 1652. Swell, L., Gustafsson, J., Schwartz, C.C., Halloran, G., Danielsson, H., Vlahcevic, Z.R., 1980. An in vivo evaluation of the quantitative significance of several potential pathways to cholic and chenodeoxycholic acid from cholesterol in man. Journal of Lipid Research 21, 455 – 466. Thurston, J.H., Hauhart, R.E., Dirgo, J.A., 1980. Taurine: A role in osmotic regulation of mammalian brain and possible clinical significances. Life Sciences 26, 1561 – 1568. Tsuji, K., Seki, T., Iwao, H., 1979. Cholesterol-lowering effects of taurine and sulfur-containing amino acids in serum and liver of rats. Sulfur-Containing Amino Acids 2, 143 – 145. Venkatesan, N., Rao, P.V., Arumugam, V., 1993. Inhibitory effect of taurine on puromycin aminonucleoside-induced hyperlipidemia in rats. Journal of Clinical Biochemistry and Nutrition 15, 203 – 210. Vlahcevic, Z.R., Eggertsen, G., Bjorkhem, I., Hylemon, P.B., Redford, K., Pandak, W.M., 2000. Regulation of sterol 12ahydroxylase and cholic acid biosynthesis in the rat. Gastroenterology 118, 599 – 607. Vlahcevic, Z.R., Pandak, W.M., Stravitz, R.T., 1999. Regulation of bile acid biosynthesis. Gastroenterological Clinics of North America 28, 1 – 25. Vlahcevic, Z.R., Schwartz, C.C., Gustafsson, J., Halloran, L.G., Danielsson, H., Swell, L., 1980. Biosynthesis of bile acids in man. Multiple pathways to cholic and chenodeoxycholic acid. Journal of Biological Chemistry 255, 2925 – 2933. Wang, H., Chen, J., Hollister, K., Sowers, L.C., Forman, B.M., 1999. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Molecular Cell 3, 543 – 553. Wright, C.E., Tallan, H.H., Lin, Y.Y., 1986. Taurine: biological update. Annual Review of Biochemistry 55, 427 – 453.
W. Chen et al. / Life Sciences 77 (2005) 746–757
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Yokogoshi, H., Mochizuki, H., Nanami, K., Hida, Y., Miyachi, F., Oda, H., 1999. Dietary taurine enhances cholesterol degradation and reduces serum and liver cholesterol concentrations in rats fed a high-cholesterol diet. Journal of Nutrition 129 (9), 1705 – 1712. Yokogoshi, H., Oda, H., 2002. Dietary taurine enhances cholesterol degradation and reduces serum and liver cholesterol concentrations in rats fed a high-cholesterol diet. Amino Acids 23 (4), 433 – 439. Zhang, E.Y., Phelps, M.A., Cheng, C., Ekins, S., Swaan, P.W., 2002. Modeling of active transport systems. Advanced Drug Delivery Reviews 54, 329 – 354.