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J. Biol. Chem., Vol. 275, Issue 50, 39685-39692, December 15, 2000
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From the
Received for publication, August 22, 2000, and in revised form, September 22, 2000
Sterol 27-hydroxylase (CYP27) participates in the
conversion of cholesterol to bile acids. We examined lipid metabolism
in mice lacking the Cyp27 gene. On normal rodent chow,
Cyp27 In mammals the conversion of cholesterol to bile acids and their
subsequent fecal excretion represents a major route for the elimination
of cholesterol from the body (1). There are two main pathways by which
cholesterol is converted to bile acids (2-4). The major pathway, also
called the classic or neutral pathway, is initiated by cholesterol
7 Despite the different pathophysiological consequences of sterol
27-hydroxylase deficiency in humans and mice, further characterization of sterol and lipid metabolism in the Cyp27 knockout mouse
is warranted for several reasons. First, there have been several recent
discoveries relating to the mechanisms through which feedback regulation of bile acid synthesis is articulated via nuclear receptors (13-16). Therefore, we wanted to study these mechanisms and related aspects of sterol metabolism in an animal model in which the rate of
bile acid synthesis was impaired. Second, in our initial evaluation of
Cyp27 Animals and Diets--
Sterol 27-hydroxylase-deficient mice were
generated as described previously (12). Breeding stock were generously
supplied by Dr. Nobuyo Maeda at the University of North Carolina. The
mutation (Cyp27 Plasma Hormone and Lipoprotein Cholesterol and Triacylglycerol
Concentrations--
Mice were exsanguinated from the vena cava under
ether anesthesia. Blood was anticoagulated with EDTA. Plasma
corticosterone, ACTH, and testosterone concentrations were determined
by a commercial facility (Endocrine Services Laboratory, Oregon
Regional Primate Research Center, Beaverton, OR). Pooled plasma samples
were fractionated by fast protein liquid chromatography and the
cholesterol and triacylglycerol content of each fraction, and of whole
plasma, was determined enzymatically using commercially available kits as was plasma glucose concentration (Roche Molecular Biochemicals and
Sigma-Aldrich).
Tissue Cholesterol and Triacylglycerol Concentrations, Fatty Acid
Composition, and Identification of "Non-cholesterol" Sterols in
Adrenals and Lungs--
Tissue total cholesterol concentrations and
hepatic fatty acid composition were determined by gas-chromatographic
methods (24). In the adrenals and lungs of the
Cyp27 RNA Analysis--
Following exsanguination of the mice under
ether anesthesia, aliquots of liver were immediately frozen in liquid
nitrogen. Five µg of poly(A+) RNA prepared from
individual livers were subjected to electrophoresis and northern blot
analysis using 32P-labeled probes as described previously
(26, 27). The amount of radioactivity in each band was quantified by
PhosphorImager and normalized to the signal generated by Parameters of Bile Acid Metabolism, Intestinal Cholesterol
Absorption, and Fecal Neutral Sterol Excretion--
Cholesterol
7 Tissue Sterol Synthesis--
The rates of sterol synthesis in
the major organs and in the whole animal were determined in
vivo using [3H]water as described (23). The rate of
synthesis in each of the extrahepatic organs was corrected for the
[3H]sterol contained in residual blood that contaminated
the tissue at the time of the excision. During the 60 min following
administration of the [3H]water, essentially all of the
[3H]sterol in blood is of hepatic origin (28). Hence,
this correction was essential, particularly for the
Cyp27 Analysis of Data--
All data are reported as the mean ± 1 S.E. in the specified number of individual animals. Differences
between mean values were tested for statistical significance
(p < 0.05) by the two-tailed Student's t
test assuming equal variance.
The data in Table I show that
although there was no difference in body weights of adult male
Cyp27 Fractionation of pooled plasma by fast protein liquid chromatography
(Fig. 1) showed that the increment in the
cholesterol (Fig. 1A) and triacylglycerol (Fig.
1B) concentrations in the Cyp27
Disruption of the Sterol 27-Hydroxylase Gene in Mice Results in
Hepatomegaly and Hypertriglyceridemia
REVERSAL BY CHOLIC ACID FEEDING*
,
,, and
Departments of Internal Medicine and
§ Molecular Genetics, Howard Hughes Medical
Institute, Department of Pharmacology, University of Texas Southwestern
Medical Center, Dallas, Texas 75390 and the ** Department of Medicine,
Center for Research, Prevention and Treatment of Atherosclerosis,
Hadassah University Hospital, 91120 Jerusalem, Israel
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/ mice have 40% larger livers, 45%
larger adrenals, 2-fold higher hepatic and plasma triacylglycerol
concentrations, a 70% higher rate of hepatic fatty acid synthesis, and
a 70% increase in the ratio of oleic to stearic acid in the liver
versus Cyp27+/+ controls. In
Cyp27/ mice, cholesterol 7
-hydroxylase
activity is increased 5-fold, but bile acid synthesis and pool size are
47 and 27%, respectively, of those in Cyp27+/+
mice. Intestinal cholesterol absorption decreases from 54 to 4% in
knockout mice, while fecal neutral sterol excretion increases 2.5-fold.
A compensatory 2.5-fold increase in whole body cholesterol synthesis
occurs in Cyp27/ mice, principally in
liver, adrenal, small intestine, lung, and spleen. The mRNA for the
cholesterogenic transcription factor sterol regulatory element-binding
protein-2 (SREBP-2) and mRNAs for SREBP-2-regulated cholesterol
biosynthetic genes are elevated in livers of mutant mice. In addition,
the mRNAs encoding the lipogenic transcription factor SREBP-1 and
SREBP-1-regulated monounsaturated fatty acid biosynthetic enzymes are
also increased. Hepatic synthesis of fatty acids and accumulation of
triacylglycerols increases in Cyp27/ mice
and is associated with hypertriglyceridemia. Cholic acid feeding
reverses hepatomegaly and hypertriglyceridemia but not adrenomegaly in
Cyp27/ mice. These studies confirm the
importance of CYP27 in bile acid synthesis and they reveal an
unexpected function of the enzyme in triacylglycerol metabolism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxylase (CYP7A1),1
located in the endoplasmic reticulum of liver cells. The alternate or
acidic pathway is initiated by mitochondrial sterol 27-hydroxylase (CYP27), which is present not only in the liver but in extrahepatic organs as well, particularly the lungs (5-7). The synthesis of bile
acids via the classic pathway also involves sterol
27-hydroxylase, which facilitates oxidation of the steroid side chain
(2, 3). Sterol 27-hydroxylase deficiency in humans results in a marked reduction in total bile acid synthesis, an increase in whole body cholesterol synthesis and the accumulation of cholestanol in tissues (8-10). Cerebrotendinous xanthomatosis, the disease arising
from this deficiency, is characterized by several conditions
including accelerated atherosclerosis (11). In Cyp27
knockout mice there is a dramatic reduction in total bile acid
synthesis, but these animals do not develop cerebrotendinous
xanthomatosis (12).
/ mice, we found that they were
hypertriglyceridemic when maintained on a low fat, conventional rodent
diet. This observation was potentially important because of earlier
findings of an association between hepatic very low density lipoprotein
(VLDL)-triacylglycerol secretion and the rate of bile acid synthesis
(17-19). Third, the measurement in vivo of the rate of
sterol synthesis in the liver and major extrahepatic organs of
Cyp27/ mice, represented a new, more
quantitative approach to further investigating the putative role of
27-hydroxycholesterol as a regulator of cholesterol biosynthesis
(20-22). Fourth, our initial investigation of
Cyp27/ mice revealed that they had a
significant enlargement of their livers and adrenal glands. Such
organomegaly is not seen in mice that lack cholesterol
7-hydroxylase, but which retain a functional alternate pathway of
bile acid synthesis (23). The studies reported here show that sterol
27-hydroxylase plays a more global role in bodily lipid metabolism than
has been documented previously. Surprisingly, the loss of the
sterol 27-hydroxylase-initiated pathway of bile acid synthesis and the
resultant marked compensatory increase in cholesterol 7-hydroxylase
activity is accompanied by dramatic changes in hepatic fatty acid
metabolism and circulating plasma VLDL-triacylglycerol concentrations.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/) was maintained in a
mixed-strain background (C57BL/6:129Sv) as were matching
Cyp27+/+ controls. The litters did not require
supplementation with cholic acid or vitamins during the nursing period.
The pups were weaned at 4 weeks and thereafter were fed ad
libitum a cereal-based rodent diet (Wayne Lab Blox, No. 8604)
(Harlan Teklad, Madison, WI), which contained 0.20 mg of cholesterol
and 50 mg of total lipid per g of diet. This chow was defined as the
basal diet. In all studies the mice were fed the meal form of this diet
without any additions except in one case when cholic acid was added
(0.1% w/w). All experiments were carried out in male mice 3-4 months of age that were housed as described (23). Mice were killed at the end
of the 12-h dark phase of the lighting cycle and were in the fed state
at the time of study except those that were fasted for 6 h before
harvesting of gallbladder bile. The adrenal glands were excised,
cleaned of adhering tissue, and weighed. Sections of adrenal tissue
were stained with hematoxylin and eosin. All experiments were approved
by the Institutional Animal Care and Research Advisory Committee.
/ mice, non-cholesterol sterols
were present. These were identified by gas chromatography-mass
spectrometry after trimethylsilyl derivatization as described elsewhere
(25). Hepatic triacylglycerol concentrations were measured as follows.
A 0.3-g aliquot of liver was extracted in chloroform:methanol (2:1,
v/v) in a final volume of 100 ml. Duplicate 2-ml aliquots of each
extract were washed with 0.5 ml of isotonic saline. After
centrifugation, the upper phase was discarded and 20 µl of
chloroform:Triton X-114 (1:1, v/v) was added to the lower phase which
was then dried completely at 45-50 °C under an air stream. Two ml
of Infinity Triglyceride reagent (number 343-25P) (Sigma-Aldrich) was
added to the residue and vortexed. After 20 min, the optical densities
of the samples were read at 520 nm against standards to which 20 µl
of chloroform:Triton X-114 had been added before drying and color development.
-actin. For
each mRNA, the fold-change for the
Cyp27/ mice was expressed relative to
matching Cyp27+/+ mice, which in each case was
arbitrarily set at 1.0.
-hydroxylase activity and bile acid pool size and composition were
determined by methods using high performance liquid chromatography as
described (23). The identity of bile acids in gallbladder bile was
established by gas chromatography-mass spectrometry (25). Fecal bile
acid and neutral sterol excretion rates, and the lipid composition of
gallbladder bile, were determined using various gas-chromatographic,
enzymatic, and colorimetric methods (23). Intestinal cholesterol
absorption was measured by a fecal isotope ratio method (23). The level
of apical sodium bile acid co-transporter protein in the distal third
of the small intestine was determined by SDS-polyacrylamide gel
electrophoresis and immunoblotting using an anti-apical sodium bile
acid co-transporter polyclonal antibody supplied by Dr. Paul Dawson,
Bowman Gray School of Medicine.
/ mice which had an exceptionally high
rate of hepatic sterol synthesis. The rate of hepatic fatty acid
synthesis was determined in the same tissue aliquots that were used for
measurement of sterol synthesis. After removal of the labeled sterols,
the aqueous phase was acidified with 1 ml of concentrated hydrochloric
acid and extracted twice with 15 ml of hexane. The combined hexane
extracts were brought to a volume of 100 ml. Ten ml of this extract was dried first under air and then in a vacuum oven before being counted.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/ and Cyp27+/+
mice, there was a marked increase in the mass of the liver (40%) and
adrenal glands (45%) in the Cyp27/ animals.
When maintained on a conventional low fat, low cholesterol rodent diet,
the Cyp27/ mice also manifest a 30%
increase in plasma total cholesterol concentration and a 2-fold
increase in plasma total triacylglycerol concentration. They maintained
normal plasma levels of glucose, corticosterone, ACTH, and testosterone
(Table II). In the
Cyp27/ mice the molar ratio of cholesterol
in gallbladder bile was double that in the
Cyp27+/+ controls. This was due to the fact that
the biliary cholesterol concentration in these animals was unchanged,
whereas the concentrations of bile acid and phospholipid fell
significantly (Table II).
Body and organ weights in Cyp27+/+ and Cyp27/ mice
/
male mice were fed ad libitum a cereal-based rodent
diet without added cholesterol. They were studied at 3 to 4 months of
age. Values are the mean ± 1 S.E. for measurements in the number
of animals given in parentheses.
Concentrations of lipids and hormones in the plasma and gallbladder
bile of Cyp27+/+ and Cyp27/ mice
/
mice was confined to VLDL. In contrast to the plasma cholesterol level,
the concentration of cholesterol in most tissues, and in the whole
animal, was the same in both genotypes (Figs.
2, A and B). There
were two exceptions. One was the adrenal gland, which in the
Cyp27/ mice had not only a 48% higher
concentration of cholesterol (Fig. 2A), but also significant
levels of two non-cholesterol sterols, the concentration of which
totaled 9.8 ± 0.6 mg/g. These sterols, which were not detected in
the adrenals of the Cyp27+/+ mice, were
identified by gas chromatography-mass spectrometry as the two forms of
lathosterol (5-cholest-7-en-3-ol and 5-cholest-8-en-3-ol). The other exception was the lungs, which had a slightly reduced cholesterol concentration in the Cyp27/ mice
(from 5.3 ± 0.1 to 4.7 ± 0.2 mg/g). Trace amounts of the two forms of lathosterol (about 4% of total tissue sterol content) were also found in lung tissue from the
Cyp27/ animals. Although the data are not
shown in Fig. 2A, the cholesterol concentration in testis
averaged 2.0 mg/g in mice of both genotypes.
View larger version (27K):
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Fig. 1.
Plasma lipoprotein profiles in
Cyp27+/+ and
Cyp27 / mice. Plasma from groups
of male mice of both genotypes that had been fed a basal rodent diet
without added cholesterol was fractionated by fast protein liquid
chromatography, and the cholesterol (A) and triacylglycerol
(B) content of each fraction was determined as described
under "Experimental Procedures." For each genotype, plasma from 5 mice was combined. LDL, low density lipoprotein;
HDL, high density lipoprotein.
View larger version (43K):
[in a new window]
Fig. 2.
Tissue cholesterol concentration in the liver
and extrahepatic organs of Cyp27+/+ and
Cyp27 / mice. Tissue total
cholesterol concentrations were measured by gas chromatography in
various organs of Cyp27+/+ and
Cyp27/ male mice fed a basal rodent diet
without added cholesterol. The concentration values (A) were
multiplied by respective organ weights to obtain whole organ
cholesterol contents normalized per 100 g of body weight
(B). These contents were added to give values for whole
animal cholesterol content per 100 g of body weight
(inset of B). Values represent the mean ± 1 S.E. of data from at least 9 animals of each genotype (for liver,
kidney, spleen, adrenal, and lung) and from 5 animals of each genotype
for the other organs. Whole body cholesterol contents were determined
in 14 mice of each genotype. *, p < 0.05 compared with
value for Cyp27+/+ animals.
A major focus of these studies was to characterize the effects of
sterol 27-hydroxylase deficiency on an array of proteins in the liver
that are involved not only in bile acid and cholesterol metabolism, but
also in fatty acid metabolism. This analysis was done by determining
the mRNA levels for each of the target proteins in concert with
measuring enzyme activities and the rates of various metabolic
processes associated with the expression of these proteins. The first
set of data relate to various enzymes and receptors involved in
regulating bile acid synthesis. As shown in Fig.
3, the Cyp27/
mice lacked sterol 27-hydroxylase, and manifest an ~10-fold increase in the mRNA for cholesterol 7-hydroxylase (CYP7A1). The
expression of oxysterol 7-hydroxylase (CYP7B1) in the
Cyp27/ mice was only 40% of that seen in
the Cyp27+/+ controls, whereas the expression of
sterol 12-hydroxylase (CYP8B1) was 2.4-fold higher in the
Cyp27/ animals. The expression of the small
heterodimer partner (SHP) in the Cyp27/ mice
was reduced to only 35% of that in the Cyp27+/+
controls, whereas the mRNA levels for both the farnesoid X receptor (FXR) and the liver receptor homologue-1 (LRH-1) showed a slight elevation in the Cyp27/ mice.
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The enzyme activity data shown in Fig. 4
agree well with the RNA measurements shown in Fig. 3, as exemplified by
the 5-fold increase in cholesterol 7-hydroxylase activity in the
Cyp27/ mice (Fig. 4A). Total bile
acid synthesis in these mice, as measured by the rate of fecal bile
acid excretion, averaged 47% of that in the
Cyp27+/+ controls (Fig. 4B). In these
same Cyp27/ mice, bile acid pool size was
only 27% of that in the Cyp27+/+ mice (Fig.
4C), and there was a marginal, but nonsignificant (p > 0.05), increase in the ratio of cholic to
muricholic acid in the pool (Fig. 4D). There was no
discernible difference in the level of the apical sodium bile acid
co-transporter protein in the ileum of
Cyp27/ and Cyp27+/+
mice (data not shown). The marked contraction in bile acid pool size
resulted in a dramatic reduction in the efficiency of intestinal cholesterol absorption (from 54 to 4%) (Fig. 4E). This
reduction in turn contributed to a 2.5-fold increase in fecal neutral
sterol excretion in the Cyp27/ mice (Fig.
4F). Although the concentration of bile acids in the gallbladder bile of Cyp27/ mice was only
37% of that in Cyp27+/+ animals (Table II), the
species of bile acid were similar in both genotypes with cholic acid
predominating in both cases (data not shown).
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The marked increase in plasma VLDL-triacylglycerol concentrations in
the Cyp27/ mice prompted us to investigate
the level of expression of several key enzymes involved in hepatic
fatty acid metabolism. As shown in the first panel of Fig.
5, there was a 50% increase in the mRNA level of sterol regulatory element-binding protein-1
(SREBP-1), which preferentially activates transcription of genes
encoding the enzymes of fatty acid synthesis. Consistent with this
change were 1.7- and 2.5-fold increases in the expression of acetyl-CoA carboxylase and fatty acid synthase, respectively. There was also a
3.7-fold increase in the mRNA level for steroyl-CoA desaturase, which catalyzes the conversion of stearic to oleic acid. Although the
expression of acyl-CoA synthase was unchanged, there was a 50%
increase in the mRNA level for acyl-CoA oxidase.
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These northern analysis data are consistent with the various metabolic
measurements described in Fig. 6. These
show that in the Cyp27/ mice there was a
2.4-fold increase in hepatic triacylglycerol concentrations (Fig.
6A), a 70% increase in hepatic fatty acid synthesis (Fig.
6B), and a marked shift in hepatic fatty acid composition
characterized by about a 70% increase in the proportion of oleic
(18:1) to stearic acid (18:0) (Fig. 6C). Together, the data
in Figs. 5 and 6 reveal a major association between sterol 27-hydroxylase and hepatic fatty acid and triacylglycerol
metabolism.
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The remaining set of mRNA measurement data defines the levels of
expression of enzymes involved in hepatic cholesterol synthesis, two of
the receptors that facilitate the uptake and intrahepatic handling of
lipoprotein cholesterol, and the liver X receptor , a nuclear
receptor that regulates the expression of several lipogenic enzymes
(Fig. 7). The first panel shows a 50%
increase in the expression of SREBP-2, which activates transcription of genes encoding the enzymes of cholesterol biosynthesis. Consistent with
this result was a 2.8- and 2.7-fold increase in the mRNA levels for
HMG-CoA synthase and HMG-CoA reductase, respectively. There was also a
50% increase in the expression of the LDL and high density lipoprotein
(scavenger receptor, class B, type 1) receptors, but little change in
the mRNA level for the liver X receptor .
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The rates of cholesterol synthesis in the liver and extrahepatic
organs, measured in vivo in matching groups of
Cyp27/ and Cyp27+/+
mice, are shown in Fig. 8. In the
Cyp27/ mice there were 4-fold increases in
cholesterol synthesis in the liver and adrenal gland (Fig.
8A). Significant increases in synthesis also occurred in the
lung (2.9-fold), spleen (2.4-fold), and small intestine (1.8-fold). In
contrast, there was little or no change in the rates of sterol
synthesis in the other extrahepatic organs, including the residual
carcass, which comprised mainly skin, muscle, adipose tissue, stomach,
and large intestine. There was a trend toward lower rates of sterol
synthesis in the kidneys of Cyp27/ mice.
Summation of the rates of synthesis per whole organ (inset Fig. 8B) showed that whole animal sterol synthesis in the
Cyp27/ mice was 2.5-fold greater than in the
Cyp27+/+ controls. About 90% of the increase in
whole body synthesis was attributable to hepatic synthesis. In contrast
to the variable effect of CYP27 deficiency on sterol synthesis in the
extrahepatic organs, little or no change in the rates of fatty acid
synthesis occurred in tissues such as the adrenal gland, lung, or small intestine (data not shown).
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The final study investigated whether feeding cholic acid to the
Cyp27/ mice would ameliorate the metabolic
changes manifest in these animals. When fed only the basal diet,
Cyp27+/+ and Cyp27/
mice had 5.2 ± 0.2 (n = 7) and 7.2 ± 0.2 (n = 9) g of liver per 100 g of body weight,
respectively. However, in matching Cyp27/
mice fed a diet supplemented with cholic acid for 10 days, relative liver weight contracted significantly to 5.9 ± 0.1 g/100 g of body weight. In contrast, adrenal gland enlargement was not reversed by
cholic acid feeding. Thus, on the basal diet, the
Cyp27+/+ and Cyp27/
mice had 14.4 ± 0.7 (n = 5) and 22.8 ± 1.3 (n = 5) mg of adrenal/100 g of body weight,
respectively. In the cholic acid-fed Cyp27/
mice this value remained at 22.8 ± 1.6 (n = 5)
mg/100 g of body weight. The hepatomegaly in the
Cyp27/ mice was also almost fully reversed
when chenodeoxycholic acid was fed in place of cholic acid (data not shown).
The metabolic effects of cholic acid supplementation in the
Cyp27/ mice are shown in Fig.
9. This treatment restored intestinal cholesterol absorption to normal levels (Fig. 9A), and
suppressed the rate of sterol synthesis in the adrenal gland (Fig.
9B) and liver (Fig. 9C) to values significantly
below those seen in Cyp27+/+ mice fed the basal
diet alone. Cholic acid supplementation also essentially normalized
hepatic fatty acid synthesis (Fig. 9D), and triacylglycerol
concentrations in both the liver (Fig. 9E) and plasma (Fig.
9F) of the Cyp27/ mice.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Several major findings from these studies warrant discussion. The
first of these relates to bile acid metabolism. In this regard, the
present data confirm and extend those of Rosen et al. (12).
Thus, we found that adult Cyp27/ mice fed a
basal rodent diet synthesized essentially the same types of bile acids
as did Cyp27+/+ controls, but at a markedly
reduced rate despite a pronounced up-regulation of cholesterol
7-hydroxylase. This diminished rate of synthesis was reflected in a
marked reduction in biliary bile acid concentration, and a decisive
contraction in bile acid pool size. From northern analyses, we showed
that the changes in the mRNA levels of several of the major enzymes
and related proteins involved in bile acid synthesis agreed well with
those seen in the metabolic measurements. In particular, it was clear
from the RNA blots that the absence of sterol 27-hydroxylase affected
gene regulation by the bile acid nuclear receptor, farnesoid X receptor (13-16). Thus, the mRNA level of small heterodimer partner, a
target gene of bile-acid activitated farnesoid X receptor (15, 16), was
reduced by 65%, and consequently the small heterodimer
partner-mediated repression of CYP7A1 (15) was relieved, resulting in a
nearly 10-fold increase in CYP7A1 mRNA levels. The northern blots
also showed a diminution of oxysterol 7-hydroxylase expression, and an appreciable rise in the mRNA level for CYP8B1, which fits well with the observed shift toward greater cholic acid enrichment of the
intestinal bile acid pool in the Cyp27/ mice.
One of the more interesting features of sterol 27-hydroxylase
deficiency in humans and mice is that it does not result in total
disruption of bile acid synthesis. This outcome is surprising, given
that the absence of sterol 27-hydroxylase affects both the alternate
and classic pathways of bile acid synthesis (2, 3). Recent studies by
Honda et al. (29) report that in
Cyp27/ mice there is a marked up-regulation
of microsomal triol 25-hydroxylase (29). While there are also
quantitatively less important increases in triol 26-hydroxylase and
tetrol 24S-hydroxylase activities in these animals, it is
apparent that the marked build up of 5-cholestane 3,7,12-triol levels in the hepatocyte that result from the compensatory increase in cholesterol 7-hydroxylase brings about a
secondary up-regulation of triol 25-hydroxylase. Although this mechanism seems not to occur in cerebrotendinous xanthomatosis patients
(29), it may account for the modest rate of bile acid synthesis found
here in the sterol 27-hydroxylase-deficient mouse. Two points about the
triol 25-hydroxylase pathway are noteworthy. One is that it must be
operational at or before birth because Cyp27/ pups, unlike those lacking
cholesterol 7-hydroxylase (30), require no dietary bile acid
supplementation. The other point is that although this pathway
apparently generates sufficient bile acids to maintain a pool large
enough to allow lipid absorption while the
Cyp27/ pups are nursing, it nevertheless
fails to expand bile acid pool size to normal levels in adulthood.
Hence, mature Cyp27/ mice, like those
missing cholesterol 7-hydroxylase (23), absorb very little
cholesterol, show marked compensatory increases in hepatic and
intestinal cholesterol synthesis, and excrete substantially greater
amounts of cholesterol in their feces. These adaptive changes allow
whole body cholesterol content to remain normal in both knockout mice,
even though there are significant changes in the concentration of
cholesterol in the adrenal and lung of the
Cyp27/ animals. These mice, unlike those
missing cholesterol 7-hydroxylase, also manifest a significant
increase in the relative cholesterol content of the bile. This
difference, and the potential for each of these models to spontaneously
develop cholesterol gallstones with age, requires additional study.
The second major finding reported here concerns the changes in hepatic
fatty acid metabolism and plasma VLDL-triacylglycerol concentrations
that accompany sterol 27-hydroxylase deficiency. While these
perturbations were fully reversed by cholic acid feeding, the
prevailing question is whether they are the product of CYP27 deficiency
per se, or ultimately are the result of the pronounced induction of cholesterol 7-hydroxylase and hepatic cholesterol synthesis. Marked up-regulation of both of these pathways accompanies cholestyramine feeding, a treatment that raises plasma triacylglycerol levels in humans (17-19) and rats (31), but not in
mice.2 One way to possibly
address this question would be to make similar measurements to those
described here in mice in which CYP27 has been overexpressed.
The third important finding relates to the putative role of
27-hydroxycholesterol as a regulator of cholesterol biosynthesis (20-22). The sterol synthesis data presented here represent the first
direct quantitation of the absolute rates of sterol synthesis in mice
deficient in sterol 27-hydroxylase. Together, these data do not support
the view that 27-hydroxycholesterol plays a physiologically important
role in regulating cholesterol biosynthesis in the intact animal. If
this were the case, then deletion of the CYP27 gene might be expected
to result in an increased rate of cholesterol synthesis in essentially
all tissues. Although whole body cholesterol synthesis was increased
2.5-fold in the Cyp 27/ mice, this
stimulation was not distributed uniformly across all organs. Thus,
while there was a marked up-regulation of sterol synthesis in the
liver, small intestine, lung, spleen, and adrenal, there was little or
no change in synthesis in the brain, testis, and bulk peripheral
organs, which were comprised mainly of muscle, skin, and adipose
tissue. In the case of the kidney, a modest, but non-significant,
suppression of sterol synthesis was evident in the
Cyp27/ animals. The same effect is seen in
the kidney of male mice deficient in the CYP7B1 oxysterol
7-hydroxylase (32). Although the activity of this enzyme was not
measured here, the mRNA level for hepatic CYP7B1 was reduced by
60% in the Cyp27/ mice. Hence, the tendency
for a lower rate of kidney sterol synthesis in these animals may be
more directly related to the reduced expression of oxysterol
7-hydroxylase rather than to the absence of sterol 27-hydroxlase
per se.
The cholic acid feeding experiments further suggest that the marked
increase in hepatic and adrenal sterol synthesis was not due to the
absence of 27-hydroxycholesterol in these tissues because the
restoration of intestinal cholesterol absorption that accompanied dietary cholic acid supplementation resulted in marked suppression of
cholesterol synthesis in these two organs to rates that were well below
those seen in the Cyp27+/+ mice fed only the
basal diet. This suppression should not have occurred if the increased
cholesterol synthesis in these organs under basal dietary conditions
was due solely to the absence of 27-hydroxycholesterol. Although it is
unclear why sterol 27-hydroxylase deficiency had such a variable effect
on cholesterol synthesis in the different organs, in the case of the
liver and small intestine, most, if not all, of the stimulation seen in
these two organs was likely just a compensatory response to the near
zero level of cholesterol absorption. A similar adaptive response is
seen in mice lacking cholesterol 7-hydroxylase, although in those animals there is no up-regulation of sterol synthesis in the adrenal, spleen, and lung (23).
Finally, perhaps the most surprising new finding concerns the effect of sterol 27-hydroxylase deficiency on adrenal mass and cholesterol homeostasis. Presumably, the increase in adrenal size, cholesterol synthesis, and cholesterol content represented a compensatory response needed to maintain plasma corticosterone concentrations within a normal range. Although cholic acid feeding abolished the increase in adrenal sterol synthesis, it did not reverse adrenal gland enlargement, which, by histological analyses was found to reflect cortical cell hypertrophy. While it is not known whether adrenal enlargement occurs in cerebrotendinous xanthomatosis patients, one study reported no evidence of adrenal insufficiency in five out of six such individuals (8).
In summary, these studies have shown that while the loss of sterol
27-hydroxylase results in qualitatively the same adaptive changes in
cholesterol balance across the whole body as those previously
documented for mice lacking cholesterol 7-hydroxylase, it also has a
significant impact on hepatic fatty acid and triacylglycerol metabolism, and adrenal cholesterol homeostasis. Clearly, further studies in the Cyp27/ mouse are needed to
determine exactly how the expression of sterol 27-hydroxylase
influences each of these processes. In addition, the deletion of other
regulatory proteins involved in sterol metabolism in mice also
deficient in sterol 27-hydroxylase should be pursued. For example,
deleting the cholesterol 7-hydroxylase gene in
Cyp27/ mice will potentially provide new
information regarding other alternative pathways of bile acid
synthesis, while removing the LDLR from mice lacking sterol
27-hydroxylase might unmask substantially greater hyperlipidemia than
is seen in mice without the CYP27 gene. It will also be
important to study both male and female animals in the future, given
the marked gender differences in some aspects of cholesterol and bile
acid metabolism that have been repeatedly documented in this species
(25, 32-34).
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ACKNOWLEDGEMENTS |
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We thank Brian Jefferson, Jeffrey Graven, Elizabeth Moore, Amanda Fletcher, Stephen Ostermann, and Monti Schneiderman for excellent technical assistance; Margrit Schwarz for measurement of apical sodium bile acid co-transporter protein in brush-border membranes; and Merikay Presley for expert preparation of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL 09610 and HL 20948, the Moss Heart Fund, Robert A. Welch Foundation Grant I-0971, the Perot Family Foundation, the William M. Keck Foundation, the Howard Hughes Medical Institute (to J. J. R.), and the American Digestive Health Foundation Industry Research Scholar Award (to J. D. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Current address: Merck Research Laboratories, Rahway, NJ 07090.
To whom correspondence should be addressed: Dept. of Internal
Medicine, The University of Texas Southwestern Medical Center at
Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-8887. Tel.: 214-648-8773; Fax: 214-648-9761; E-mail:
stephen.turley@utsouthwestern.edu.
Published, JBC Papers in Press, September 22, 2000, DOI 10.1074/jbc.M007653200
2 S. D. Turley and J. M. Dietschy, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: CYP, cytochrome P450; ACTH, adrenocorticotrophic hormone; SREBP-1 and SREBP-2, sterol regulatory element-binding protein-1 and -2; VLDL, very low density lipoprotein; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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