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

Hjalmar Gullberg, Mats Rudling, Douglas Forrest, Bo Angelin and Björn Vennström

Laboratory of Developmental Biology (H.G., B.V.) Department of Cell and Molecular Biology (CMB) Karolinska Institute S-171 77 Stockholm Sweden
Centers for Metabolism and Endocrinology and Nutrition and Toxicology (M.R., B.A.) Department of Medicine Karolinska Institute at Huddinge University Hospital S-141 86 Stockholm, Sweden
Department of Human Genetics (D.F.) Mount Sinai School of Medicine New York, New York 10029


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyroid hormone (T3) influences hepatic cholesterol metabolism, and previous studies have established an important role of this hormone in the regulation of cholesterol 7{alpha}-hydroxylase (CYP7A), the rate-limiting enzyme in the synthesis of bile acids. To evaluate the respective contribution of thyroid hormone receptors (TR) {alpha}1 and ß in this regulation, the responses to 2% dietary cholesterol and T3 were studied in TR{alpha}1 and TRß knockout mice under hypo- and hyperthyroid conditions. Our experiments show that the normal stimulation in CYP7A activity and mRNA level by T3 is lost in TRß-/- but not in TR{alpha}1-/- mice, identifying TRß as the mediator of T3 action on CYP7A and, consequently, as a major regulator of cholesterol metabolism in vivo. Somewhat unexpectedly, T3-deficient TRß-/- mice showed an augmented CYP7A response after challenge with dietary cholesterol, and these animals did not develop hypercholesterolemia to the extent as did wild-type (wt) controls. The latter results lend strong support to the concept that TRs may exert regulatory effects in vivo independent of T3.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyroid hormone is important in cholesterol metabolism, and hypothyroidism is a prototype disease illustrating the role of this hormone in the negative regulation of serum cholesterol (1, 2, 3). Hypothyroidism is frequently associated with an elevation of serum cholesterol, generally reflecting increased low-density lipoprotein (LDL) cholesterol, which can be normalized after substitution therapy with thyroid hormone (4). Regulation of LDL cholesterol is controlled by the liver, which balances cholesterol uptake from the circulation with cholesterol synthesis and degradation in this organ (5, 6, 7, 8). All these processes are influenced by T3; thus, it has been shown that T3 affects the hepatic synthesis and uptake of cholesterol, largely controlled by hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase and LDL receptors, respectively (9, 10, 11, 12, 13). T3 is also important for the hepatic degradation of cholesterol into bile acids, and the rate limiting enzyme in this process, cholesterol 7{alpha}-hydroxylase (CYP7A), is induced by T3 at the transcriptional level (9, 11, 12, 14, 15). Mice and rats are relatively resistant to development of hypercholesterolemia after dietary cholesterol, and there are also differences between strains in this respect (16, 17). After challenge with a cholesterol-rich diet, these rodents respond with an increased formation and secretion of bile acids, leading to an increased excretion of body cholesterol (18, 19). This response is blunted in hormonally deficient animals, such as thyroidectomized or hypophysectomized rats (20, 21, 22, 23).

The responses to T3 in vivo are mediated by thyroid hormone receptors (TRs), being ligand-dependent transcription factors of the family of nuclear hormone receptors (24). TRs have a dual function in gene regulation. The ligand-receptor complex up-regulates many target genes, whereas the unliganded receptor often suppresses the basal level of transcription in a gene-specific manner (25, 26); thus TRs may have regulatory effects even in the absence of ligand. There are four variants of TRs, {alpha}1, {alpha}2, ß1, and ß2, encoded by two different genes. In mammals, all but TR{alpha}2 bind T3 in their C-terminal domains. Previously, we generated mice with null mutations of TR{alpha}1 (27) and TRß (28). We found that TR{alpha}1-/- mice have lowered body temperature and reduced heart rate, whereas TRß -/- mice show impaired auditory function and overproduce TSH and thyroid hormones.

TR{alpha}1 and TRß1 are the dominant TRs in the liver, whereas TRß2 is limited to the pituitary. In rat liver, 80% of the T3 binding proteins consist of TRß1 and 20% of TR{alpha}1 (29), with almost identical values in the mouse liver (30). To unveil the relative role of these receptors in the T3-mediated control of serum cholesterol levels we used TR{alpha}1- and TRß-deficient mice. We also challenged TR{alpha}1-/-, TRß-/-, and the respective wild-type (wt) mice with a cholesterol-rich diet under hypo- and hyperthyroid conditions and characterized their serum lipoprotein pattern and hepatic cholesterol metabolism. Our experiments show that the normal stimulation in CYP7A activity and mRNA level by T3 is lost in TRß-/- but not in TR{alpha}1-/- mice, identifying TRß as the mediator of T3 action on CYP7A and consequently as a major regulator of cholesterol metabolism in vivo. Somewhat unexpectedly, T3-deficient TRß-/- mice showed an augmented CYP7A response after challenge with dietary cholesterol, and these animals did not develop hypercholesterolemia to the extent as did wt controls. The latter results lend strong support to the concept that TRs may exert regulatory effects in vivo independent of T3.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Since thyroid hormone potently lowers cholesterol levels in blood, we aimed to determine the relative role of the TR isoforms in this regulation. The experimental setup is shown in Fig. 1Go. To achieve maximal responses to T3 treatment, TR{alpha}1-/ -, TRß-/-, and their respective wt mouse strains were made hypothyroid before injection of the hormone. Mice were thus placed on a synthetic low iodine diet (LID), and the drinking water was supplemented with methimazole (MMI) + perchlorate (LID+MMI). This regimen lowered free T4 in serum in all mice (P < 0.001, compared with normal diet) to hypothyroid levels, without inducing severe hypothyroidism (Table 1Go). The reduction in T4 was more pronounced in TRß wt and TRß-/- mice as compared with TR{alpha}1-/- and TR{alpha}1 wt mice. To induce hyperthyroidism, T3 was injected daily the last 5 days (LID+MMI+T3); this reduced serum T4 levels to less than 0.2 pM (P < 0.001, compared with normal diet) in all mice and resulted in an increase in serum T3 to hyperthyroid levels irrespective of whether the animals had received cholesterol or not. The serum T3 levels 24 h after the last injection were at least 11 pmol/liter, which is well above the 5–8 pmol/liter found in the untreated control mice.



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Figure 1. Experimental Setup

Altogether, 117 mice (28 TRß-/- and 30 wt plus 29 TR{alpha}1-/- and 30 wt mice were used). Before the experiment 71 animals (18 TRß-/-, 18 wt TRß, and 18 TR{alpha}1-/-, and 17 wt TR{alpha}1) were bled from the tail to obtain basal thyroid hormone and serum cholesterol levels. Thereafter, four to six knockout (KO) and six of the respective wt animals were assigned to five groups as shown. {dagger}, Killing of animals for collection of serum and tissue.

 

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Table 1. Free T4 (pmol/liter) at Baseline (Normal Diet and LID), during Thyroid Hormone Deprivation (LID+MMI) and T3 Treatment (LID+MMI+T3)

 
On normal chow, serum total cholesterol levels in the TRß-/- mice were identical to those in TRß wt mice (Fig. 2Go, upper panel). However, these levels were marginally increased (1.4-fold; P < 0.001) in the TRß-/- mice upon thyroid hormone deprivation as compared with (P < 0.01) the control animals (2.5-fold; P < 0.001). In addition, the TRß-/ - mice failed to lower their cholesterol levels after T3 treatment, whereas a more than 5-fold reduction (P < 0.001) was seen in the controls.



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Figure 2. Total Serum Cholesterol (mmol/liter) in TRß-/-, TR{alpha} 1-/-, and Corresponding wt Mice Described in Table 1Go and Fig. 1Go

Determinations were done on serum obtained before treatment (NORMAL DIET) and after a LID diet (LID) fed for 14 days followed by the additional inclusion of both 0.05% MMI and 1% potassium perchlorate in the drinking water (LID+MMI) for a further 21 days, resulting in hypothyroidism. Hyperthyroidism was induced by daily injections of T3 for the last 5 days of the regimen (LID+MMI+T3). The two last animal groups (LID+MMI+CHOLESTEROL and LID+MMI+CHOLESTEROL+T3) were treated the same way as above, except that their food was supplemented with 2% cholesterol from day 14. Results are expressed as mean ± SEM. Number of animals in each group is indicated to the left of each bar. *, Statistical differences between wt and knockout mice within each treatment. ¤, Statistical differences in total serum cholesterol between hypothyroid mice (LID+MMI and LID+MMI+CHOLESTEROL) and hyperthyroid (LID+MMI+T3 and LID+MMI+T3+CHOLESTEROL) mice. * and ¤, P < 0.05; ** and ¤¤, P < 0.01; *** and ¤¤¤, P < 0.001).

 
The differences between the two strains of mice were further enhanced by inclusion of cholesterol in the diet during thyroid hormone deprivation. Here, the TRß-/- and the control mice increased serum cholesterol levels 2-fold (P < 0.001) and 4-fold (P < 0.001), respectively, as compared with that seen with normal diet. Treatment with T3 reduced the cholesterol levels only marginally in the TRß-/- mice (P < 0.01), contrasting (P < 0.05 for difference between wt and knockout) the 4-fold reduction (P < 0.001) to below baseline values in the controls.

To better understand the nature of the increase in serum cholesterol, analysis of the lipoprotein patterns by forced pressure liquid chromatography (FPLC) was done (Fig. 3Go). The data show that all mice but the TRß -/- mice had a strong increase in the large high-density lipoprotein (HDL) and LDL fractions during T3 deprivation (red lines). Cholesterol in the diet only marginally increased the HDL fractions whereas the LDL and very low-density lipoprotein (VLDL) increased significantly in all strains tested (yellow lines). Finally, T3 decreased the LDL and HDL fractions significantly in all strains but the TRß -/- mice (blue lines). The data thus corroborate the serum cholesterol measurements.



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Figure 3. Lipoprotein Patterns of All Animal Groups Presented in Fig. 1Go

Serum (10 µl) from each individual was subjected to FPLC separation, and the cholesterol content was continuously monitored on line as described in Materials and Methods. Each chromatogram shows the mean for each group of animals.

 
The lack of effect of T3 in TRß-/- mice indicated that TRß is the main mediator of T3 actions on serum cholesterol levels. To further test this hypothesis, the same experiment as above was done with TR{alpha}1-/- mice and their respective control mice. In agreement with our hypothesis, serum total cholesterol levels were similar in TR{alpha}1-/- and TR{alpha}1 wt mice, both of which responded similarly to changes in T3 levels (Fig. 2Go, lower panel). Analysis by FPLC (Fig. 3Go, lower panel) indicated that the response to T3 in LDL and HDL cholesterol was similar in wt and TR{alpha}1-/ - animals.

Comparison of total serum cholesterol levels in TR{alpha}1 wt and TRß wt mice showed that TR{alpha}1 wt mice had a lower baseline level of serum total cholesterol, a lower increase of cholesterol in response to T3 deprivation, and a blunted response to T3 compared with the TRß wt mice (Figs. 2Go and 3Go, lower panels). This difference between the TR{alpha}1 and TRß wt mice is likely to be due to their different genetic backgrounds, in consonance with previous studies (16, 31), and the milder hypothyroidism attained in the former strain (Table 1Go). Nevertheless, we conclude that TRß is required for mediating the cholesterol-lowering effects of T3.

To understand the mechanism responsible for the failure to respond to T3 in the TRß--/ -- mice, the expression of putative target genes for TRs was surveyed in TR{alpha}1-/-, TRß-/-, and corresponding wt mice under eu-, hypo-, and hyperthyroid conditions (Fig. 4Go). The enzyme 5'-deiodinase-I (5'DI-I), which is under positive T3 control, was used to monitor the responses to hypo- and hyperthyroidism. Figure 4Go shows that 5'-DI-I steady state mRNA levels were strongly decreased under hypothyroid conditions (LID+MMI and LID+MMI+CHOLESTEROL) and increased by T3 (LID+MMI+T3 and LID+MMI+T3+CHOLESTEROL) in wt and TR{alpha}1-/- mice. However, as we report elsewhere, TRß-/- mice are deficient in induction of 5'-DI-I (L. L. Amma, A. Campos-Barros, B. Vennstrom, and D. Forrest, submitted). This indicates that the experimental procedure was valid and that the respective treatments rendered the animals functionally hypo- and hyper- thyroid.



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Figure 4. Hepatic mRNA Levels in TRß-/-, TR{alpha}1-/-, and Corresponding wt Mice

Effect of thyroid hormone deprivation (LID+MMI) and T3 treatment (LID+MMI+T3), in the presence or absence of 2% cholesterol in the diet, of TRß-/- and wt TRß mice (Fig. 4AGo) or TR{alpha}1-/- and wt TR{alpha}1 mice (Fig. 4BGo), on the abundance of mRNA for 5'DI-I, CYP7A, LXR{alpha}, LDL receptor (LDL-R), HMG CoA reductase (HMGR), Apo AI, and Apo AII. Levels of mRNA expression were normalized to that of G3PDH, and results are expressed as mean correlated to the expression in wt mice on LID, which was set to 1.0. For the TRß liver mRNA, each mRNA preparation represented an equal amount of tissue from two mice, whereas for the TR{alpha}1 mice each mRNA preparation was from one animal.

 
CYP7A, the rate limiting enzyme in cholesterol degradation to bile acids, is a central regulator of cholesterol homeostasis. The results shown in Figs. 4Go and 5Go demonstrate that the mRNA levels for CYP7A were reduced under hypothyroid conditions and increased by T3 and cholesterol in the two wt and the TR{alpha}1-/- mice, whereas no change was seen with the TRß -/- mice. Assay of the CYP7A enzyme activity (Fig. 5Go) showed a similar response pattern, where the CYP7A enzyme activity was unchanged in the TRß-/ - mice while being up-regulated by T3 (LID+MMI+T3) in the wt TRß (P < 0.001) animals. Surprisingly, CYP7A mRNA and enzyme activity was induced under hypothyroid conditions by cholesterol in the TRß-/- mice: a 10-fold increase in RNA levels and a 5-fold higher enzyme activity was observed (LID+MMI+CHOLESTEROL; Fig. 5Go, upper panels). Furthermore, Fig. 6Go shows that hepatic total cholesterol was increased by 10-fold (P < 0.01) in response to cholesterol feeding in hypothyroid TRß-/- mice compared with approximately 3-fold (not significant) in hypothyroid TRß wt mice. The addition of T3 increased hepatic cholesterol by more than 2-fold (P < 0.05) in cholesterol-fed hypothyroid TRß wt animals, whereas it had no effect in the corresponding TRß-/ - mice (P < 0.01 for difference). Taken together, these data support the conclusion that TRß is important for both the ligand- independent suppression and the T3-induced elevation of CYP7A activity.



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Figure 5. Quantitation of Liver CYP7A mRNA Abundance and Enzyme Activity in TRß-/-, TR{alpha}1-/-, and Corresponding wt Mice

CYP7A mRNA abundance data were obtained as described in the legend to Fig. 4Go. The enzymatic activity of CYP7A was determined in liver microsomes as described in Materials and Methods. Three separate pools of liver were prepared from each animal group containing four to six animals. Bars indicate means and error bars indicate SEM. *, Statistical differences in CYP7A activity between wt and knockout mice within each treatment. ¤, Statistical differences between hypothyroid mice (LID+MMI and LID+MMI+CHOLESTEROL) and hyperthyroid (LID+MMI+T3 and LID+MMI+T3+CHOLESTEROL) mice. #, Statistical differences between hypothyroid mice before and after addition of 2% cholesterol in the diet. *, ¤, and #, P < 0.05; **, ¤¤, and ##, P < 0.01; ***, ¤¤¤, and ###, P < 0.001.

 


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Figure 6. Total Cholesterol in Liver Homogenates from TRß-/- and TRß wt Mice

Total hepatic tissue cholesterol was determined in pooled liver tissue (three separate liver pools per group) from four to six animals in each group. Cholesterol was determined as described in Material and Methods. Bars indicate means and error bars indicate SEM. *, Statistical differences in total liver cholesterol between wt and knockout mice within each treatment. ¤, Statistical differences between hypothyroid mice (LID+MMI and LID+MMI+CHOLESTEROL) and hyperthyroid (LID+MMI+T3 and LID+MMI+T3+CHOLESTEROL) mice. #, Statistical differences between hypothyroid mice before and after addition of 2% cholesterol in the diet. *, ¤, and #, P < 0.05; **, ¤¤, and ##, P < 0.01.

 
The oxysterol-binding transcription factor LXR{alpha} has been proposed to be an important regulator in the response in CYP7A to dietary cholesterol (18, 24). Thus, LXR{alpha}-deficient mice fail to induce CYP7A transcription in response to 2% dietary cholesterol (32). As shown in Fig. 4AGo, LXR{alpha} mRNA levels were unaffected by T3 and/or cholesterol feeding in the TRß wt and TRß-/- mice, suggesting that the abnormal expression of CYP7A in TRß-/- mice is not simply caused by dysregulation of LXR{alpha} gene expression.

Removal of LDL and intermediate density lipoprotein (IDL) from serum is mediated by hepatic LDL receptors. LDL receptor mRNA levels were induced by T3 in both TRß wt and TR{alpha}1 wt mice (Fig. 4Go). In contrast, TRß-/- and TR{alpha}1-/- mice had higher LDL receptor mRNA levels under hypothyroid conditions, whereas there was no effect of T3 treatment. In response to dietary cholesterol, the LDL receptor mRNA levels were reduced in both TR{alpha}1-/- and TRß-/- mice but not in the respective wt animals. These results clearly show that the transcriptional regulation of the LDL receptor gene by T3 is different from that of CYP7A.

HMG CoA reductase is transcriptionally down- regulated by the accumulation of hepatic cholesterol through the inactivation of release of membrane-bound transcription factors (33). HMG CoA reductase mRNA is also sensitive to T3 (10, 11, 34). As seen in Fig. 4AGo, HMG CoA reductase mRNA was reduced by cholesterol feeding in TRß wt mice, but not in TRß-/- mice. Furthermore, T3 stimulated expression of the HMG CoA reductase gene only in TRß wt animals. Since HMG CoA reductase activity may also be down-regulated by cholesterol by posttranscriptional means (5), it is possible that a less pronounced reduction of endogenous cholesterol synthesis contributed to the greater accumulation of cholesterol in the livers of cholesterol-fed TRß-/- mice (Fig. 6Go).

HDLs participate in reverse cholesterol transport, a pathway for cholesterol transfer from peripheral tissues to the liver for excretion (35). The major structural proteins of HDL are apolipoproteins AI and AII (apo AI and apo AII). Apo AI mRNA levels seem to be positively, and apo AII mRNA negatively regulated by T3 (11, 13, 34, 36, 37). Our results (Fig. 4AGo) show that in agreement with previous work (11, 13) hepatic apo AI mRNA levels were increased by cholesterol feeding (LID+MMI+CHOLESTEROL), and further increased by T3 treatment (LID+MMI+T3+CHOLESTEROL) in TRß wt mice. In the TRß-/- mice, however, apo AI mRNA levels were increased by cholesterol but not by T3. Apo AII mRNA levels were unaffected by T3 in the TRß-/- mice, although it was decreased by T3 in all other mice. These results indicate that TRß mediates the T3-dependent regulation of apo AI and apo AII mRNA in the liver.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyroid hormones exert profound control over many functions in development and homeostasis. In the present work, we have characterized the role of the two predominant T3 receptors in the liver, TR{alpha}1 and TRß, on lipoprotein and cholesterol metabolism. Our studies clearly show that TRß is a major mediator of TR effects on serum lipoproteins and is specifically involved in the transcriptional regulation of the CYP7A and the apo AI and apo AII genes.

Hypothyroidism is a well established cause for hypercholesterolemia and elevated serum LDL (2). The response to a cholesterol-enriched diet is exaggerated in hypothyroid rats (20, 21), with the accumulation of lipoprotein particles within the VLDL/IDL range, representing chylomicron and VLDL remnants. We also observed this response in the wt and the TR{alpha}1-/- mice, whereas the response in TRß-/- mice was considerably blunted with regard to HDL. However, the VLDL and LDL patterns in cholesterol-treated TRß-/- mice were like those seen with cholesterol and T3-treated wt control animals. This highlights a distinction between thyroid hormone deficiency vs. a lack of TRß receptor protein, an observation supported by a recent report (30) which shows that TRß-deficient mice are resistant to T3-induced changes in serum cholesterol levels.

When fed excess amounts of cholesterol in the diet, both rats and mice respond with an increase in CYP7A activity (18, 19). This response is blunted in hypothyroid normal animals, a phenomenon that has been suggested to be caused by a lack of activation of the CYP7A promoter by ligand-bound TR (14, 15). Here, we show that T3 treatment resulted in increased CYP7A mRNA levels and microsomal enzyme activities in all animals except those lacking TRß, consistent with the view that this receptor is the major mediator of T3 effects on CYP7A expression.

The transcriptional regulation of CYP7A is now becoming unveiled (38). The classical regulation of bile acid biosynthesis by product inhibition is critically dependent on suppression of CYP7A transcription by the recently characterized bile acid receptor, FXR (39, 40). FXR acts as a negative transcription factor on the CYP7A reporter gene (39, 40). This inactivation may be, at least in part, exerted via antagonism of the positive action of LXR{alpha}, an oxysterol binding transcription factor that directly activates CYP7A transcription. LXR{alpha} is necessary to induce the CYP7A transcription and enzyme activity in response to cholesterol feeding in mice, since LXR{alpha}-/- mice fed high-cholesterol diets fail to induce enzyme activity and accumulate large amounts of cholesterol in the liver (32). In addition, the specific expression of CYP7A in the liver is dependent on liver-specific response elements such as the recently characterized CYP7A promoter-binding factor (CPF) (41).

Our results suggest that TRß, in the mouse, in a T3-independent fashion exerts a negative control on CYP7A expression that is accompanied by elevated serum cholesterol levels, and that it, as a response to ligand, induces CYP7A activity, which in turn results in reduced cholesterol levels. Accordingly, our data are consistent with the hypothesis that in TRß-deficient mice, regardless of their serum T3 levels, cholesterol feeding allows LXR{alpha} to induce CYP7A expression, thus preventing accumulation of excess levels of cholesterol. The vast stimulation of CYP7A mRNA and enzyme activity in response to cholesterol feeding in hypothyroid TRß-/- mice is compatible with the hypothesis that, in hypothyroid control mice, unliganded TRß acts as a repressor that counteracts the inductive effect of cholesterol on CYP7A expression. This repression mediated by unliganded TRß is obviously absent in the TRß-/- mice. Moreover, there was no difference in LXR{alpha} mRNA levels between the different groups of animals, a fact that also supports the hypothesis that increased activation of LXR{alpha} by oxy-sterols in hypothyroid TRß-/- mice during cholesterol feeding is the cause for the resistance to hypercholesterolemia. The fact that hepatic cholesterol was increased in these mice despite an increased CYP7A activity indicates that also other TRß-dependent regulatory steps in cholesterol metabolism may be involved in creating an enhanced amount of hepatic oxysterols. The lack of suppression of HMG CoA reductase mRNA in these animals could be one such factor, as discussed below.

A mechanism for how the ligand free TRß represses CYP7A expression cannot be detailed based on the available data, although several different possibilities can be suggested: TRß could compete with LXR{alpha} for binding to the same regulatory DNA sequence in the CYP7A promoter; it could exert its negative effect through binding to a distinct site in the promoter; or it could interfere with LXR or other factors necessary for CYP7A expression in a DNA-independent manner.

TRß also appeared to be of major importance for the regulation of HMG CoA reductase transcription by T3. Preliminary studies (not shown) indicate that a negative effect on cholesterol 12{alpha}-hydroxylase, an enzyme regulating the relative synthesis of the primary bile acids, cholic acid and chenodeoxycholic acid, was exerted via TRß. Thus, TRß may be involved in several important regulatory steps in cholesterol synthesis and degradation.

Reduced binding activity of hepatic LDL receptors is generally considered as a major mechanism of hyperlipidemia in hypothyroidism (42). In the present work, there were clearly effects of T3 on LDL receptor mRNA, but they could not be distinctly ascribed to TR{alpha}1 or TRß. Although T3 rapidly regulates the transcription of the LDL receptor gene (43), no specific TRE (thyroid response element) has so far been described in the LDL receptor gene promoter. Suppression of CYP7A activity would lead to down-regulation of LDL receptor mRNA, and we can therefore not conclude from the present work that T3 directly regulates the LDL receptor transcription.

Thyroid hormone is also known to stimulate transcription of the apo AI gene and the secretion of apo AI in HDL (13, 36, 42). In contrast, apo AII mRNA is reduced upon treatment with T3. Since apo AI overexpression has been shown to protect, and apo AII overexpression to aggravate, the atherosclerosis process in cholesterol-fed animals (17, 35), the effects of T3 on HDL metabolism would appear to be beneficial. From our results, it can be concluded that these effects are mediated by TRß, and not by TR{alpha}1, in vivo. The stimulating effects on CYP7A and apo AI mediated by TRß would lend support for the concept of designing TRß-specific agonists, which would avoid the side effects which make T3 therapy to be of limited clinical value, such as the TR{alpha}1-mediated increase in heart rate and body temperature. Preliminary reports indicate that liver-specific TR agonists may indeed result in positive effects on lipoprotein pattern (44). In this context, it is of interest to consider the relevance of the present findings for the in vivo effects of T3 in humans.

As mentioned, hypothyroidism is a well established hypercholesterolemic condition in humans (1, 2, 3). However, there is no good evidence that T3 affects bile acid production or CYP7A in humans (45, 46, 47). Considering the apparent lack of an LXR{alpha}-responsive element in the human CYP7A gene (48), this difference between mice and rats, on the one hand, and humans, on the other, may indirectly support the concept that LXR{alpha} is involved in the resistance to cholesterol feeding in the TRß-/-mice. Future studies should further analyze these interesting species differences, which will be important in the evaluation of possible therapeutic trials of TRß agonists in humans.

In conclusion, the present studies further elucidate the complex regulation of cholesterol and lipoprotein metabolism by T3. The in vivo experiments identify a specific TR, namely TRß, in the transcriptional regulation of CYP7A and apo AI. The results also clearly highlight the importance of T3-independent actions of T3 receptors in vivo.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RIAs
Free T3 and T4 were measured using the Amerlex-MAB kit as described previously (27).

Serum and Lipoprotein Cholesterol Determination
Size fractionation of lipoproteins by miniaturized on-line FPLC was performed using a micro-FPLC column (30 x 0.32 cm Superose 6B) coupled to a system for on-line separation and subsequent detection of cholesterol. Ten microliters of serum were injected from every animal, and the cholesterol content in lipoproteins was determined on line using the commercially available MPR 2 1 442 350 cholesterol assay kit (Roche Molecular Biochemicals ,Indianapolis, IN), which was continuously mixed with the separated lipoproteins at a flow rate of 40 + 40 µl/min. Absorbance was measured at 500 nM, and the data were collected using EZ Chrom software (Scientific Software, San Ramon, CA).

Serum total cholesterol was assayed with the Roche Molecular Biochemicals cholesterol assay kit, using a 5.2 mmol/liter cholesterol standard from Merck & Co., Inc., Darmstadt, Germany (catalog no. 14164).

Determination of mRNA Levels in Liver
Polyadenylated mRNA was prepared from frozen liver (49). For the TRß liver mRNA each preparation represented an equal amount of tissue from two mice whereas for the TR{alpha}1 mice each mRNA preparation was from one animal. Northern blots were hybridized with cDNA probes for mouse 5'-deiodinase-I, rat cholesterol 7{alpha}-hydroxylase, rat LDL receptor, human apo AI, mouse apo AII, human HMG CoA reductase, and human LXR{alpha}. Hybridization with glyceraldehyde-3-phosphate dehydrogenase (G3PDH) served as a control for equivalent loading of mRNA. Levels of mRNA expression were normalized to that of G3PDH using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) for quantification.

Enzymatic Activity of CYP7A
Hepatic microsomes were prepared by differential ultracentrifugation of liver homogenates as described previously (50). Three separate pools were prepared from each animal group. The activity of CYP7A was determined from the formation of 7{alpha}-hydroxycholesterol (picomoles/mg protein/min) from endogenous microsomal cholesterol by the use of isotope dilution-mass spectrometry as described in detail elsewhere (51). Assays were carried out in duplicate.

Total Hepatic Cholesterol
Pooled mouse liver samples were homogenized with a Polytron in chloroform-methanol (2:1, vol/vol) to obtain lipid fractions that were subsequently dried under nitrogen. Cholesterol was assayed with the Roche Molecular Biochemicals cholesterol assay kit. Three separate pools were prepared from each animal group.

Statistical Analysis
Data are presented as means ± SEM. P values were calculated by a two- sample Student’s t test (assuming equal variance) using Excel software (Microsoft Corp., Redmond, WA). P values < 0.05 were considered significant.

Animals and Experimental Setup
Altogether, 117 male mice aged 2 to 3.5 months (28 TRß-/ -, 30 TRß wt mice, 29 TR{alpha}1-/-, and 30 TR{alpha}1 wt mice) were used. The studies were approved by the Institutional Animal Care and Use Committee. TR{alpha}1- and TRß-deficient mice were genotyped by Southern blot or PCR analysis using PCR primers specific for the mutant TR{alpha}1 and TRß alleles, as described previously (27, 28). The genetic background of the TRß-/- mice is a hybrid of 129/Sv x C57Bl/6J and that of TR{alpha}1 -/- mice is a hybrid of 129/OlaHsd x Balb/c. The mice and their housing conditions were recently described (52). The wt control and the respective knockout strains used in this study were derived from crosses between heterozygotes for the respective knockout strains. The control strains therefore had the same, mixed genetic background as their respective receptor-deficient strain. To minimize the risk for genetic drift between the wt and the knockout strains, no more than three generations of breeding was done until new intercrosses were set up for regeneration of the respective wt and knockout strains. The experimental setup is described in Fig. 1Go. Blood for serum cholesterol and thyroid hormone analyses was collected before the start of the experiment and at the time of kill.

The mice were divided into five groups, each consisting of six wt (TR{alpha}1 wt or TRß wt) and four to six knockout (TR{alpha}1-/- or TRß-/-) animals. All groups of animals were at the onset of the experiment provided a LID (R584, AnalyCen Nordic AB, Lidköping, Sweden) for 14 days to accustom them to the synthetic chow. This limited time period resulted only in minor decreases in their serum levels of T3 or T4 (data not shown and Table 1Go). The mice were then made hypothyroid by inclusion of 0.05% methimazole (MMI) and 1% potassium perchlorate in the drinking water (LID+MMI) for 21 days when on the LID diet. From day 35 one group of animals were injected daily with 5 µg T3 for an additional 5-day period to induce hyperthyroidism. Two other groups of mice were treated the same way as described above except that the chow was supplemented with 2% cholesterol (23) from day 14. At the time of kill, trunk blood was collected after decapitation. Livers were immediately frozen on solid CO2.


    ACKNOWLEDGMENTS
 
We thank Kristina Nordström for animal setup, handling, and analyses and Mrs. Ingela Arvidsson for technical assistance. We also thank Dr. K. I. Okuda for providing the cDNA probe for rat cholesterol 7{alpha}-hydroxylase and Dr. G. Ness for the rat LDL receptor, human apo AI, and human HMG CoA reductase cDNA probes.


    FOOTNOTES
 
Address requests for reprints to: Björn Vennström, Laboratory of Developmental Biology, Department of Cell and Molecular Biology (CMB), Karolinska Institute, Box 285, Von Eulers väg 3, S-171 77 Stockholm, Sweden. E-mail: bjorn.vennstrom{at}cmb.ki.se

This study was supported by grants from the Medical Research Council (03X-7137), Strategiska stiftelsen, the Swedish Society for Medical Research, the Ax:son Johnson, the Ruth and Richard Julin, and the Swedish Heart Lung Foundations, and by grants from the Karolinska Institute and the Nordic Insulin Fund (M.R and B.A.), Human Frontiers Science Program and NIH Grant DC-03441 (D.F.), the Swedish Cancer Foundation, Hedlund’s Stiftelse, and the Karolinska Institute (B.V. and H.G.).

Received for publication April 24, 2000. Revision received July 12, 2000. Accepted for publication August 4, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Havel RJ 1982 Approach to the patient with hyperlipidemia. Med Clin North Am 66:319–333[Medline]
  2. Mahley RW, Weisgraber KH, Farese RVJ 1998 Disorders of lipid metabolism. In: Wilson JD, Foster EW, Kronenberg HM, Larsen PR (eds) Williams Textbook of Endocrinology. W.B. Saunders, Philadelphia, pp 1099–1153
  3. Stone NJ 1994 Secondary causes of hyperlipidemia. Med Clin North Am 78:117–141[Medline]
  4. O’Brien T, Young WF, Jr, Palumbo PJ, O’Brien PC, Service FJ 1993 Hyperlipidemia in patients with primary and secondary hypothyroidism. Mayo Clin Proc 68:860–866[Medline]
  5. Brown MS, Goldstein JL 1986 A receptor-mediated pathway for cholesterol homeostasis. Science 232:34–47[Medline]
  6. Grundy SM, Vega GL, Bilheimer DW 1986 Causes and treatment of hypercholesterolemia. Atheroscler Rev 15:13–39
  7. Dietschy JM, Turley SD, Spady DK 1993 Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans. J Lipid Res 34:1637–1659[Medline]
  8. Angelin B 1995 Studies on the regulation of hepatic cholesterol metabolism in humans. Eur J Clin Invest 25:215–224[Medline]
  9. Mathe D, Chevallier F 1976 Effects of the thyroid state on cholesterol metabolism in the rat. Biochim Biophys Acta 441:155–164[Medline]
  10. Ness GC, Dugan RE, Lakshmanan MR, Nepokroeff CM, Porter JW 1973 Stimulation of hepatic ß-hydroxy-ß-methylglutaryl coenzyme A reductase activity in hypophy-sectomized rats by L-triiodothyronine. Proc Natl Acad Sci USA 70:3839–3842[Abstract]
  11. Ness GC, Pendleton LC, Li YC, Chiang JY 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[Medline]
  12. Ness GC, Zhao Z 1994 Thyroid hormone rapidly induces hepatic LDL receptor mRNA levels in hypophysectomized rats. Arch Biochem Biophys 315:199–202[CrossRef][Medline]
  13. 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]
  14. Crestani M, Karam WG, Chiang JY 1994 Effects of bile acids and steroid/thyroid hormones on the expression of cholesterol 7 {alpha}-hydroxylase mRNA and the CYP7 gene in HepG2 cells. Biochem Biophys Res Commun 198:546–553[CrossRef][Medline]
  15. Pandak WM, Heuman DM, Redford K, Stravitz RT, Chiang JY, Hylemon PB, Vlahcevic ZR 1997 Hormonal regulation of cholesterol 7{alpha}-hydroxylase specific activity, mRNA levels, and transcriptional activity in vivo in the rat. J Lipid Res 38:2483–2491[Abstract]
  16. Paigen B, Ishida BY, Verstuyft J, Winters RB, Albee D 1990 Atherosclerosis susceptibility differences among progenitors of recombinant inbred strains of mice. Arteriosclerosis 10:316–323[Abstract]
  17. Paigen B, Plump AS, Rubin EM 1994 The mouse as a model for human cardiovascular disease and hyperlipidemia. Curr Opin Lipidol 5:258–264[Medline]
  18. Russell DW, Setchell KDR 1992 Bile acid biosynthesis. Biochemistry 31:4737–4749[Medline]
  19. Björkhem I, Lund E, Rudling M 1997 Coordinate regulation of HMG CoA reductase and cholesterol 7{alpha}-hydroxylase. In: Bittman R (ed) Subcellular Biochemistry. Plenum Press, New York, vol 28:23–55
  20. DeLamatre JG, Roheim PS 1981 Effect of cholesterol feeding on apo B and apo E concentrations and distributions in euthyroid and hypothyroid rats. J Lipid Res 22:297–306[Abstract]
  21. Kris-Etherton PM, Fosmire MA, Mela DJ, Etherton TD 1982 Studies on the etiology of increased tissue cholesterol concentration in cholesterol-fed hypothyroid rats. J Nutr 112:2324–2332[Medline]
  22. Rudling M, Parini P, Angelin B 1997 Growth hormone and bile acid synthesis: Key role for the activity of hepatic microsomal cholesterol 7{alpha}-hydroxylase in the rat. J Clin Invest 99:2239–2245[Abstract/Free Full Text]
  23. Rudling M, Angelin B 1993 Loss of resistance to dietary cholesterol in the rat following hypophysectomy: Importance of growth hormone for the expression of hepatic low density lipoprotein receptors. Proc Natl Acad Sci USA 90:8851–8855[Abstract]
  24. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[Medline]
  25. Damm K, Thompson CC, Evans RM 1989 Protein encoded by v-erbA functions as a thyroid-hormone receptor antagonist. Nature 339:593–597[CrossRef][Medline]
  26. Sap J, Munoz A, Schmitt J, Stunnenberg H, Vennström B 1989 Repression of transcription mediated at a thyroid hormone response element by the v-erb-A oncogene product. Nature 340:242–244[CrossRef][Medline]
  27. Wikström L, Johansson C, Salto C, Barlow C, Campos Barros A, Baas F, Forrest D, Thoren P, Vennström B 1998 Abnormal heart rate and body temperature in mice lacking thyroid hormone receptor {alpha}1. EMBO J 17:455–461[Abstract/Free Full Text]
  28. Forrest D, Hanebuth E, Smeyne RJ, Everds N, Stewart CL, Wehner JM, Curran T 1996 Recessive resistance to thyroid hormone in mice lacking thyroid hormone receptor beta: evidence for tissue-specific modulation of receptor function. EMBO J 15:3006–3015[Abstract]
  29. Schwartz HL, Strait KA, Ling NC, Oppenheimer JH 1992 Quantitation of rat tissue thyroid hormone binding receptor isoforms by immunoprecipitation of nuclear triiodothyronine binding capacity. J Biol Chem 267:11794–11799[Abstract/Free Full Text]
  30. Weiss RE, Murata Y, Cua K, Hayashi Y, Seo H, Refetoff S 1998 Thyroid hormone action on liver, heart, and energy expenditure in thyroid hormone receptor ß-deficient mice. Endocrinology 139:4945–4952[Abstract/Free Full Text]
  31. Paigen B, Morrow A, Brandon C, Mitchell D, Holmes P 1985 Variation in susceptibility to atherosclerosis among inbred strains of mice. Atherosclerosis 57:65–73[Medline]
  32. Peet DJ, Turley SD, Ma W, Janowski BA, Lobaccaro JM, Hammer RE, Mangelsdorf DJ 1998 Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR {alpha}. Cell 93:693–704[Medline]
  33. Brown MS, Goldstein JL 1997 The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89:331–340[Medline]
  34. Ness GC, Lopez D, Chambers CM, Newsome WP, Cornelius P, Long CA, Harwood HJ, Jr 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]
  35. Tall AR, Breslow JL 1996 Plasma high-density lipoproteins and atherosclerosis. In: Fuster V, Ross R, Topol E (eds) Atherosclerosis and Coronary Artery Disease. Lippincott-Raven, Philadelphia, pp 105–128
  36. Wilcox HG, Frank RA, Heimberg M 1991 Effects of thyroid status and fasting on hepatic metabolism of apolipoprotein A-I [published erratum appears in J Lipid Res 1991 May;32(5):886]. J Lipid Res 32:395–405[Abstract]
  37. Hargrove GM, Junco A, Wong NC 1999 Hormonal regulation of apolipoprotein AI. J Mol Endocrinol 22:103–111[Abstract/Free Full Text]
  38. Russell DW 1999 Nuclear orphan receptors control cholesterol catabolism. Cell 97:539–542[Medline]
  39. Wang H, Chen J, Hollister K, Sowers LC, Forman BM 1999 Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol Cell 3:543–553[Medline]
  40. Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, Luk A, Hull MV, Lustig KD, Mangelsdorf DJ, Shan B 1999 Identification of a nuclear receptor for bile acids. Science 284:1362–1365[Abstract/Free Full Text]
  41. Nitta M, Ku S, Brown C, Okamoto AY, Shan B 1999 CPF: an orphan nuclear receptor that regulates liver-specific expression of the human cholesterol 7{alpha}-hydroxylase gene. Proc Natl Acad Sci USA 96:6660–6665[Abstract/Free Full Text]
  42. Myant NB 1990 Cholesterol Metabolism, LDL, and the LDL Receptor. Academic Press, San Diego, CA
  43. 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]
  44. Taylor AH, Stephan ZF, Steele RE, Wong NC 1997 Beneficial effects of a novel thyromimetic on lipoprotein metabolism. Mol Pharmacol 52:542–547[Abstract/Free Full Text]
  45. Abrams JJ, Grundy SM 1981 Cholesterol metabolism in hypothyroidism and hyperthyroidism in man. J Lipid Res 22:323–238[Abstract]
  46. 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]
  47. 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]
  48. Cohen JC, Cali JJ, Jelinek DF, Mehrabian M, Sparkes RS, Lusis AJ, Russel DW, Hobbs HH 1992 Cloning of the human cholesterol7{alpha}-hydroxylase gene (CYP7) and localization to chromosome 8q11–q12. Genomics 14:153–161[Medline]
  49. Vennstrom B, Bishop JM 1982 Isolation and characterization of chicken DNA homologous to the two putative oncogenes of avian erythroblastosis virus. Cell 28:135–143[Medline]
  50. Angelin B, Einarsson K, Liljeqvist L, Nilsell K, Heller RA 1984 3-hydroxy-3-methylglutaryl coenzyme A reductase in human liver microsomes: active and inactive forms and cross-reactivity with antibody against rat liver enzyme. J Lipid Res 25:1159–1166[Abstract]
  51. Einarsson K, Angelin B, Ewerth S, Nilsell K, Björkhem I 1986 Bile acid synthesis in man: assay of hepatic microsomal cholesterol 7 {alpha}-hydroxylase activity by isotope dilution-mass spectrometry. J Lipid Res 27:82–88[Abstract]
  52. Göthe S, Wang Z, Ng L, Kindblom JM, Barros AC, Ohlsson C, Vennström B, Forrest D 1999 Mice devoid of all known thyroid hormone receptors are viable but exhibit disorders of the pituitary-thyroid axis, growth, and bone maturation. Genes Dev 13:1329–1341[Abstract/Free Full Text]