help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2003-0993
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Drover, V. A. B.
Right arrow Articles by Agellon, L. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Drover, V. A. B.
Right arrow Articles by Agellon, L. B.
Endocrinology Vol. 145, No. 2 574-581
Copyright © 2004 by The Endocrine Society

Regulation of the Human Cholesterol 7{alpha}-Hydroxylase Gene (CYP7A1) by Thyroid Hormone in Transgenic Mice

Victor A. B. Drover and Luis B. Agellon

Canadian Institutes of Health Research Group in Molecular and Cell Biology of Lipids and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2S2

Address all correspondence and requests for reprints to: L. B. Agellon, Department of Biochemistry, 327 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2S2. E-mail: luis.agellon{at}ualberta.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid hormones exert significant changes in the metabolism of bile acids. However, in humans, the effect of thyroid hormone on cholesterol 7{alpha}-hydroxylase (cyp7a), the rate- controlling enzyme in the classical bile acid biosynthetic pathway, remains poorly understood and has been difficult to study directly in vivo. Previous studies from our laboratory have shown that the activity of the human cholesterol 7{alpha}-hydroxylase gene promoter is repressed by T3 in cultured cells. Accordingly, we hypothesized that T3 would negatively regulate human CYP7A1 gene expression in vivo. We tested this hypothesis by inducing hypo- and hyperthyroidism in transgenic mice expressing the human CYP7A1 gene. Hypothyroidism did not affect the abundance of human cyp7a mRNA in transgenic mice. In hyperthyroid male mice, human cyp7a mRNA abundance was decreased. No significant change in cyp7a mRNA abundance was observed in hyperthyroid female mice. Gender differences in the amount of cholesterol and bile acids in gallbladder bile were also observed. The data indicate that thyroid hormone can repress the human CYP7A1 gene in transgenic mice, but this effect is dependent on gender in this in vivo model.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE LIVER-SPECIFIC ENZYME known as cyp7a (cholesterol 7{alpha}-hydroxylase) catalyzes the first step in the classical bile acid biosynthetic pathway (1, 2). Cholesterol catabolism via this pathway produces 7{alpha}-hydroxy-4- cholesten-3-one intermediates from which cholic acid (CA) is synthesized (1). In mammals, the synthesis of bile acids via the classical pathway accounts for the majority of the bile acids produced by the liver and represents the primary method of terminal cholesterol catabolism.

T3 is an important regulator of bile acid metabolism. In rodents, T3 stimulates the expression of the gene encoding cyp7a, as reflected by increases in cyp7a mRNA abundance and activity (3, 4, 5, 6, 7). In humans, the effect of T3 on cyp7a function is less understood because of the difficult nature of obtaining samples for assessing cyp7a enzyme activity and mRNA abundance. Rather, studies have largely relied on indirect parameters, such as kinetic analysis or plasma concentration of bile acid intermediates, to estimate the efficiency of bile acid biosynthesis in the human liver. CA synthesis, CA pool size, and the output of bile acids into the duodenum are reduced during hyperthyroidism and increased by treatments that normalize plasma T3 levels (8, 9). In contrast, other studies indicate that T3 status does not affect CA synthesis (10, 11). Sauter et al. (12) reported that plasma concentrations of 7{alpha}-hydroxy-4-cholesten-3-one, which presumably reflect the hepatic cyp7a activity, were comparable in hypothyroid and hyperthyroid patients.

The addition of T3 to the medium of primary cultures of human hepatocytes tends to decrease the synthesis of bile acids (13). The activity of the human CYP7A1 gene promoter is inhibited when cotransfected into HepG2 hepatoblastoma cells with a plasmid encoding the thyroid hormone receptor-{alpha} (14). Recently, we demonstrated that repression of the human CYP7A1 gene promoter activity in response to T3 treatment requires the direct interaction of rodent thyroid hormone receptor with an element in the CYP7A1 gene promoter (15). To determine the effects of T3 on human CYP7A1 gene expression in vivo, we induced hypothyroid and hyperthyroid states in a transgenic mouse strain that expresses the human CYP7A1 gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, treatments, and sample collection
All procedures were performed in accordance with the guidelines of the University of Alberta Health Sciences Animal Policy and Welfare Committee. Wild-type and transgenic mice expressing the human CYP7A1 gene but not the endogenous mouse Cyp7a1 gene (16) were maintained on a normal rodent chow diet ad libitum. The transgenic mice carry a human genomic fragment spanning approximately 125 kb and containing the CYP7A1 gene flanked by more than 17 and 6 kb of sequence in the 5' and 3' regions, respectively. Age-matched mice (8 and 12 wk) were switched to a reverse light cycle and acclimated for 1 wk before the experiment. Alteration of thyroid status was accomplished according to the following procedure. Mice were separated into three treatment groups (n = 7–10 per group) and maintained on a low-iodine diet (ICN Biomedicals, Inc., Aurora, OH) for the remainder of the experiment. On d 7, two treatment groups received oral administration of 0.05% 2-mercapto-1-methylimidazole (methimazole) and 1% potassium perchlorate (Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada). On d 21, 0.1 ml of carrier (0.9% sodium chloride and 0.005 N sodium hydroxide) or T3 (0.05 mg/ml in carrier; Sigma-Aldrich) was administered by ip injection at the end of the light cycle. Single daily injections continued for 5 d. On d 25, food was removed midway through the light cycle. Animals were killed the following morning, 3 h after the beginning of the dark cycle, and plasma, bile, and tissue samples were collected. Plasma samples were stored at 4 C whereas bile and liver samples were flash-frozen in liquid nitrogen and stored at -70 C.

Measurement of cholesterol and free T3
Biliary and plasma cholesterol were measured with the Infinity cholesterol reagent (Sigma-Aldrich) using the manufacturer’s protocol. Lipoprotein cholesterol profiles were obtained from pooled plasma samples (equal volumes from each mouse) by first separating plasma lipoproteins by size exclusion chromatography on a HPLC apparatus (Beckman Instruments, Inc., Mississauga, Ontario, Canada) fitted with a Sepharose 6 gel filtration column (Amersham Pharmacia Biotech, Inc., Baie d’Urfé, Quebec, Canada). The column effluent was mixed directly with the Infinity cholesterol reagent using a post-column T-connector and passed through a post-column reactor. Reaction products were monitored in real-time at 500 nm using a visible-light detector (Beckman Instruments, Inc.). The amount of cholesterol associated with each of the lipoprotein fractions was determined by calculating the area of the respective peaks in the HPLC profile using the System GOLD software (Beckman Instruments, Inc.). The concentration of T3 in the plasma was measured using the ACTIVE free T3 enzyme immunassay kit (Diagnostic Systems Laboratories, Webster, TX) following the manufacturer’s protocol.

Measurement of mRNA abundance
Total RNA was prepared from frozen liver as described previously (17). Complementary DNA was synthesized from 10 µg of total RNA with Superscript II reverse transcriptase (Invitrogen Canada, Inc., Burlington, Ontario, Canada) using the manufacturer’s protocol. PCRs were done in the presence of 1x SYBR Green I (Sigma-Aldrich) with REDTaq DNA polymerase (Sigma-Aldrich) and intron-spanning, gene-specific oligonucleotides [human CYP7A1 sense primer 5'-AGAAGGCAAACGGGTGAACC-3' and antisense primer 5'-GGGTCAATGCTTCTGTGCCC-3'; murine apolipoprotein A-I (apoA-I) sense primer 5'-GAAAGCTGTGGTGCTGGCCG-3' and antisense primer 5'-CCTTGTTCATCTCCTGTCTCACCC-3'; murine cyclophilin sense primer 5'-TCCAAAGACAGCAGAAAACTTTCG-3' and antisense primer 5'-TCTTCTTGCTGGTCTTGCCATTCC-3'; and murine sterol 12{alpha}-hydroxylase (cyp8b1) sense primer 5'-GTCACTCCATGGCTTTCCGG-3' and antisense primer 5'-CTTTAGGCCCTAGCATCACC-3']. The annealing temperature of each oligonucleotide pair was optimized using a T-Gradient thermal cycler (Biometra, Göttingen, Germany) to ensure the synthesis of only one DNA product. Cycle number was also optimized to determine the range of cycles in which amplification remained linear. Amplicon production was monitored by green fluorescence using a LightCycler (Roche Diagnostics Canada, Laval, Ontario, Canada). Melting curve analysis was used to distinguish between specific and nonspecific fluorescence, and amplicon mass was quantitated from the melting curve by calculating the area under the peak corresponding to the expected amplicon using the LightCycler software package (Roche). All reactions were confirmed by agarose gel electrophoresis. Cyclophilin mRNA abundance varied less than 10% among all samples and was used to normalize cyp7a and apoA-I mRNA levels.

Biochemical analyses
Microsomes were prepared from frozen liver samples, and cyp7a enzyme activity was measured as described previously (18). The mass of bile acids in gallbladder bile was measured with the bile acid reagent (Sigma-Aldrich) following the manufacturer’s protocol. Bile acid speciation was determined by HPLC as described previously (19).

Statistical analysis
Differences between treatment groups were evaluated using ANOVA, Bartlett’s test for equal variances, and Bonferroni’s multiple comparison test. Differences within and between genders were determined by one-way and two-way ANOVA, respectively. Differences were considered significant when P < 0.05. Coefficients of correlation (r) were determined using the Pearson product moment method.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of hypo- and hyperthyroidism in mice
The concentration of T3 in the plasma of male and female mice maintained on the low-iodine diet was similar (0.86 ± 0.17 pg/ml and 0.84 ± 0.21 pg/ml, respectively). Oral administration of methimazole/perchlorate, in addition to a low-iodine diet, reduces plasma T3 levels in mice (7). We used this treatment without or with T3 replacement to induce hypo- and hyperthyroid states, respectively, in transgenic mice expressing the human CYP7A1 gene (16) (Fig. 1AGo). Plasma T3 was almost undetectable in male hypothyroid mice (0.02 ± 0.10 pg/ml; Fig. 1BGo), whereas hyperthyroid males displayed a plasma T3 concentration of 1.65 ± 0.18 pg/ml, almost 2-fold higher than controls. In female hypothyroid mice, the plasma T3 concentration was reduced to 0.21 ± 0.10 pg/ml, whereas in hyperthyroid females it was 4.1-fold higher than in controls (3.52 ± 0.59 vs. 0.86 ± 0.59 pg/ml, respectively). Thus, the effects of methimazole/perchlorate treatment and T3 replacement were similar in both the male and female mice, but the plasma T3 concentrations in hyperthyroid females were 2-fold higher than treatment-matched males.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 1. Induction of hypo- and hyperthyroidism in mice. A, All mice were maintained on a low-iodine diet throughout the 26-d treatment period. Control mice were injected with saline only. The remaining mice received methimazole and perchlorate by oral administration. The hypothyroid mice received saline injections, whereas the hyperthyroid mice received injections of T3. B, Free T3 was measured in fresh plasma samples from all mice. The data shown are the mean ± SEM from two experiments. Bars, Control; open bars, hypothyroid; filled bars, hyperthyroid. ANOVA results: F51 = 19.37, P < 0.0001.

 
T3 reduces plasma cholesterol levels and repartitions cholesterol into the high-density lipoprotein (HDL) fraction in transgenic mice
Hypothyroidism in humans is typically associated with elevated total plasma cholesterol concentration (TPC), a condition that can be reversed by hormone replacement (reviewed in Ref.20). In rodents, plasma cholesterol is also sensitive to plasma T3 levels (21). Because the liver plays a critical role in modulating plasma cholesterol, we used this parameter to gauge the impact of altered T3 status on hepatic cholesterol metabolism in transgenic mice. As shown in Fig. 2AGo, both male and female hypothyroid mice had higher TPC compared with control mice. Administration of T3 reduced TPC significantly and to similar levels in both sexes. The distribution of lipoprotein cholesterol is also affected by T3 status. As shown in Fig. 2BGo, hypothyroidism resulted in a dramatic increase in plasma low-density lipoprotein (LDL) cholesterol concentration that was reversed by T3 treatment. Similarly, plasma HDL cholesterol concentration was increased in hypothyroid mice and reduced in hyperthyroid mice. Analysis of the distribution of cholesterol in LDL and HDL revealed that the percentage of cholesterol associated with HDL was reduced in hypothyroid mice whereas T3 replacement reversed this effect (in males, control was 83%, hypothyroid 54%, and hyperthyroid 71%; in females, control was 77%, hypothyroid 53%, and hyperthyroid 73%).



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 2. Analysis of cholesterol in the plasma of hypo- and hyperthyroid mice. A, Total plasma cholesterol concentration. The data shown are the mean ± SEM from two experiments. Hatched bars, Control; open bars, hypothyroid; filled bars, hyperthyroid. ANOVA results: F51 = 31.45, P < 0.0001. B, Lipoprotein-cholesterol profile.

 
Human cyp7a mRNA abundance is reduced by T3 in male but not in female mice
We showed previously that the activity of the human CYP7A1 gene promoter is repressed by T3 in cultured cells (15). To determine whether the same effect occurs in vivo, we examined the relative abundance of human cyp7a mRNA in transgenic mice after induction of hypo- and hyperthyroid states. As shown in Fig. 3AGo, human cyp7a mRNA levels in male mice were similar in the control and hypothyroid groups but was reduced by 37% in hyperthyroid mice. In contrast, cyp7a mRNA abundance did not differ significantly in any of the female treatment groups. The apoA-I gene is known to be stimulated by T3 (22, 23); thus, we also examined changes in apoA-I mRNA abundance to determine whether the differential expression of the human CYP7A1 gene in male and female transgenic mice was specific to this gene. As shown in Fig. 3BGo, hypothyroidism and hyperthyroidism decreased and increased, respectively, apoA-I mRNA levels in both male and female transgenic mice. A strong correlation between plasma T3 and apoA-I mRNA levels was also observed in both genders (Fig. 3CGo; r = 0.39; R2 = 0.15; P = 0.0024; n = 50). Thus, gender has a specific influence on the expression of the human CYP7A1 gene in transgenic mice.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 3. The effect of T3 on the abundance of human CYP7A1 mRNA (A) and murine apoA-I mRNA (B) in the liver. The data shown are the mean ± SEM from two experiments and separately normalized to the values seen in the male and female control groups. Hatched bars, Control; open bars, hypothyroid; filled bars, hyperthyroid. ANOVA results for A: F51 = 2.527, P = 0.0397; for B: F49 = 5.747, P = 0.0004. The Pearson correlation between apoA-I mRNA abundance and plasma T3 concentration is shown in C.

 
T3 increases cyp7a enzyme activity in male but not female transgenic mice
We next determined whether cyp7a enzyme activity covaried with mRNA abundance in response to T3 status in transgenic mice. Control and hypothyroid transgenic mice had comparable activities regardless of gender (Fig. 4AGo). However, cyp7a enzyme activity was increased 2-fold in hyperthyroid male mice, whereas no significant change was observed in hyperthyroid female mice. Thus, changes in cyp7a mRNA abundance and enzyme activity in hyperthyroid male transgenic mice are not correlated. In addition, cyp7a activity in male, but not female, mice was tightly correlated to plasma T3 concentration (Fig. 4BGo; r = 0.68; R2 = 0.46; P < 0.0001; n = 25) and TPC (Fig. 4CGo; r = -0.78; R2 = 0.60; P < 0.0001; n = 26) levels.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 4. A, Cyp7a enzyme activity was measured in hepatic microsomes from transgenic mice. The data shown are the mean ± SEM and were separately normalized to the values seen in the male and female control groups. For male control mice, 100% cyp7a activity = 0.96 ± 0.21 pmol/min·mg protein; for female control mice, 100% cyp7a activity = 1.13 ± 0.22 pmol/min·mg protein. Hatched bars, Control; open bars, hypothyroid; filled bars, hyperthyroid. ANOVA results: F51 = 5.781, P = 0.0003. Pearson correlations between cyp7a enzyme activity (B) and plasma T3/cholesterol concentration (C) are shown.

 
T3-dependent changes in bile composition are gender specific
We measured the amount of bile acids in gallbladder bile to estimate the bile acid pool size in an effort to explain the discrepancy between changes in human cyp7a mRNA abundance and enzyme activity in male hyperthyroid transgenic mice. The total mass of bile acids in gallbladder bile was significantly reduced in male hyperthyroid mice compared with male hypothyroid mice (Fig. 5AGo). The concentration of bile acids was also reduced in male hyperthyroid mice (control, 53.46 ± 9.48 mM, vs. hyperthyroid, 32.85 ± 5.21 mM; P = 0.037) and a significant inverse correlation with cyp7a activity was observed (Fig. 5CGo; r = -0.52; R2 = 0.27; P = 0.0066; n = 22). No significant change in total bile acid mass or concentration in the gallbladder was observed in any of the female treatment groups, consistent with the inability of T3 to alter cyp7a activity (r = -0.38; R2 = 0.14; P = 0.0423; n = 22).



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 5. The effect of T3 on gallbladder lipids. A, Total bile acids; B, total cholesterol. The data shown are the mean ± SEM from two experiments. Hatched bars, Control; open bars, hypothyroid; filled bars, hyperthyroid. ANOVA results for A: F43 = 2.539, P = 0.0445; for B: F41 = 4.339, P = 0.0034. Pearson correlations between cyp7a enzyme activity and biliary bile acid (C) or cholesterol concentration (D) are shown.

 
Additional gender-specific effects were observed in the bile of transgenic mice. The total mass of cholesterol in gallbladder bile was reduced in hyperthyroid males but not in females (Fig. 5BGo). Moreover, the amount of cholesterol in the bile of female transgenic mice tended to be greater than that in male transgenic mice. This was also reflected by the concentration of cholesterol in gallbladder bile (control male, 1.22 ± 0.21 mM, vs. control female, 2.83 ± 0.53 mM; P = 0.009). Regardless of gender, biliary cholesterol concentration and cyp7a activity were inversely correlated (Fig. 5DGo; r = -0.37; R2 = 0.14; P < 0.0083; n = 42).

Gender differences in bile acid speciation are independent of cyp7a activity
To determine whether the T3-mediated gender differences in the response of the human CYP7A1 gene, cyp7a activity and bile composition were related to the transgene or other factors inherent in mice, we first examined the speciation of bile acids in gallbladder bile of wild-type mice. As shown in Fig. 6Go, the ratio of taurocholic acid (TCA) to tauromuricholic acid (TMCA) was reduced by T3 treatment in wild-type male mice but not in female mice. This gender disparity was not due to differences in cyp7a activity as T3 increased enzyme activity to a comparable level in both males (hypothyroid, 1.18 ± 0.17 pmol/min·mg protein; hyperthyroid, 8.53 ± 0.61 pmol/min·mg protein) and females (hypothyroid, 3.12 ± 0.36 pmol/min·mg protein; hyperthyroid, 7.29 ± 0.75 pmol/min·mg protein). Thus, differences in bile acid speciation in male and female wild-type mice are due to factors inherent in the murine species.



View larger version (10K):
[in this window]
[in a new window]
  		FIG. 6. T3 alters biliary bile acid composition in male wild-type mice. The data shown are the mean ± SEM (n = 4). Open bars, Hypothyroid; filled bars, hyperthyroid. ANOVA results: F15 = 48.25, P < 0.0001.

 
In transgenic mice, the TCA/TMCA ratio was not significantly altered by T3 status in males. In contrast, hypothyroid females displayed a significantly increased TCA/TMCA ratio, and this effect was reversed with T3 replacement (Fig. 7AGo). As cyp7a activity is increased in hyperthyroid males but unchanged in hyperthyroid females, the TCA/TMCA ratio cannot be accounted for by changes in cyp7a activity. Sterol 12{alpha}-hydroxylase (cyp8b1) is the enzyme that catalyzes the 12{alpha}-hydroxylation of bile acid intermediates and is required for CA synthesis. Thus, cyp8b1 activity could affect the TCA/TMCA ratio. T3 treatment had no significant effect on cyp8b1 mRNA abundance in male or female mice (Fig. 7BGo).



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 7. T3 alters biliary bile acid composition in female transgenic mice. A, TCA/TMCA ratio in gallbladder bile; B, relative abundance of cyp8b1 mRNA. The data shown are the mean ± SEM from two experiments and separately normalized to the values seen in the male and female control groups. Hatched bars, Control; open bars, hypothyroid; filled bars, hyperthyroid. ANOVA results for A: F43 = 8.149, P < 0.0001; for B: F50 = 2.431, P = 0.0493.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The consequences of thyroid dysfunction on the synthesis of bile acids in humans are poorly understood. Direct analysis of CYP7A1 gene expression and hepatic cyp7a enzyme activity under normal and pathological states have been difficult due to the invasive techniques involved in obtaining liver tissue. In the present study, we evaluated the effect of thyroid hormone on human CYP7A1 gene expression in vivo using a newly developed transgenic mouse model (16). This mouse strain does not express the endogenous murine Cyp7a1 gene, thus allowing human CYP7A1 gene expression and cyp7a activity to be surveyed without interference from the functional consequences of the endogenous enzyme. Moreover, the expression of the human CYP7A1 gene in this model is under the control of its natural regulatory regions. Previous studies have documented fundamental differences in the array of trans-acting factors that bind to the cyp7a1 gene promoters in different species in vitro (16, 24). This new strain is thus an ideal model in which to evaluate the in vivo consequences of these differences.

Thyroid hormones alter plasma cholesterol metabolism in predictable patterns (reviewed in Ref.20). Two well-characterized effects of elevated plasma T3 are increased apoA-I and low density lipoprotein receptor gene expression resulting in reduced TPC and a relative enrichment of cholesterol in HDL. However, the effect of altered thyroid status on these markers of T3 action has not been examined in transgenic mice carrying the human CYP7A1 gene. Induction of hypothyroidism and hyperthyroidism in the transgenic mice carrying the human CYP7A1 gene resulted in changes in plasma cholesterol concentrations similar to those observed in human patients and experimental animals with thyroid dysfunction (7, 20, 22). The hypothyroid transgenic mice exhibited increased TPC and repartitioning of cholesterol into the LDL fraction. Thyroid hormone replacement reduced TPC and caused cholesterol to shift back to the HDL fraction. Furthermore, apoA-I mRNA abundance was tightly correlated to plasma T3 concentrations. These data illustrate that the effects of T3 on hepatic cholesterol metabolism appear to be intact in this new mouse strain.

Previous studies using in vitro model systems have documented the repression of the human CYP7A1 gene by T3 (13, 14, 15). Our finding that the abundance of the human cyp7a mRNA was reduced by T3 in male transgenic mice indicates that human CYP7A1 gene expression can be repressed by T3 in vivo. These data are consistent with a direct interaction of thyroid hormone receptors with the human CYP7A1 gene promoter as we have documented previously (15). Unexpectedly, however, this effect was not evident in female transgenic mice. The gender dimorphic response of the human CYP7A1 gene to T3 regulation in vivo was a surprising feature. The data available in the literature do not reveal a gender disparity in bile acid synthesis or output in male and female patients treated for hypo- or hyperthyroidism (8, 9, 12). The importance of gender in regulating cyp7a activity or CA synthesis by T3 in humans will need to be examined in a prospective study. In contrast, a number of differences in the metabolism of bile acids in male and female mice have been documented (e.g. Refs.25, 26, 27, 28). It is possible that gender differences inherent in this species (such as biliary cholesterol secretion; see below) are responsible for the inability of T3 to affect cyp7a mRNA levels in female transgenic mice.

The stimulatory effect of thyroid hormones on rodent cyp7a appears to be mediated primarily at the level of transcription as cyp7a activity is tightly linked to mRNA abundance and the rate of Cyp7a1 gene transcription (4, 5, 6, 7). However, our finding that the activity of human cyp7a was increased by T3 in male transgenic mice, despite the reduction of cyp7a mRNA abundance, suggests the involvement of posttranscriptional mechanisms in regulating cyp7a activity. The correlations of cyp7a activity with both plasma cholesterol and biliary cholesterol levels in males may also indicate the presence of a sterol-dependent posttranscriptional feed-forward pathway. In female mice, the high concentration of cholesterol in the gallbladder bile may be indicative of enhanced cholesterol secretion. This alternate mode of cholesterol disposal may alleviate the need for enhanced cyp7a activity, thus masking the presence of posttranscriptional mechanisms in this gender.

The stimulation of cyp7a activity by T3 in transgenic mice is not consistent with the decreased rate of CA synthesis/output observed clinically in some hyperthyroid patients (8, 9) or the reduced biliary bile acids observed in hyperthyroid males. It is possible that T3 affects biliary bile acid secretion in these mice. Additional work is required to determine the effects of T3 on bile acid secretion and bile formation in male and female transgenic mice.

The formation of CA in mice requires hepatic cyp8b1 (sterol 12{alpha}-hydroxylase) activity as demonstrated by mice lacking a functional Cyp8b1 gene (29). Previously, the inhibition of the Cyp8b1 gene by thyroid hormone was noted in male mice (30) as well as in male rats (31, 32). In our study, T3 administration did not significantly change cyp8b1 mRNA abundance in transgenic mice, although there was a tendency for a decrease and increase in males (P = 0.08) and females (P = 0.11), respectively. Surprisingly, the changes in TCA/TMCA ratio induced by thyroid hormone status were not correlated with the changes in cyp8b1 mRNA abundance. It was reported previously that hepatic cyp8b1 activity and the ratio of CA and chenodeoxycholic acid are not correlated in humans (33). It is likely that additional factors operate to influence the speciation of bile acids in bile, and these may be accentuated by thyroid hormone status. Hyperthyroid females also displayed significantly higher cyp8b1 mRNA levels and TCA/TMCA ratio compared with hyperthyroid males, further highlighting the gender dimorphic response to the regulation of bile acid metabolism by T3 in this mouse strain.

In summary, we report that the human CYP7A1 gene can be repressed by thyroid hormone in vivo. The activity of the cyp7a enzyme encoded by the human CYP7A1 gene was influenced by gender in transgenic mice and was correlated with plasma and biliary cholesterol. These findings suggest that both species-specific and gender-specific mechanisms operate to regulate the efficiency of the classical bile acid biosynthetic pathway.


   Acknowledgments
 
We thank Jody Seewalt for excellent technical assistance.


   Footnotes
 
This work was supported by Grant MOP-14812 from the Canadian Institutes of Health Research.

Present address for V.A.B.D.: Department of Physiology and Biophysics, 5-141 Basic Science Tower, State University of New York at Stony Brook, Stony Brook, New York 11794.

Abbreviations: apoA-I, Apolipoprotein A-I; CA, cholic acid; cyp7a, cholesterol 7{alpha}-hydroxylase; cyp8b1, sterol 12{alpha}-hydroxylase; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TCA, taurocholic acid; TMCA, tauromuricholic acid; TPC, total plasma cholesterol.

Received August 4, 2003.

Accepted for publication October 21, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bjorkhem I 1985 Mechanism of bile acid biosynthesis is mammalian liver. In: Danielsson H, Sjovall J, eds. Sterols and bile acids. Amsterdam: Elsevier Science Publishers; 231–278
  2. Agellon LB 2002 Metabolism and function of bile acids. In: Vance DE, Vance JE, eds. Biochemistry of lipids, lipoproteins and membranes. 4th ed. Amsterdam: Elsevier; 433–448
  3. Takeuchi N, Ito M, Uchida K, Yamamura Y 1975 Effect of modification of thyroid function on cholesterol 7{alpha}-hydroxylation in rat liver. Biochem J 148:499–503[Medline]
  4. Ness GC, Pendleton LC, Li YC, Chiang JYL 1990 Effect of thyroid hormone on hepatic cholesterol 7{alpha}-hydroxylase, LDL receptor, HMG-CoA reductase, farnesyl pyrophosphate synthetase and apolipoprotein A-I mRNA levels in hypophysectomized rats. Biochem Biophys Res Commun 172:1150–1156[CrossRef][Medline]
  5. Ness GC, Pendelton LC, Zhao Z 1994 Thyroid hormone rapidly increases cholesterol 7{alpha}-hydroxylase mRNA levels in hypophysectomized rats. Biochim Biophys Acta 1214:229–233[Medline]
  6. Ness GC, Lopez D 1995 Transcriptional regulation of rat hepatic low-density lipoprotein receptor and cholesterol 7{alpha}-hydroxylase by thyroid hormone. Arch Biochem Biophys 323:404–408[CrossRef][Medline]
  7. Gullberg H, Rudling M, Forrest D, Angelin B, Vennstrom B 2000 Thyroid hormone receptor ß-deficient mice show complete loss of the normal cholesterol 7{alpha}-hydroxylase (CYP7A) response to thyroid hormone but display enhanced resistance to dietary cholesterol. Mol Endocrinol 14:1739–1749[Abstract/Free Full Text]
  8. Miller LJ, Owyang C, Malagelada JR, Gorman CA, Go VL 1980 Gastric, pancreatic, and biliary responses to meals in hyperthyroidism. Gut 21:695–700[Abstract/Free Full Text]
  9. Pauletzki J, Stellaard F, Paumgartner G 1989 Bile acid metabolism in human hyperthyroidism. Hepatology 9:852–855[Medline]
  10. Abrams JJ, Grundy SM 1981 Cholesterol metabolism in hypothyroidism and hyperthyroidism in man. J Lipid Res 22:323–338[Abstract]
  11. Angelin B, Einarsson K, Leijd B 1983 Bile acid metabolism in hypothyroid subjects: response to substitution therapy. Eur J Clin Invest 13:99–106[Medline]
  12. Sauter G, Weiss M, Hoermann R 1997 Cholesterol 7{alpha}-hydroxylase activity in hypothyroidism and hyperthyroidism in humans. Horm Metab Res 29:176–179[Medline]
  13. Ellis E, Goodwin B, Abrahamsson A, Liddle C, Mode A, Rudling M, Bjorkhem I, Einarsson C 1998 Bile acid synthesis in primary cultures of rat and human hepatocytes. Hepatology 27:615–620[CrossRef][Medline]
  14. Wang D-P, Stroup D, Marrapodi M, Crestani M, Galli G, Chiang JYL 1995 Transcriptional regulation of the human cholesterol 7{alpha}-hydroxylase gene (CYP7A) in HepG2 cells. J Lipid Res 37:1831–1841
  15. Drover VA, Wong NC, Agellon LB 2002 A distinct thyroid hormone response element mediates repression of the human cholesterol 7{alpha}-hydroxylase (CYP7A1) gene promoter. Mol Endocrinol 16:14–23[Abstract/Free Full Text]
  16. Agellon LB, Drover VA, Cheema SK, Gbaguidi GF, Walsh A 2002 Dietary cholesterol fails to stimulate the human cholesterol 7{alpha}-hydroxylase gene (CYP7A1) in transgenic mice. J Biol Chem 277:20131–20134[Abstract/Free Full Text]
  17. Cheema SK, Cikaluk D, Agellon LB 1997 Dietary fats modulate the regulatory potential of dietary cholesterol on cholesterol 7{alpha}-hydroxylase gene expression. J Lipid Res 38:315–323[Abstract]
  18. Agellon LB 1997 Partial transfection of liver with a synthetic cholesterol 7{alpha}-hydroxylase transgene is sufficient to stimulate the reduction of cholesterol in the plasma of hypercholesterolemic mice. Biochem Cell Biol 75:255–262[CrossRef][Medline]
  19. Torchia EC, Labonte ED, Agellon LB 2001 Separation and quantitation of bile acids using an isocratic solvent system for high performance liquid chromatography coupled to an evaporative light scattering detector. Anal Biochem 298:293–298[CrossRef][Medline]
  20. Duntas LH 2002 Thyroid disease and lipids. Thyroid 12:287–293[CrossRef][Medline]
  21. Ness GC, Lopez D, Chambers CM, Newsome WP, Cornelius P, Long CA, Harwood Jr HJ 1998 Effects of L-triiodothyronine and the thyromimetic L-94901 on serum lipoprotein levels and hepatic low-density lipoprotein receptor, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and apo A-I gene expression. Biochem Pharmacol 56:121–129[CrossRef][Medline]
  22. Staels B, Van Tol A, Chan L, Will H, Verhoeven G, Auwerx J 1990 Alterations in thyroid status modulate apolipoprotein, hepatic triglyceride lipase, and low density lipoprotein receptor in rats. Endocrinology 127:1144–1152[Abstract]
  23. Romney JS, Chan J, Carr FE, Mooradian AD, Wong NC 1992 Identification of the thyroid hormone-responsive messenger RNA spot 11 as apolipoprotein-AI messenger RNA and effects of the hormone on the promoter. Mol Endocrinol 6:943–950[Abstract]
  24. Cheema SK, Agellon LB 2000 The murine and human cholesterol 7{alpha}-hydroxylase gene promoters are differentially responsive to regulation by fatty acids via peroxisome proliferator-activated receptor {alpha}. J Biol Chem 275:12530–12536[Abstract/Free Full Text]
  25. Alexander M, Portman OW 1987 Different susceptibilities to the formation of cholesterol gallstones in mice. Hepatology 7:257–265[Medline]
  26. Turley SD, Schwarz M, Spady DK, Dietschy JM 1998 Gender-related differences in bile acid and sterol metabolism in outbred CD-1 mice fed low- and high-cholesterol diets. Hepatology 28:1088–1094[CrossRef][Medline]
  27. Wang DQ, Lammert F, Paigen B, Carey MC 1999 Phenotypic characterization of lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice. Pathophysiology of biliary lipid secretion. J Lipid Res 40:2066–2079[Abstract/Free Full Text]
  28. Schwarz M, Russell DW, Dietschy JM, Turley SD 2001 Alternate pathways of bile acid synthesis in the cholesterol 7{alpha}-hydroxylase knockout mouse are not upregulated by either cholesterol or cholestyramine feeding. J Lipid Res 42:1594–1603[Abstract/Free Full Text]
  29. Li-Hawkins J, Gafvels M, Olin M, Lund EG, Andersson U, Schuster G, Bjorkhem I, Russell DW, Eggertsen G 2002 Cholic acid mediates negative feedback regulation of bile acid synthesis in mice. J Clin Invest 110:1191–1200[CrossRef][Medline]
  30. Gullberg H, Rudling M, Salto C, Forrest D, Angelin B, Vennstrom B 2002 Requirement for thyroid hormone receptor ß in T3 regulation of cholesterol metabolism in mice. Mol Endocrinol 16:1767–1777[Abstract/Free Full Text]
  31. Andersson U, Yang YZ, Bjorkhem I, Einarsson C, Eggertsen G, Gafvels M 1999 Thyroid hormone suppresses hepatic sterol 12{alpha}-hydroxylase (CYP8B1) activity and messenger ribonucleic acid in rat liver: failure to define known thyroid hormone response elements in the gene. Biochim Biophys Acta 1438:167–174[Medline]
  32. Vlahcevic ZR, Eggertsen G, Bjorkhem I, Hylemon PB, Redford K, Pandak WM 2000 Regulation of sterol 12{alpha}-hydroxylase and cholic acid biosynthesis in the rat. Gastroenterology 118:599–607[CrossRef][Medline]
  33. Bjorkhem I, Eriksson M, Einarsson K 1983 Evidence for a lack of regulatory importance of the 12{alpha}-hydroxylase in formation of bile acids in man: an in vivo study. J Lipid Res 24:1451–1456[Abstract]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
I. Tancevski, A. Wehinger, E. Demetz, P. Eller, K. Duwensee, J. Huber, K. Hochegger, W. Schgoer, C. Fievet, F. Stellaard, et al.
Reduced Plasma High-Density Lipoprotein Cholesterol in Hyperthyroid Mice Coincides with Decreased Hepatic Adenosine 5'-Triphosphate-Binding Cassette Transporter 1 Expression
Endocrinology, July 1, 2008; 149(7): 3708 - 3712.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
A. Berkenstam, J. Kristensen, K. Mellstrom, B. Carlsson, J. Malm, S. Rehnmark, N. Garg, C. M. Andersson, M. Rudling, F. Sjoberg, et al.
From the Cover: The thyroid hormone mimetic compound KB2115 lowers plasma LDL cholesterol and stimulates bile acid synthesis without cardiac effects in humans
PNAS, January 15, 2008; 105(2): 663 - 667.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
I. Klein and S. Danzi
Thyroid Disease and the Heart
Circulation, October 9, 2007; 116(15): 1725 - 1735.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
D.-J. Shin, M. Plateroti, J. Samarut, and T. F. Osborne
Two uniquely arranged thyroid hormone response elements in the far upstream 5' flanking region confer direct thyroid hormone regulation to the murine cholesterol 7{alpha} hydroxylase gene
Nucleic Acids Res., September 1, 2006; 34(14): 3853 - 3861.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
L. Johansson, M. Rudling, T. S. Scanlan, T. Lundasen, P. Webb, J. Baxter, B. Angelin, and P. Parini
Selective thyroid receptor modulation by GC-1 reduces serum lipids and stimulates steps of reverse cholesterol transport in euthyroid mice
PNAS, July 19, 2005; 102(29): 10297 - 10302.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Drover, V. A. B.
Right arrow Articles by Agellon, L. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Drover, V. A. B.
Right arrow Articles by Agellon, L. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals