Thyroid Hormone Receptor ß-Deficient Mice Show Complete Loss of the Normal Cholesterol 7
-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
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ABSTRACT
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Thyroid hormone (T3)
influences hepatic cholesterol metabolism, and previous studies have
established an important role of this hormone in the regulation of
cholesterol 7
-hydroxylase (CYP7A), the rate-limiting enzyme in the
synthesis of bile acids. To evaluate the respective contribution of
thyroid hormone receptors (TR)
1 and ß in this regulation, the
responses to 2% dietary cholesterol and T3
were studied in TR
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
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.
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INTRODUCTION
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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
-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,
1,
2, ß1,
and ß2, encoded by two different genes. In mammals, all but TR
2
bind T3 in their C-terminal domains. Previously,
we generated mice with null mutations of TR
1 (27) and TRß (28). We
found that TR
1-/- mice have lowered body temperature and reduced
heart rate, whereas TRß -/- mice show impaired auditory function
and overproduce TSH and thyroid hormones.
TR
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
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
1- and TRß-deficient mice. We also challenged
TR
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
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.
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RESULTS
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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. 1
. To achieve maximal responses to
T3 treatment, TR
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 1
).
The reduction in T4 was more pronounced in TRß
wt and TRß-/- mice as compared with TR
1-/- and TR
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 58 pmol/liter found
in the untreated control mice.
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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)
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On normal chow, serum total cholesterol levels in the TRß-/- mice
were identical to those in TRß wt mice (Fig. 2
, 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.
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. 3
).
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. 1
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.
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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
1-/- mice and their
respective control mice. In agreement with our hypothesis, serum total
cholesterol levels were similar in TR
1-/- and TR
1 wt mice, both
of which responded similarly to changes in T3
levels (Fig. 2
, lower panel). Analysis by FPLC (Fig. 3
, lower panel) indicated that the response to
T3 in LDL and HDL cholesterol was similar in wt
and TR
1-/ - animals.
Comparison of total serum cholesterol levels in TR
1 wt and TRß wt
mice showed that TR
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. 2
and 3
, lower panels). This difference between the TR
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 1
).
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
1-/-, TRß-/-, and corresponding wt mice under eu-, hypo-,
and hyperthyroid conditions (Fig. 4
). 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 4
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
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 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. 4A ) or TR 1-/-
and wt TR 1 mice (Fig. 4B ), on the abundance of mRNA for 5'DI-I,
CYP7A, LXR , 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 1 mice each mRNA preparation was from one animal.
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CYP7A, the rate limiting enzyme in cholesterol degradation to
bile acids, is a central regulator of cholesterol homeostasis. The
results shown in Figs. 4
and 5
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
1-/- mice, whereas no change was seen with the TRß -/- mice.
Assay of the CYP7A enzyme activity (Fig. 5
) 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. 5
, upper
panels). Furthermore, Fig. 6
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 1-/-, and Corresponding wt Mice
CYP7A mRNA abundance data were obtained as described in the legend to
Fig. 4 . 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.
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The oxysterol-binding transcription factor LXR
has been proposed to
be an important regulator in the response in CYP7A to dietary
cholesterol (18, 24). Thus, LXR
-deficient mice fail to induce CYP7A
transcription in response to 2% dietary cholesterol (32). As shown in
Fig. 4A
, LXR
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
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
1 wt
mice (Fig. 4
). In contrast, TRß-/- and TR
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
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. 4A
, 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. 6
).
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. 4A
) 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.
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DISCUSSION
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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
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
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
, an oxysterol
binding transcription factor that directly activates CYP7A
transcription. LXR
is necessary to induce the CYP7A transcription
and enzyme activity in response to cholesterol feeding in mice, since
LXR
-/- 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
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
mRNA
levels between the different groups of animals, a fact that also
supports the hypothesis that increased activation of LXR
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
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
-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
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
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
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
-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
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.
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MATERIALS AND METHODS
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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
1 mice each mRNA preparation was
from one animal. Northern blots were hybridized with cDNA probes for
mouse 5'-deiodinase-I, rat cholesterol 7
-hydroxylase, rat LDL
receptor, human apo AI, mouse apo AII, human HMG CoA reductase, and
human LXR
. 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
-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 Students
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
1-/-, and 30 TR
1 wt mice)
were used. The studies were approved by the Institutional Animal Care
and Use Committee. TR
1- and TRß-deficient mice were genotyped by
Southern blot or PCR analysis using PCR primers specific for the mutant
TR
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
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. 1
. 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
1 wt or TRß wt) and four to six knockout (TR
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 1
). 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
-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, Hedlunds 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
|
---|
-
Havel RJ 1982 Approach to the patient with
hyperlipidemia. Med Clin North Am 66:319333[Medline]
-
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
10991153
-
Stone NJ 1994 Secondary causes of hyperlipidemia. Med Clin
North Am 78:117141[Medline]
-
OBrien T, Young WF, Jr, Palumbo PJ, OBrien PC, Service FJ 1993 Hyperlipidemia in patients with primary and secondary
hypothyroidism. Mayo Clin Proc 68:860866[Medline]
-
Brown MS, Goldstein JL 1986 A receptor-mediated pathway for
cholesterol homeostasis. Science 232:3447[Medline]
-
Grundy SM, Vega GL, Bilheimer DW 1986 Causes and treatment of
hypercholesterolemia. Atheroscler Rev 15:1339
-
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 34:16371659[Medline]
-
Angelin B 1995 Studies on the regulation of hepatic
cholesterol metabolism in humans. Eur J Clin Invest 25:215224[Medline]
-
Mathe D, Chevallier F 1976 Effects of the thyroid state on
cholesterol metabolism in the rat. Biochim Biophys Acta 441:155164[Medline]
-
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 70:38393842[Abstract]
-
Ness GC, Pendleton LC, Li YC, Chiang JY 1990 Effect of thyroid
hormone on hepatic cholesterol 7
hydroxylase, LDL receptor, HMG-CoA
reductase, farnesyl pyrophosphate synthetase and apolipoprotein A-I
mRNA levels in hypophysectomized rats. Biochem Biophys Res Commun 172:11501156[Medline]
-
Ness GC, Zhao Z 1994 Thyroid hormone rapidly induces hepatic
LDL receptor mRNA levels in hypophysectomized rats. Arch Biochem
Biophys 315:199202[CrossRef][Medline]
-
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 127:11441152[Abstract]
-
Crestani M, Karam WG, Chiang JY 1994 Effects of bile acids and
steroid/thyroid hormones on the expression of cholesterol 7
-hydroxylase mRNA and the CYP7 gene in HepG2 cells. Biochem Biophys
Res Commun 198:546553[CrossRef][Medline]
-
Pandak WM, Heuman DM, Redford K, Stravitz RT, Chiang JY,
Hylemon PB, Vlahcevic ZR 1997 Hormonal regulation of cholesterol
7
-hydroxylase specific activity, mRNA levels, and transcriptional
activity in vivo in the rat. J Lipid Res 38:24832491[Abstract]
-
Paigen B, Ishida BY, Verstuyft J, Winters RB, Albee D 1990 Atherosclerosis susceptibility differences among progenitors of
recombinant inbred strains of mice. Arteriosclerosis 10:316323[Abstract]
-
Paigen B, Plump AS, Rubin EM 1994 The mouse as a model for
human cardiovascular disease and hyperlipidemia. Curr Opin Lipidol 5:258264[Medline]
-
Russell DW, Setchell KDR 1992 Bile acid biosynthesis.
Biochemistry 31:47374749[Medline]
-
Björkhem I, Lund E, Rudling M 1997 Coordinate regulation
of HMG CoA reductase and cholesterol 7
-hydroxylase. In: Bittman
R (ed) Subcellular Biochemistry. Plenum Press, New York, vol 28:2355
-
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 22:297306[Abstract]
-
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 112:23242332[Medline]
-
Rudling M, Parini P, Angelin B 1997 Growth hormone and bile
acid synthesis: Key role for the activity of hepatic microsomal
cholesterol 7
-hydroxylase in the rat. J Clin Invest 99:22392245[Abstract/Free Full Text]
-
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 90:88518855[Abstract]
-
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 83:835839[Medline]
-
Damm K, Thompson CC, Evans RM 1989 Protein encoded by v-erbA
functions as a thyroid-hormone receptor antagonist. Nature 339:593597[CrossRef][Medline]
-
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 340:242244[CrossRef][Medline]
-
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
1. EMBO J 17:455461[Abstract/Free Full Text]
-
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 15:30063015[Abstract]
-
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 267:1179411799[Abstract/Free Full Text]
-
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 139:49454952[Abstract/Free Full Text]
-
Paigen B, Morrow A, Brandon C, Mitchell D, Holmes P 1985 Variation in susceptibility to atherosclerosis among inbred strains of
mice. Atherosclerosis 57:6573[Medline]
-
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
. Cell 93:693704[Medline]
-
Brown MS, Goldstein JL 1997 The SREBP pathway: regulation of
cholesterol metabolism by proteolysis of a membrane-bound transcription
factor. Cell 89:331340[Medline]
-
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 56:121129[CrossRef][Medline]
-
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
105128
-
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 32:395405[Abstract]
-
Hargrove GM, Junco A, Wong NC 1999 Hormonal regulation of
apolipoprotein AI. J Mol Endocrinol 22:103111[Abstract/Free Full Text]
-
Russell DW 1999 Nuclear orphan receptors control cholesterol
catabolism. Cell 97:539542[Medline]
-
Wang H, Chen J, Hollister K, Sowers LC, Forman BM 1999 Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol
Cell 3:543553[Medline]
-
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 284:13621365[Abstract/Free Full Text]
-
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
-hydroxylase gene. Proc Natl Acad Sci USA 96:66606665[Abstract/Free Full Text]
-
Myant NB 1990 Cholesterol Metabolism, LDL, and the LDL
Receptor. Academic Press, San Diego, CA
-
Ness GC, Pendelton LC, Zhao Z 1994 Thyroid hormone rapidly
increases cholesterol 7
-hydroxylase mRNA levels in hypophysectomized
rats. Biochim Biophys Acta 1214:229233[Medline]
-
Taylor AH, Stephan ZF, Steele RE, Wong NC 1997 Beneficial
effects of a novel thyromimetic on lipoprotein metabolism. Mol
Pharmacol 52:542547[Abstract/Free Full Text]
-
Abrams JJ, Grundy SM 1981 Cholesterol metabolism in
hypothyroidism and hyperthyroidism in man. J Lipid Res 22:323238[Abstract]
-
Angelin B, Einarsson K, Leijd B 1983 Bile acid metabolism in
hypothyroid subjects: response to substitution therapy. Eur J Clin
Invest 13:99106[Medline]
-
Sauter G, Weiss M, Hoermann R 1997 Cholesterol 7
-hydroxylase activity in hypothyroidism and hyperthyroidism in
humans. Horm Metab Res 29:176179[Medline]
-
Cohen JC, Cali JJ, Jelinek DF, Mehrabian M, Sparkes RS,
Lusis AJ, Russel DW, Hobbs HH 1992 Cloning of the human
cholesterol7
-hydroxylase gene (CYP7) and localization to chromosome
8q11q12. Genomics 14:153161[Medline]
-
Vennstrom B, Bishop JM 1982 Isolation and characterization of
chicken DNA homologous to the two putative oncogenes of avian
erythroblastosis virus. Cell 28:135143[Medline]
-
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 25:11591166[Abstract]
-
Einarsson K, Angelin B, Ewerth S, Nilsell K,
Björkhem I 1986 Bile acid synthesis in man: assay of hepatic
microsomal cholesterol 7
-hydroxylase activity by isotope
dilution-mass spectrometry. J Lipid Res 27:8288[Abstract]
-
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 13:13291341[Abstract/Free Full Text]