|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 273, Issue 24, 14805-14812, June 12, 1998
From the Sterol 27-hydroxylase is important for the
degradation of the steroid side chain in conversion of cholesterol into
bile acids and has been ascribed a regulatory role in cholesterol
homeostasis. Its deficiency causes the autosomal recessive disease
cerebrotendinous xanthomatosis (CTX), characterized by progressive
dementia, xanthomatosis, and accelerated atherosclerosis.
Mice with a disrupted cyp27
(cyp27 It is evident that sterol 27-hydroxylase is more important for bile
acid synthesis in mice than in humans. The results do not support the
contention that 27-hydroxylated steroids are critical for
maintenance of cholesterol homeostasis or levels of vitamin D
metabolites in the circulation.
Sterol 27-hydroxylase is a mitochondrial species of cytochrome
P-450 with a broad tissue and organ distribution and with a broad
substrate specificity (for a review, see Ref. 1). The enzyme is
important for bile acid biosynthesis but has also been ascribed a role
in connection with cholesterol removal from extrahepatic tissues, in
regulation of cholesterol homeostasis, and in metabolism of vitamin
D.
Sterol 27-hydroxylase is responsible for the first step in the
degradation of the steroid side chain in connection with bile acid
biosynthesis in the liver. In the major pathway to bile acids in
mammalian liver, 7 Sterol 27-hydroxylase has also 25-hydroxylase activity toward vitamin D
(4) and 1 27-Hydroxycholesterol, formed from cholesterol by the sterol
27-hydroxylase, is a potent down-regulator of cholesterol synthesis in
cultured cells (for reviews see Refs. 7 and 8). On the basis of this,
and on the basis of studies with sterol 27-hydroxylase inhibitors,
sterol 27-hydroxylase has been suggested to have a regulatory role in
cholesterol homeostasis. The relative importance of this mechanism is
controversial, however. Based on studies with cholesterol specifically
deuterium-labeled in the 27-position, which retards the rate of
27-hydroxylation, we showed that sterol 27-hydroxylase activity is of
little or no direct importance for cholesterol-induced suppression of
cholesterol synthesis in mouse liver (9).
It is now well documented that patients with the rare disease
cerebrotendinous xanthomatosis
(CTX)1 have a deficiency of
sterol 27-hydroxylase (for a review, see Ref. 10). Recently, a number
of mutations have been defined in the sterol 27-hydroxylase gene of
these patients (11-20). As a result of the enzymatic defect, patients
with CTX have a reduced synthesis of bile acids, in particular
chenodeoxycholic acid (10). Cholic acid is formed in these patients by
a pathway involving 25-hydroxylated bile alcohols as intermediates.
Under normal conditions this alternative pathway is of little or no
importance, both in rats and man (21, 22). Due to the reduced formation
of bile acids, the negative feedback of the cholesterol
7 Patients with CTX have normal cholesterol levels in the circulation
(17). Despite this they develop xanthomas and premature atherosclerosis. The possibility has been discussed that this may be
due to the reduced elimination of cholesterol from macrophages by the
sterol 27-hydroxylase (1-3).
Disturbances in vitamin D metabolism have been described in a few cases
of CTX (23), but this does not seem to be a general finding.
Studies on patients with CTX have provided important information about
the role of the sterol 27-hydroxylase in man. The importance of this
enzyme for bile acid formation, cholesterol homeostasis, and vitamin D
metabolism may, however, be different in different species. A lack of
an enzyme may activate compensatory mechanisms that may be different in
different species. In order to evaluate further the role of the sterol
27-hydroxylase in mammals, we have produced and characterized mice
deficient of the enzyme.
Cloning of the Mouse cyp27 and Construction of the Targeting
Plasmid--
Oligonucleotides complementary to the putative exon 3 of
the rat cyp27 were prepared (24-26). Oligonucleotides e
(5'-AGGACAGCAGTGGTACCATCTGCG-3') and f
(5'-CTTCCAAGGCAAGGTGGTAAAGAAGA-3') were used to amplify 197 bp
containing the putative mouse exon 3 (Fig. 1). The PCR product was used
to probe a mouse 129SV Genomic Library (Lambda Fix2, Stratagene, La
Jolla, CA). A positive clone designated 24(1), which includes 13.1-kb
mouse cyp27 gene sequences, was isolated. A targeting
construct was made to replace a 71-bp BamHI fragment in exon
8 with a neo sequence (see Ref. 27, Fig. 1). To do so, a 7-kb
BamHI fragment, which includes sequences homologous to the
human CYP27 exons 2-8, was subcloned into a unique
BamHI site of the pPNT plasmid and designated pPNT-7. A
1.1-kb BamHI/NotI fragment, which includes the 3'
end of the putative exon 8, exon 9, and the 3'-untranslated sequences,
was purified from clone 24(1), subcloned into pSC301 plasmid, and
designated pSC301-S. The BamHI/SalI 1.1-kb band
of pSC301-S was then subcloned into the pPNT-7 plasmid that was
previously digested by XhoI/NotI to make
pCYP27T1. In the targeting construct the deletion of a 71-bp fragment
from the putative mouse cyp27 exon 8 is located upstream to
the putative heme-binding site, highly conserved in human (28) and
rabbit (29), and crucial for the activity of P-450 enzymes (30). For
verification, the junctions between the neo and the cyp27 sequences were sequenced and compared with the
published cyp27 rat cDNA (24).
Gene Targeting and Screening of the Homologous
Recombinants--
The targeting plasmid was linearized by cleavage
with NotI. ES cell cultures and electroporations were
performed as described (31). Ten to twelve days after electroporation,
colonies resistant to 200 µg/ml G418 and to 2 µM
ganciclovir (Syntex, Palo Alto, CA) were picked and passaged in clonal
fashion. To screen the cells with correct targeting, a probe that
identifies the 1.1-kb short arm was prepared by PCR using
oligonucleotide a (5'-TGGTTCCCACAAACTCCCGGATCAT-3'), which is
complementary to the putative rat 5' exon 7 sequences, and
oligonucleotide T3, which is complementary to the vector short arm
sequence. Southern blot analysis resulted in identification of a 9- and
a 12-kb XbaI fragment for the targeted and for the non-targeted alleles, respectively. To verify the planned disruption of
the gene, a probe from the putative mouse exon 3 was prepared and used
in Southern blotting to identify a 5- and a 16-kb XbaI fragment from the targeted and non-targeted alleles, respectively (Fig.
2a).
Generation of Germ Line Competent Chimeras and Mouse
Breeding--
Approximately 10 ES cells were injected into the
blastocyte cavity of C57BL/6J embryos. Surviving blastocytes were
transferred to the uteri of pseudo-pregnant CD-1 females. An average of
two to three transfers were made per cell line. Chimeric animals were further bred to C57BL/6J animals to determine their germ line competency. F1 animals heterozygote for the cyp27 disruption
were crossed to produce mice homozygote to the knock-out
cyp27.
PCR Genotyping of the F2 Siblings--
To genotype the F2
siblings, a double PCR reaction was performed. PCR was carried out
using oligonucleotide a (5'-TGGTTCCCACAAACTCCCGGATCAT-3') mapped to the
5' end of the putative exon 7, oligonucleotide b (5'-CCATAGCCAAAGGGCACAGAGCCAA-3') mapped to the 3' end of the putative
exon 8, and oligonucleotide c (5'- ATCGCATCGAGCGAGCACGTACT-3') complementary to neo sequences (Fig. 1). The PCR products of
the targeted allele and the normal gene were 1- and 0.3-kb,
respectively. To avoid the out competition of the 1-kb by the 0.3-kb
PCR product, a triple amount of oligonucleotide a was used. The PCR
conditions were as follows: denaturation for 5 min at 94 °C followed
by 35 cycles of 94 °C for 1 min, 65 °C for 1 min, and 75 °C
for 1 min. To avoid PCR artifacts in genotyping, two additional,
independent, reactions were carried out to identify the targeted allele
and the normal allele. The oligonucleotides used for these reactions were c and d (5'-CCACCATGATATTCGGCAAGCAGG-3', and a and b,
respectively) (Fig. 3).
RNA Blot Analysis--
For visualization of cyp27
mRNA the following protocol was used. RNA was extracted from mice
livers using Trizol reagent (Life Technologies, Inc.), quantified by
spectrophotometry and visualized following electrophoresis on a 1.5%
agarose gel stained by ethidium bromide. Twenty µg of total RNA were
loaded on a 1.6% agarose gel, electrophoresed, and blotted as
described (32). The blots were hybridized using a mouse
cyp27 exon 3 or a Syrian Hamster HMG-CoA reductase cDNA
probe labeled by nick translation (Life Technologies, Inc.).
Immunoblot Analysis--
Livers were homogenized in a lysis
buffer containing 20 mmol of Tris-HCl, pH 7.7, 1 mmol of EDTA, and 0.25 M sucrose and centrifuged at 1000 × g for
5 min. The supernatant was precipitated at 5000 × g
for 10 min and the mitochondrial fraction resuspended in the lysis
buffer. The proteins were quantified using the Bio-Rad protein assay
(Bio-Rad, Munchen, Germany). A total of 75 µg of protein was loaded
on a 10% SDS-polyacrylamide gel and electrophoresed for 12 h at
15 mA. Electroblotting to cellulose nitrate (Schleicher & Schuell,
Dassel, Germany) was carried out overnight at 20 V. Blocking of the
membrane was carried out in a buffer containing 1.34 M
NaCl, 0.03 M KCl, and 0.25 M Tris (TBS buffer)
for 5 h at 4 °C. Incubation with primary polyclonal antibody
directed against the mouse sterol 27-hydroxylase, extracted from rabbit serum (a gift from Dr. David W. Russell), was diluted 1/1000 in the
blocking buffer (TBS) that also contained 0.05% Nonidet P-40 (Sigma).
Incubation was carried out overnight at 4 °C. The filter was washed
four times for 15 min each in a TBS buffer containing 0.1% SDS, 0.1%
Nonidet P-40, and 0.5% deoxycholic acid sodium salt (Sigma-Aldrich
Chemie, GmbH). Incubation with 1/2000 secondary antibody, anti-rabbit
IgG horseradish peroxidase-linked whole antibody from donkey (Amersham
Pharmacia Biotech, Buckinghamshire, UK) was carried-out for 2 h at
room temperature. Development was carried out using the Amersham ECL
detection reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK).
The film was exposed for 3 s and developed.
Pathological Analysis--
Animals were maintained at the
Hadassah-Hebrew University animal facility in a specific pathogen-free
unit. Breeding pairs were set up to provide normal heterozygote and
homozygote siblings of the targeted cyp27
Markedly Reduced Bile Acid Synthesis but Maintained Levels of
Cholesterol and Vitamin D Metabolites in Mice with Disrupted Sterol
27-Hydroxylase Gene*
§,
,,, and Department of Molecular Virology, Department
of Pathology and Laboratory Medicine, University of North Carolina,
Chapel Hill, North Carolina 37599-7525, the ** Department of
Nephrology, Hypertension and Genetics, Max-Delbrück-Center,
13122 Berlin, Germany, and the Department
of Medical Laboratory Sciences and Technology, Division of Clinical
Chemistry, Karolinska Institute, Huddinge University Hospital,
SE-141 86 Huddinge, Sweden
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
/) had normal plasma levels of
cholesterol, retinol, tocopherol, and 1,25-dihydroxyvitamin D. Excretion of fecal bile acids was decreased (<20% of normal), and
formation of bile acids from tritium-labeled 7
-hydroxycholesterol was less than 15% of normal. Compensatory up-regulation of hepatic cholesterol 7-hydroxylase and hydroxymethylglutaryl-CoA reductase (9- and 2-3-fold increases in mRNA levels, respectively) was
found. No CTX-related pathological abnormalities were observed. In CTX, there is an increased formation of 25-hydroxylated bile alcohols and
cholestanol. In bile and feces of the
cyp27/ mice only traces of bile alcohols
were found, and there was no cholestanol accumulation.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-hydroxylation of cholesterol is the first and
rate-limiting step, and 27-hydroxylation occurs at a later stage with a
7-hydroxylated intermediate as substrate. In an alternative pathway,
27-hydroxylation of cholesterol is the first step in the sequence,
followed by a 7-hydroxylation catalyzed by a specific oxysterol
7-hydroxylase (1). In a recently discovered minor pathway,
extrahepatic sterol 27-hydroxylase converts cholesterol into
27-hydroxycholesterol or 3
-hydroxy-5-cholestenoic acid (2, 3). The
latter compounds are transported to the liver and converted into bile
acids.
-hydroxylase activity toward 25-hydroxyvitamin D (5). Since
there is also a microsomal cytochrome P-450 that catalyzes
25-hydroxylation of vitamin D (6), the relative importance of the
sterol 27-hydroxylase is not known.
-hydroxylase is reduced, resulting in an up-regulation of this
enzyme. As a consequence, gram amounts of bile alcohols are formed and
excreted in bile and feces. Another consequence of the disease is
excess formation and accumulation of cholestanol (10). Most of this formation seems to be secondary to the accumulation of an intermediate in bile acid biosynthesis, 7-hydroxy-4-cholesten-3-one. This steroid
can be converted into cholestanol by hepatic enzymes.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
70 °C. Semi-quantitative analysis of the
relative amount of mRNA was estimated by densitometry.
/
mice. Weaning was carried out at the age of 3 weeks. The animals were
genotyped and grouped. Each group consisted of four to six male
littermates. At the end of the experiment the animals were sacrificed,
and their plasma and bile were collected and gross pathology and
histology were performed.
water, tap
water (5 min), counterstained with hemalaun, rinsed in tap water,
dehydrated, and coverslipped as described above. Heart and aorta were
processed separately for lipid staining with Sudan III. The heart was
taken out, fixed in 10% buffered formalin for at least 48 h,
incubated at 37 °C in 5% gelatin for 2.5 h, in 10% gelatin
for 2.5 h and in 20% gelatin overnight. The hearts were put into
a gelatin block and frozen in isopentane (35 °C) for further
processing in a cryostat (Leica, Frigocut). In brief, the hearts were
cut in 10-µm thickness until the 3-valve cusps at the junction of the
aorta to the heart and an aorta that was round in shape was seen.
Several sections were taken at this level, stained with Sudan III, and
counterstained with hemalaun. The sections were examined under a light
microscope. The whole aorta was fixed in 10% buffered formalin, rinsed
in tap water, cut open, blocked with needles, and stained with Sudan
III. The aortas were examined for atherosclerotic plaques. A total of
six animals were examined by Pathology Associates Int. (Frederick, MD,
project no. 3116-101 and 97-111-09), and 18 mice were analyzed at the Max-Delbrück-Center (Berlin, Germany).
Vitamin Analysis-- Vitamin A and vitamin E levels were analyzed by high pressure liquid chromatography as described (35). 25-Hydroxyvitamin D was analyzed by a radioimmunoassay (36) using a kit from Nichols Institute Diagnostics. The accuracy of this method has been ascertained by a method based on isotope dilution-mass spectrometry (37). 1,25-Dihydroxyvitamin D was analyzed with a radioreceptor method (38) using a kit from Incstar (Incstar Corp., Stillwater, MN).
Lipid and Steroid Analysis--
Cholesterol and triglycerides in
serum were analyzed with standard enzymatic photometric methods with
use of commercial kits (Boehringer Mannheim, Germany). Cholesterol in
feces was analyzed by isotope dilution-mass spectrometry with
2H6-labeled cholesterol as internal standard
(39). Cholesterol levels in tissue extract were analyzed by the same
method. The tissues were frozen in liquid nitrogen, pulverized
mechanically, and extracted with chloroform/methanol (2:1, v/v). In
both cases, a hydrolysis step was included, and thus the method detects
both free and esterified cholesterol. Oxysterols
(7-hydroxycholesterol, 24-hydroxycholesterol, and
27-hydroxycholesterol in serum and in various organs) were analyzed by
isotope dilution-mass spectrometry with use of deuterium-labeled
internal standards as described previously (40). The procedure includes
a saponification step and thus the method includes both free and
esterified steroid. The interassay variation in this assay is below 5%
(40).
Bile Acid Analysis--
Hydrolyzed bile acids in bile and in
feces were analyzed by combined gas chromatography under the conditions
described previously (41) using a Hewlett-Packard 5970 mass specific
detection instrument equipped with a 0.33-µm phase HP-ultra 1 column.
All samples were hydrolyzed prior to extraction, methylated with
diazomethane, and trimethylsialylated prior to analysis (41). The
samples were analyzed by the repetitive scanning method for
identification of all bile acids present in the samples (cf.
Fig. 5). In addition cholic acid, chenodeoxycholic acid, deoxycholic
acid, and lithocholic acid were quantitated by isotope dilution-mass
spectrometry with use of deuterium-labeled internal standards and
selected ion monitoring as described previously (41). The other bile
acids were quantitated from the chromatogram (total ion current)
obtained in the analysis of material to which no internal deuterated
standards had been added (Fig. 5). The peak area of the trihydroxy bile
acids was compared with the peak area of cholic acid. The amount of
cholic acid had been quantitated by isotope dilution-mass spectrometry in a separate analysis as described above. The peak area of the dihydroxy bile acids was compared with the peak area of deoxycholic acid. Deoxycholic acid had been quantitated by isotope dilution-mass spectrometry in a separate experiment. In a few cases the gas chromatographic peak was found to contain contaminating interfering compounds (this was the case in the assay of the small amounts of some
muricholic acid isomers in the material from the
cyp27/ mice). In these cases a specific ion
at m/z 195, specific for 6-hydroxylated bile acids, was used
for assay.
Assay of Lathosterol-- This assay was performed with isotope dilution-mass spectrometry and utilization of deuterium labeled lathosterol as internal standard as described previously (43).
Experiments with 7-3H-Labeled
7-Hydroxycholesterol--
7-3H-labeled
7-hydroxycholesterol with a specific radioactivity of 70 × 106 cpm/mg was synthesized as described previously (44).
The steroid, about 9 × 106 cpm, was dissolved in 0.2 ml of ethanol, and this solution was added to 1 ml of 0.9% NaCl (w/v)
containing 1% bovine serum albumin (w/v). The solution was injected
intraperitoneally in one cyp27+/+ and one
cyp27/ mouse. Feces was collected during
three 24-h periods after this injection. The different fecal portions
were refluxed with ethanol for 24 h. The ethanol was evaporated,
and the material was hydrolyzed with KOH in aqueous ethanol (41). The
hydrolyzed alkaline material was diluted with water and extracted three
times with hexane. After acidification with diluted HCl, the water
phase was extracted three times with diethyl ether. Radioactivity was
measured in aliquots of the hexane phase containing neutral steroids
and of the ether phase containing bile acids.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Procedures
Results
Discussion
References
|
|---|
Cloning of the Mouse cyp27 and Construction of the Targeting
Plasmid--
Southern blot analysis on genomic DNA extracted from
SV129 female mice resulted in a partial restriction map of the mouse cyp27 and revealed the presence of a single copy gene (data
not shown). We disrupted the cyp27 gene by inserting the
neo gene and deleting 71-bp BamHI fragment from
exon 8 (Fig. 1b) which is
adjacent to the putative heme-binding site. Electroporation of the
targeting vector to ES cells resulted in colonies from which extracted
DNA revealed a ~5-kb fragment that corresponded to the targeted
allele by Southern blot analysis (Fig.
2a). These cells were used to
establish cyp27 knock-out mice (Figs. 1c and 2a). Northern blot analysis of RNA extracted from wild type
and mutant mice revealed a 1.9-2.4-kb band in the normal mice and 3.9-kb band in the homozygote cyp27/ animals
(Fig. 2b). Immunoblot analysis revealed no
immunoprecipitable protein in the homozygote mice (Fig. 2c).
Mutant mice were generated and genotyped by using three independent PCR
reactions (Fig. 3).
|
|
DNA digested with HindIII restriction endonuclease.
b, blot analysis of RNA obtained from livers of control
(1) and homozygote (2) mice. M,
molecular weight marker, Kodak IB 76200 (Eastman Kodak), the location
of the migration of ribosomal RNA is shown. Quantification (1 µg/lane) is verified using ethidium bromide staining (lower
panel). c, immunoblotting of total liver mitochondrial
protein obtained from control mouse (1) and from a
homozygote mouse (2) with polyclonal antibody is shown. The
lower panel represents Ponceau (Sigma) staining of the
filter used for hybridization and reveals the presence of similar
quantities of protein in both lanes.
|
X, HaeIII-digested DNA. The results for the wild type
(WT) and targeted (T) alleles are shown.
Pathological Analysis-- At the age of 3 weeks, the mice were weaned, and at the age of 4 weeks littermates in groups of 4-6 animals were allocated to the different cages. A total of 18 animals were used for these experiments and were fed a normal chow diet for 3 or 6 months. In general, all animals gained weight and behaved normally. No gross abnormalities were detected. Also a detailed histological analysis of all major organs and the vascular system revealed no abnormal findings. Diffuse hepatocytic cytoplasmic pallor grade 2-3 was observed, which is a normal finding in well-nourished animals.
Formation of Bile Acids in cyp27/ Mice--
With
the use of combined gas chromatography-mass spectrometry as described
under "Experimental Procedures," the concentration of bile acids in
a sample of hydrolyzed bile from a cyp27/
mouse was found to be only about 0.9 mg/ml as compared with about 8 mg/ml in a sample from a cyp27+/+ mouse. As
shown in Table I, cholic acid was the
dominating bile acid, and the amount of trihydroxy bile acids was
almost 20-fold higher than the amount of dihydroxy bile acids in both
bile samples.
|
/
mouse were also analyzed by electrospray mass spectrometry. As shown in
Fig. 4, the major peak both in the
analysis of the cyp27+/+ and in the analysis of
the cyp27/ appeared with a m/z at
514, corresponding to taurine conjugate of trihydroxy bile acids. In
addition, there was a minor peak at m/z 498, corresponding
to taurine conjugate of dihydroxy bile acids. In the analysis of the
bile sample from the cyp27/ mouse, small
peaks also appeared at m/z 531 and m/z 545. Most likely, these peaks correspond to sulfate ester of dihydroxy bile acid
and monodihydroxy bile acid. Interestingly, trace amounts of compounds
with m/z 611, 627, and 643 were present in the bile sample
from the cyp27/ mouse. These peaks
correspond to quasimolecular ions of glucuronides of bile alcohols
containing 27-carbon atoms and four, five, and six hydroxyl groups
(42). No such compounds could be detected in the analysis of the bile
sample from the cyp27+/+ mouse.
|
/ mice was also confirmed by combined
gas chromatography-mass spectrometry of feces obtained from four
cyp27+/+ and four
cyp27/ mice (Fig.
5 and Table I). As shown in Table I, the
excretion of bile acids in feces was about 78 µg/g in the
cyp27/ mouse and about 430 µg/g in the
cyp27+/+ mouse. The composition of the fecal
bile acids was similar in the two groups, although the relative amount
of dihydroxy bile acids was somewhat higher in the
cyp27/ group (about 50%) as compared with
the cyp27+/+ group (about 30%).
|
-muricholic acid
(3 mouse. The amount of bile acids (about
400 µg/g feces) as well as the composition was very similar to those
found in feces of cyp27+/+ mice.
In order to further ascertain that there is a reduced formation of bile
acids in cyp27/ mice, one
cyp27/ mouse and one
cyp27+/+ mouse was injected with the same amount
of 7-3H-labeled 7-hydroxycholesterol. Since
7-hydroxycholesterol is an intermediate in bile acid biosynthesis
occurring after the rate-limiting step, a rapid formation of bile acids
from this compound can be expected in normal mice. In accordance with
this, 1.6 × 106 cpm were found in the bile acid
containing ether extract of the feces collected during 72 h after
the injection of 9 × 106 cpm in the
cyp27+/+ mouse. The hexane extract containing
neutral steroids contained 0.4 × 106 cpm. Only
0.2 × 106 cpm were found in the bile acid containing
extract from the cyp27/ mouse and 1.0 × 106 cpm in the extract containing neutral steroids. The
formation of radioactive bile acids from 7-3H-labeled
7-hydroxycholesterol in the cyp27/ mouse
was thus only about 13% of that in the
cyp27+/+ mouse. It should be pointed out,
however, that due to the expanded pool of 7-hydroxycholesterol in
the cyp27/ mice (Table III), the conversion
of the trace amount of labeled intermediate may not be an adequate
reflection of the conversion of the endogenous compound.
The possibility that bile acids were excreted in urine was also tested
with electrospray mass spectrometry of un-hydrolyzed urine samples from
cyp27+/+ and cyp27/
mice. No significant amounts of bile acids were seen. Small peaks at
m/z 627, 643, and 657 were seen in the analysis of urine
from cyp27/ mice but not from
cyp27+/+ mice. The latter peaks are likely to be
due to glucuronides of bile alcohols containing 27 carbon atoms and
five, six, and seven hydroxyl groups (42) (cf. above).
The low production of bile acids is likely to result in an
up-regulation of the cholesterol 7-hydroxylase due to a reduced negative feedback suppression of this enzyme. In accordance with this,
the concentration of 7-hydroxycholesterol in the liver, the kidney,
and the brain was markedly higher in the
cyp27/ mice than in the
cyp27+/ and in the
cyp27+/+ mice (Table
II). It is well established that
circulating levels of 7-hydroxycholesterol reflect the activity of
the cholesterol 7-hydroxylase in the liver (45). The concentration
of circulating 7-hydroxycholesterol was found to be 4-10-fold
higher in the cyp27/ mice than in the two
other genotypes (Table III).
|
|
-hydroxylase was up-regulated was also
confirmed by RNA blot analysis (data not shown). Thus the ratio between
the cyp7 and the actin mRNA was 2.7 ± 0.5 (n = 7) and 0.3 ± 0.1 (n = 6), in
the livers of the cyp27/ and the
cyp27+/+ mice, respectively.
Cholesterol Synthesis--
The reduced synthesis of bile acids in
the knock-out mice leads to reduced absorption of cholesterol, which
may lead to a compensatory increased synthesis. The up-regulation of
the cholesterol 7-hydroxylase can also be expected to lead to a
secondary up-regulation of the rate-limiting enzyme in cholesterol
synthesis, HMG-CoA reductase (46-48). In accordance with this, the
hepatic levels of HMG-CoA reductase mRNA was found to be 2-3-fold
higher in the knock-out mice than in the wild type (data not
shown).
/ mice was found to be 769 ± 56 ng/ml (mean ± S.E.) as compared with 231 ± 90 ng/ml in
three cyp27+/+ mice (p < 0.01, Student's t test).
Circulating Levels of Fat-soluble Vitamins--
As shown in Table
IV, the levels of vitamin A were about
the same in the wild type and in the cyp27/
mice. Vitamin E levels in the cyp27/ mice
were somewhat lower than those of the wild type, but this difference
was not significant from a statistical point of view. Levels of
25-hydroxyvitamin D were clearly higher in the
cyp27/ mice than in the wild type
(p < 0.01, Student's t test). Levels of
1,25-dihydroxyvitamin D were similar in the two groups of mice.
|
Circulating Levels of Cholesterol and Triglycerides, Fecal
Excretion of Cholesterol--
The circulating levels of cholesterol
(total cholesterol) varied between 1.5 and 3.0 mmol/liter in both the
wild type and the cyp27/ mice on a normal
diet. The triglyceride levels in the three groups of mice varied
between 0.2 and 0.8 mmol/liter.
/ mice could be expected to result in
increased fecal excretion of fat in feces. The excretion of cholesterol
was 3.4 ± 0.3 (n = 5) and 1.9 ± 0.2 mg/g
(n = 5) feces in the cyp27/
and the cyp27+/+ mice, respectively
(p = 0.05).
Levels of Cholestanol in Liver and Circulation--
The ratio
between cholestanol and cholesterol was found to be 0.009 ± 0.003, 0.011 ± 0.003, and 0.016 ± 0.006 in livers of the
cyp27/, the
cyp27+/, and the
cyp27+/+ mice, respectively. Similar ratios were
found in the circulation (not shown).
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Procedures
Results
Discussion
References
|
|---|
Consequences of the Lack of Sterol 27-Hydroxylase for Bile Acid
Synthesis--
It is evident that a lack of the sterol 27-hydroxylase
has a more dramatic effect on production of bile acids in mice than in
humans. In patients with CTX the enzyme deficiency leads to a markedly
reduced production of chenodeoxycholic acid, whereas production of
cholic acid is normal or almost normal (10). The latter production is
most probably due to a microsomal 25-hydroxylase, active on
5-cholestane-3,7,12-triol. The product
5-cholestane-3,7,12,25-tetrol can be converted into cholic
acid in a pathway involving cleavage of acetone from the steroid side
chain (cf. above). The microsomal 25-hydroxylase is
considerably more active in human liver microsomes than in rat liver
microsomes (51). In preliminary experiments the activity of this enzyme
was found to be about equal in rat and mouse liver. The relatively low
activity of the microsomal 25-hydroxylase in mouse liver may thus
explain why cyp27/ mice are unable to
compensate for the lack of the sterol 27-hydroxylase by formation of
bile acids through the alternative 25-hydroxylase mechanism.
/ mice might in part have been formed
by the sterol 25-hydroxylase pathway. Small amounts of bile alcohols
were found in bile and urine of cyp27/ mice,
and trace amounts of 5-cholestane-3,7,12,25-tetrol could in
fact be identified in feces of one of the
cyp27/ mice.
In contrast to humans, rat liver contains a microsomal 26-hydroxylase
that is able to catalyze 26-hydroxylation of
5-cholestane-3,7,12-triol (52). The product,
5-cholestane-3,7,12,26-tetrol, being an isomer to
5-cholestane-3,7,12,27-tetrol, might well be further
converted into cholic acid. Whether or not mouse liver microsomes
contain such a 26-hydroxylase is not known with certainty. If such
enzyme activity exists, it may also explain part of the small amounts
of bile acids formed in the cyp27/ mice. At
the present state of knowledge, we also cannot exclude that a sterol
24-hydroxylase present in the brain (53) may be of some importance for
the conversion of cholesterol into bile acids in the
cyp27/ mice.
In accordance with the findings in patients with CTX, the lack of the
sterol 27-hydroxylase resulted in a compensatory up-regulation of the
cholesterol 7-hydroxylase in the cyp27/
mice. The circulating levels of 7-hydroxycholesterol were thus markedly elevated, and RNA blot analysis showed about 9-fold increased concentrations of cholesterol 7-hydroxylase mRNA in the
liver.
Formation of Cholestanol in cyp27/ Mice--
One
of the characteristics of CTX is an increased production of cholestanol
(10). This production is most probably linked to the accumulation of
7-hydroxylated intermediates in bile acid biosynthesis, in
particular 7-hydroxy-4-cholesten-3-one. Conversion of
7-hydroxy-4-cholesten-3-one into cholestanol requires a critical dehydration of this steroid by a microsomal dehydratase (54). We have
shown that the activity of this enzyme is severalfold higher in human
liver than in rat liver (54). In preliminary experiments we showed that
the activity of this dehydratase is about similar in rat and in mouse
liver. The relatively low activity of this enzyme may thus explain why
the cyp27/ mice did not have higher levels
of cholestanol than the cyp27+/+ mice.
Consequences of the Lack of Sterol 27-Hydroxylase for Transport of
Cholesterol from Peripheral Tissues--
Patients with CTX are known
to develop premature atherosclerosis despite normal circulating levels
of cholesterol (10). It has been suggested that this could be a
consequence of the lack of the sterol 27-hydroxylase in macrophages in
these patients (1-3). The sterol 27-hydroxylase is thus able to
eliminate cholesterol from macrophages by converting it into
27-hydroxycholesterol and 3-hydroxy-5-cholestenoic acid that are
easily excreted from the cells. The general importance of this
mechanism is reflected in the very high levels of sterol 27-hydroxylase
in human macrophages (1, 2) and in the high flux of 27-oxygenated
C27 steroids from extrahepatic tissues to the liver in
humans (3). The latter flux is likely to be of similar magnitude in
mice and in humans, as judged from the relatively high levels of
circulating 27-hydroxycholesterol (Table III). Under the conditions
employed, however, the cyp27/ mice did not
produce visible xanthomas or atheromas. At the present state of
knowledge, it cannot be excluded that the accumulation of cholestanol
and/or C27 bile alcohols that are seen in CTX patients but
not in the cyp27/ mice is the most critical
factor for the development of xanthomas and atheromas in CTX
patients.
Consequences of the Lack of the Sterol 27-Hydroxylase for
Cholesterol Synthesis and Homeostasis--
27-Hydroxycholesterol is
known to be a potent down-regulator of HMG-CoA reductase in cultured
cells (7, 8). The importance of this mechanism is, however,
controversial. If the sterol 27-hydroxylase has a key role in
cholesterol homeostasis, a lack of the enzyme would be expected to be
associated with a markedly increased synthesis of cholesterol and
elevated circulating levels of cholesterol in the circulation. CTX
patients are, however, known to have normal circulating levels of
cholesterol, and this was found to be the case also in the present
cyp27/ mice. Patients with CTX are known to
have an overall increased cholesterol synthesis, most probably
secondary to the up-regulated cholesterol 7-hydroxylase (10). That
the increased cholesterol synthesis in the present sterol
27-hydroxylase-deficient mice is secondary to the up-regulated
cholesterol 7-hydroxylase seems likely in view of the finding that
the synthesis of the latter enzyme was increased much more than the
synthesis of HMG-CoA reductase. The up-regulated hepatic HMG-CoA
reductase may be a compensation for a consumption of cholesterol due to
the highly up-regulated cholesterol 7-hydroxylase (cf.
Ref. 48).
Consequences of the Lack of the Sterol 27-Hydroxylase for
Metabolism of Vitamin D--
Sterol 27-hydroxylase is able to
25-hydroxylate vitamin D (4). Since the liver mitochondrial fraction of
a patient with CTX has been found to possess some 25-hydroxylase
activity toward vitamin D (55) and since the microsomal fraction of
liver homogenate also contains this activity (6), the relative
importance of the sterol 27-hydroxylase for this reaction is difficult
to evaluate. Reduced bone density and reduced circulating levels of
25-hydroxyvitamin D and 24,25-dihydroxyvitamin D have also been
reported in some CTX patients (23). Other patients have normal levels
of 25-hydroxyvitamin D, however
(10).2 A serum pool from
three cyp27/ mice had somewhat higher
concentrations of 25-hydroxyvitamin D than a serum pool from three
cyp27+/+ mice. In view of this, sterol
27-hydroxylase seems to be of little importance for 25-hydroxylation of
vitamin D in mice. Recently it was shown that sterol 27-hydroxylase has
some 1-hydroxylase activity toward 25-hydroxyvitamin D3
(5). The circulating concentration of 25-dihydroxyvitamin D was,
however, somewhat higher in cyp27/ mice than
in cyp27+/+ mice, whereas the corresponding
concentrations of 1,25-dihydroxyvitamin D were similar in the two
groups of mice. Thus it seems less likely that sterol 27-hydroxylase is
of importance in formation of 25-hydroxyvitamin D and
1,25-dihydroxyvitamin D in this species.
Consequences of the Reduced Formation of Bile Acids in
cyp27/ Mice--
Surprisingly, the markedly reduced
production of bile acids in the cyp27/ mice
was not associated with any symptoms of malabsorption. The content of
cholesterol and fatty acids in feces was thus only moderately
increased, and the circulating levels of the fat-soluble vitamins A and
E were normal or only slightly reduced. It is interesting to compare
this situation with the situation in mice with a disruption of the
cyp7 (56). During the first 4 weeks of life, these mice had
a severe fat malabsorption with very low circulating levels of
25-hydroxyvitamin D3 and vitamin E and high content of
lipids in stool. After this period a normalization occurred, most
probably due to induction of an oxysterol 7-hydroxylase in the
liver. The adult cyp27/ mice had an
excretion of bile acids in stool that was reduced by about 80% as
compared with the wild type. Evidently, this low degree of production
of bile acids is sufficient to prevent fat malabsorption.
Is the Cyp 27/ Mouse Suitable as an Animal Model
for CTX?--
It is evident that a disruption of the
cyp27/ gene in a mouse on a normal diet does
not lead to the metabolic, neurologic, and vascular disturbances found
in patients with a corresponding genetic defect. The compensatory
mechanism in humans (activation of the 25-hydroxylase pathway) and the
consequences of accumulation of bile acid intermediates (accumulation
of cholestanol) were not observed in the
cyp27/ mice. It is possible that the
enzymatic defect in the cyp27/ mice may be
more important when increasing the dietary intake of cholesterol. In
preliminary experiments an increased death rate was observed for
cyp27/ mice when fed an atherogenic diet. No
explanation for this finding has been obtained thus far, however.
Experiments with the goal of clarifying the importance of the sterol
27-hydroxylase in mice with additional genetic defects and under
different dietary conditions are now in progress.
| |
ACKNOWLEDGEMENTS |
|---|
We gratefully acknowledge the skillful technical assistance of Anita Lövgren and Manfred Held. We thank Kim Kluckman and Denise Lee for technical help.
| |
FOOTNOTES |
|---|
* This work was supported by grants from the Sarah and Moshe Mayer Foundation for Research, the Swedish Medical Research Council Project 3141, and Hjärt-Lungfonden and by United States Public Health Service Grant HL42630.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.
§ The first two authors contributed equally to this study.
§§ To whom correspondence should be addressed: Dept. of Medicine, Center for Research, Prevention and Treatment of Atherosclerosis, Hadassah University Hospital, 91120 Jerusalem, Israel. Tel.: 972-2-6778029; Fax: 972-2-6411136; E-mail: eranl{at}hadassah.org.il.
1 The abbreviations used are: CTX, cerebrotendinous xanthomatosis; PCR, polymerase chain reaction; bp, base pair; kb, kilobase pair; HMG-CoA, hydroxymethylglutaryl-CoA.
2 I. Björkhem and E. Leitersdorf, unpublished studies.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Procedures
Results
Discussion
References
|
|---|
- Björkhem, I. (1992) J. Lipid Res. 33, 455-471[Medline] [Order article via Infotrieve]
-
Björkhem, I.,
Andersson, O.,
Diczflusy, U.,
Sevastik, B.,
Xiu, R. J.,
Duan, C.,
and Lund, E.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
8592-8596
[Abstract/Free Full Text] -
Lund, E.,
Andersson, O.,
Zhang, J.,
Babiker, A.,
Ahlborg, G.,
Diczfalusy, U.,
Einarsson, K.,
Sjövall, J.,
and Björkhem, I.
(1996)
Arterioscler. Thromb. Vasc. Biol.
16,
208-212
[Abstract/Free Full Text] -
Ohyama, Y.,
Masumoto, O.,
Usui, E.,
and Okuda, K.
(1991)
J. Biochem. (Tokyo)
109,
389-393
[Abstract/Free Full Text] -
Axén, E.,
Postlind, H.,
Sjöberg, H.,
and Wikvall, K.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
10014-10018
[Abstract/Free Full Text] - Björkhem, I., Hansson, R., Holmberg, I., and Wikvall, K. (1979) Biochem. Biophys. Res. Commun. 90, 615-622[CrossRef][Medline] [Order article via Infotrieve]
- Javitt, N. B. (1990) J. Lipid Res. 31, 1527-1533[Abstract]
- Lund, E., and Björkhem, I. (1995) Acc. Chem. Res. 28, 241-249[CrossRef]
-
Lund, E.,
Breuer, O.,
and Björkhem, I.
(1992)
J. Biol. Chem.
267,
25092-25097
[Abstract/Free Full Text] - Björkhem, I., and Muri-Boberg, K. (1994) in The Metabolic Basis of Inherited Diseases (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valee, D., eds), pp. 2073-2100, McGraw-Hill Inc., New York
-
Cali, J. J.,
Hsieh, C. L.,
Francke, U.,
and Russell, D. W.
(1991)
J. Biol. Chem.
266,
7779-7783
[Abstract/Free Full Text] - Leitersdorf, E., Reshef, A., Meiner, V., Levitzki, R., Pressman-Schwartz, S., Dann, E. J., Berkman, N., Cali, J. J., Klapholz, L., and Berginer, V. M. (1993) J. Clin. Invest. 91, 2488-2496
-
Meiner, V.,
Marais, D. A.,
Reshef, A.,
Björkhem, I.,
and Leitersdorf, E.
(1994)
Hum. Mol. Genet.
3,
193-194
[Free Full Text] - Kim, K. S., Kubota, S., Kuriyama, M., Fujiyama, J., Björkhem, I., Eggertsen, G., and Seyama, Y. (1994) J. Lipid Res. 35, 1031-1039[Abstract]
-
Meiner, V.,
Meiner, Z.,
Reshef, A.,
Björkhem, I.,
and Leitersdorf, E.
(1994)
Neurology
44,
288-290
[Abstract/Free Full Text] - Reshef, A., Meiner, V., Berginer, V. M., and Leitersdorf, E. (1994) J. Lipid Res. 35, 478-483[Abstract]
- Leitersdorf, E., Safadi, R., Meiner, V., Reshef, A., Bjorkhem, I., Friedlander, Y., Morkos, S., and Berginer, V. M. (1994) Am. J. Hum. Genet. 55, 907-915[Medline] [Order article via Infotrieve]
- Segev, H., Reshef, A., Clavey, V., Delbart, C., Routier, G., and Leitersdorf, E. (1995) Hum. Mol. Genet. 95, 238-240
-
Watts, G. F.,
Mitchell, W. D.,
Bending, J. J.,
Reshef, A.,
and Leitersdorf, E.
(1996)
Q. J. Med.
89,
55-63
[Abstract/Free Full Text] - Nakashima, N., Sakai, Y., Sakai, H., Yanese, T., Haji, M., Umeda, F., Koga, S., Hoshita, T., and Nawata, H. (1994) J. Lipid Res. 35, 663-668[Abstract]
- Duane, W. E., Björkhem, I., Hamilton, J. N., and Mueller, S. M. (1988) Hepatology 8, 613-618[Medline] [Order article via Infotrieve]
- Duane, W. E., Pooler, P. A., and Hamilton, J. N. (1988) J. Clin. Invest. 82, 82-85
- Berginer, V. M., Salen, G., and Shefer, S. (1989) Neurol. Clin. 7, 55-64[Medline] [Order article via Infotrieve]
- Usui, E., Noshiro, M., and Okuda, K. (1990) FEBS Lett. 262, 135-138[CrossRef][Medline] [Order article via Infotrieve]
- Su, P., Rennert, H., Shayiq, R. M., Yamamoto, R., Zheng, Y. M., Addya, S., Strauss, J. F., III, and Avadhani, N. G. (1990) DNA Cell Biol. 9, 657-665[Medline] [Order article via Infotrieve]
- Tybulewicz, V. L., Crawford, C. E., Jackson, P. K., Bronson, R. T., and Mulligan, R. C. (1991) Cell 65, 1153-1163[CrossRef][Medline] [Order article via Infotrieve]
- Beck, E., Ludwig, G., Auerswald, E. A., Riess, B., and Schaller, H. (1982) Gene (Amst.) 19, 327-336[CrossRef][Medline] [Order article via Infotrieve]
-
Cali, J. J.,
and Russell, D. W.
(1991)
J. Biol. Chem.
266,
7774-7778
[Abstract/Free Full Text] -
Andersson, S.,
Davis, D. L.,
Dahlback, H.,
Jornvall, H.,
and Russell, D. W.
(1989)
J. Biol. Chem.
264,
8222-8229
[Abstract/Free Full Text] - Gonzales, F. J. (1989) Pharmacol. Rev. 40, 243-288[Medline] [Order article via Infotrieve]
-
Piedrahita, J. A.,
Zhang, S. H.,
Hagaman, J. R.,
Oliver, P. M.,
and Maeda, N.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
4471-4475
[Abstract/Free Full Text] -
McMaster, G. K.,
and Carmichael, G. G.
(1977)
Proc. Natl. Acad. Sci. U. S. A.
74,
4835-4838
[Abstract/Free Full Text] - Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
-
Gehring, M.,
Shiels, B. R.,
Northemann, W.,
de Bruijn, M. H. L.,
Kan, C. C.,
Chain, A. C.,
Noonan, D. J.,
and Fey, C. C.
(1987)
J. Biol. Chem.
262,
446-454
[Abstract/Free Full Text] -
Catagnani, G. L.,
and Bieri, J. G.
(1983)
Clin. Chem.
29,
708-712
[Free Full Text] - Belsey, R., Clark, M. B., Bernat, M., Glowacki, J., Holick, M., DeLuca, H. F., and Potts, J. T. (1974) Am. J. Med. 57, 50-56[CrossRef][Medline] [Order article via Infotrieve]
-
Lindbäck, B.,
Berlin, T.,
and Björkhem, I.
(1987)
Clin. Chem.
33,
1226-1227
[Abstract/Free Full Text] -
Hollis, B. W.
(1986)
Clin. Chem.
32,
2060-2065
[Abstract/Free Full Text] - Björkhem, I., Blomstrand, R., and Svensson, L. (1974) Clin. Chim. Acta 54, 185-193[CrossRef][Medline] [Order article via Infotrieve]
- Dzeletovic, S., Breuer, O., Lund, E., and Diczfalusy, U. (1995) Anal. Biochem. 225, 73-80[CrossRef][Medline] [Order article via Infotrieve]
- Björkhem, I., and Falk, O. (1983) Scand. J. Clin. Lab. Invest. 43, 163-170[Medline] [Order article via Infotrieve]
- Egestad, B., Pettersson, P., Skrede, S., and Sjövall, J. (1985) Scand. J. Clin. Lab. Invest. 45, 443-446[Medline] [Order article via Infotrieve]
- Lund, E., Sisfontes, L., Reihner, E., and Björkhem, I. (1989) Scand. J. Clin. Lab. Invest. 49, 165-171[Medline] [Order article via Infotrieve]
-
Danielsson, H.,
and Einarsson, K.
(1966)
J. Biol. Chem.
241,
1449-1454
[Abstract/Free Full Text] - Björkhem, I., Reihnér, E., Angelin, B., Ewerth, S., Äkerlund, J.-E., and Einarsson, K. (1987) J. Lipid Res. 28, 889-894[Abstract]
- Sudjana-Sugiaman, E., Eggertsen, G., and Björkhem, I. (1994) J. Lipid Res. 35, 319-327[Abstract]
- Äkerlund, J.-E., and Björkhem, I. (1990) J. Lipid Res. 31, 2159-2166[Abstract]
- Sudjana-Sugiaman, E., Eggertsen, G., Sjöblom, P., Maeda, Y., Okuda, K., and Björkhem, I. (1994) Biochem. Biophys. Res. Commun. 202, 896-901[CrossRef][Medline] [Order article via Infotrieve]
- Spady, D. K., Cuthbert, J. A., Willard, M. N., and Meidell, R. S. (1995) J. Clin. Invest. 96, 700-709
- Björkhem, I., Miettinen, T. A., Reihner, E., Ewerth, S., Angelin, B., and Einarsson, K. (1987) J. Lipid Res. 28, 1137-1143[Abstract]
- Björkhem, I., Gustafsson, J., Johansson, G., and Person, B. (1975) J. Clin. Invest. 55, 478-486
- Björkhem, I., and Gustafsson, J. (1973) Eur. J. Biochem. 36, 201-212[Medline] [Order article via Infotrieve]
-
Lutjohann, D.,
Breuer, O.,
Ahlborg, G.,
Nennesmo, I.,
Siden, A.,
Diczfalusy, U.,
and Björkhem, I.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
9799-9804
[Abstract/Free Full Text] - Skrede, S., Buchmann, M. S., and Björkhem, I. (1988) J. Lipid Res. 29, 157-164[Abstract]
- Oftebro, H., Björkhem, I., Skerede, S., Schreiner, A., and Pedersen, J. (1980) J. Clin. Invest. 65, 1418-1430
-
Schwarz, M.,
Lund, E. G.,
Setchell, K. D. R.,
Kayden, H. J.,
Zerwekh, J. E.,
Björkhem, I.,
Herz, J.,
and Russell, D. W.
(1996)
J. Biol. Chem.
271,
18024-18031
[Abstract/Free Full Text]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  I. Bjorkhem Are side-chain oxidized oxysterols regulators also in vivo? J. Lipid Res., April 1, 2009; 50(Supplement): S213 - S218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Heverin, S. Meaney, A. Brafman, M. Shafir, M. Olin, M. Shafaati, S. von Bahr, L. Larsson, A. Lovgren-Sandblom, U. Diczfalusy, et al. Studies on the Cholesterol-Free Mouse: Strong Activation of LXR-Regulated Hepatic Genes When Replacing Cholesterol With Desmosterol Arterioscler. Thromb. Vasc. Biol., October 1, 2007; 27(10): 2191 - 2197. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Ikonen Mechanisms for cellular cholesterol transport: defects and human disease. Physiol Rev, October 1, 2006; 86(4): 1237 - 1261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Yang, J. G. McDonald, A. Patel, Y. Zhang, M. Umetani, F. Xu, E. J. Westover, D. F. Covey, D. J. Mangelsdorf, J. C. Cohen, et al. Sterol Intermediates from Cholesterol Biosynthetic Pathway as Liver X Receptor Ligands J. Biol. Chem., September 22, 2006; 281(38): 27816 - 27826. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Inagaki, A. Moschetta, Y.-K. Lee, L. Peng, G. Zhao, M. Downes, R. T. Yu, J. M. Shelton, J. A. Richardson, J. J. Repa, et al. Regulation of antibacterial defense in the small intestine by the nuclear bile acid receptor PNAS, March 7, 2006; 103(10): 3920 - 3925. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Inoue, A.-M. Yu, S. H. Yim, X. Ma, K. W. Krausz, J. Inoue, C. C. Xiang, M. J. Brownstein, G. Eggertsen, I. Bjorkhem, et al. Regulation of bile acid biosynthesis by hepatocyte nuclear factor 4{alpha} J. Lipid Res., January 1, 2006; 47(1): 215 - 227. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Meaney Is C-26 hydroxylation an evolutionarily conserved steroid inactivation mechanism? FASEB J, August 1, 2005; 19(10): 1220 - 1224. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Dusso, A. J. Brown, and E. Slatopolsky Vitamin D Am J Physiol Renal Physiol, July 1, 2005; 289(1): F8 - F28. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Honda, G. Salen, Y. Matsuzaki, A. K. Batta, G. Xu, T. Hirayama, G. S. Tint, M. Doy, and S. Shefer Disrupted coordinate regulation of farnesoid X receptor target genes in a patient with cerebrotendinous xanthomatosis J. Lipid Res., February 1, 2005; 46(2): 287 - 296. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Dubrac, S. R. Lear, M. Ananthanarayanan, N. Balasubramaniyan, J. Bollineni, S. Shefer, H. Hyogo, D. E. Cohen, P. J. Blanche, R. M. Krauss, et al. Role of CYP27A in cholesterol and bile acid metabolism J. Lipid Res., January 1, 2005; 46(1): 76 - 85. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. F DeLuca Overview of general physiologic features and functions of vitamin D Am. J. Clinical Nutrition, December 1, 2004; 80(6): 1689S - 1696S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nishimaki-Mogami, M. Une, T. Fujino, Y. Sato, N. Tamehiro, Y. Kawahara, K. Shudo, and K. Inoue Identification of intermediates in the bile acid synthetic pathway as ligands for the farnesoid X receptor J. Lipid Res., August 1, 2004; 45(8): 1538 - 1545. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Yamasaki, S. Izumi, H. Ide, and Y. Ohyama Identification of a Novel Rat Microsomal Vitamin D3 25-Hydroxylase J. Biol. Chem., May 28, 2004; 279(22): 22848 - 22856. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. B. Cheng, M. A. Levine, N. H. Bell, D. J. Mangelsdorf, and D. W. Russell Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase PNAS, May 18, 2004; 101(20): 7711 - 7715. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Savolainen, T. J. Kotti, W. Schmitz, T. I. Savolainen, R. T. Sormunen, M. Ilves, S. J. Vainio, E. Conzelmann, and J. K. Hiltunen A mouse model for {alpha}-methylacyl-CoA racemase deficiency: adjustment of bile acid synthesis and intolerance to dietary methyl-branched lipids Hum. Mol. Genet., May 1, 2004; 13(9): 955 - 965. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Kirby, P. N. Howles, and D. Y. Hui Rate of gastric emptying influences dietary cholesterol absorption efficiency in selected inbred strains of mice J. Lipid Res., January 1, 2004; 45(1): 89 - 98. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. B. Cheng, D. L. Motola, D. J. Mangelsdorf, and D. W. Russell De-orphanization of Cytochrome P450 2R1: A MICROSOMAL VITAMIN D 25-HYDROXYLASE J. Biol. Chem., September 26, 2003; 278(39): 38084 - 38093. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Sehayek, R. Wang, J. G. Ono, V. S. Zinchuk, E. M. Duncan, S. Shefer, D. E. Vance, M. Ananthanarayanan, B. T. Chait, and J. L. Breslow Localization of the PE methylation pathway and SR-BI to the canalicular membrane: evidence for apical PC biosynthesis that may promote biliary excretion of phospholipid and cholesterol J. Lipid Res., September 1, 2003; 44(9): 1605 - 1613. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Xie, E. G. Lund, S. D. Turley, D. W. Russell, and J. M. Dietschy Quantitation of two pathways for cholesterol excretion from the brain in normal mice and mice with neurodegeneration J. Lipid Res., September 1, 2003; 44(9): 1780 - 1789. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Escher, Z. Krozowski, K. D. Croft, and D. Sviridov Expression of Sterol 27-Hydroxylase (CYP27A1) Enhances Cholesterol Efflux J. Biol. Chem., March 21, 2003; 278(13): 11015 - 11019. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Y. L. Chiang Bile Acid Regulation of Hepatic Physiology: III. Bile acids and nuclear receptors Am J Physiol Gastrointest Liver Physiol, March 1, 2003; 284(3): G349 - G356. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Pineda Torra, T. Claudel, C. Duval, V. Kosykh, J.-C. Fruchart, and B. Staels Bile Acids Induce the Expression of the Human Peroxisome Proliferator-Activated Receptor {alpha} Gene via Activation of the Farnesoid X Receptor Mol. Endocrinol., February 1, 2003; 17(2): 259 - 272. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Goodwin, K. C. Gauthier, M. Umetani, M. A. Watson, M. I. Lochansky, J. L. Collins, E. Leitersdorf, D. J. Mangelsdorf, S. A. Kliewer, and J. J. Repa Identification of bile acid precursors as endogenous ligands for the nuclear xenobiotic pregnane X receptor PNAS, January 7, 2003; 100(1): 223 - 228. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Theodoropoulos, C. Demers, J.-L. Petit, and M. Gascon-Barre High sensitivity of rat hepatic vitamin D3-25 hydroxylase CYP27A to 1,25-dihydroxyvitamin D3 administration Am J Physiol Endocrinol Metab, January 1, 2003; 284(1): E138 - E147. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Meir, D. Kitsberg, I. Alkalay, F. Szafer, H. Rosen, S. Shpitzen, L. B. Avi, B. Staels, C. Fievet, V. Meiner, et al. Human Sterol 27-Hydroxylase (CYP27) Overexpressor Transgenic Mouse Model. EVIDENCE AGAINST 27-HYDROXYCHOLESTEROL AS A CRITICAL REGULATOR OF CHOLESTEROL HOMEOSTASIS J. Biol. Chem., September 6, 2002; 277(37): 34036 - 34041. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Y. L. Chiang Bile Acid Regulation of Gene Expression: Roles of Nuclear Hormone Receptors Endocr. Rev., August 1, 2002; 23(4): 443 - 463. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Bjorkhem and U. Diczfalusy Oxysterols: Friends, Foes, or Just Fellow Passengers? Arterioscler. Thromb. Vasc. Biol., May 1, 2002; 22(5): 734 - 742. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Schwarz, D. L. Davis, B. R. Vick, and D. W. Russell Genetic analysis of intestinal cholesterol absorption in inbred mice J. Lipid Res., November 1, 2001; 42(11): 1801 - 1811. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Fu, J. G. Menke, Y. Chen, G. Zhou, K. L. MacNaul, S. D. Wright, C. P. Sparrow, and E. G. Lund 27-Hydroxycholesterol Is an Endogenous Ligand for Liver X Receptor in Cholesterol-loaded Cells J. Biol. Chem., October 12, 2001; 276(42): 38378 - 38387. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Schwarz, D. W. Russell, J. M. Dietschy, and S. D. Turley Alternate pathways of bile acid synthesis in the cholesterol 7{alpha}-hydroxylase knockout mouse are not upregulated by either cholesterol or cholestyramine feeding J. Lipid Res., October 1, 2001; 42(10): 1594 - 1603. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Sehayek, J. G. Ono, E. M. Duncan, A. K. Batta, G. Salen, S. Shefer, L. B. Neguyen, K. Yang, M. Lipkin, and J. L. Breslow Hyodeoxycholic acid efficiently suppresses atherosclerosis formation and plasma cholesterol levels in mice J. Lipid Res., August 1, 2001; 42(8): 1250 - 1256. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Hall, P. Hylemon, Z. Vlahcevic, D. Mallonee, K. Valerie, N. Avadhani, and W. Pandak Overexpression of CYP27 in hepatic and extrahepatic cells: role in the regulation of cholesterol homeostasis Am J Physiol Gastrointest Liver Physiol, July 1, 2001; 281(1): G293 - G301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Honda, G. Salen, Y. Matsuzaki, A. K. Batta, G. Xu, E. Leitersdorf, G. S. Tint, S. K. Erickson, N. Tanaka, and S. Shefer Differences in hepatic levels of intermediates in bile acid biosynthesis between Cyp27-/- mice and CTX J. Lipid Res., February 1, 2001; 42(2): 291 - 300. [Abstract] [Full Text]
|
|
|
|
 |
|
 C. Song and S. Liao Cholestenoic Acid Is a Naturally Occurring Ligand for Liver X Receptor {alpha} Endocrinology, November 1, 2000; 141(11): 4180 - 4184. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Yu, F. Wang, M. Kan, C. Jin, R. B. Jones, M. Weinstein, C.-X. Deng, and W. L. McKeehan Elevated Cholesterol Metabolism and Bile Acid Synthesis in Mice Lacking Membrane Tyrosine Kinase Receptor FGFR4 J. Biol. Chem., May 12, 2000; 275(20): 15482 - 15489. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Verrips, L. H. Hoefsloot, G. C. H. Steenbergen, J. P. Theelen, R. A. Wevers, F. J. M. Gabreels, B. G. M. van Engelen, and L. P. W. J. van den Heuvel Clinical and molecular genetic characteristics of patients with cerebrotendinous xanthomatosis Brain, May 1, 2000; 123(5): 908 - 919. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Honda, G. Salen, S. Shefer, Y. Matsuzaki, G. Xu, A. K. Batta, G. S. Tint, and N. Tanaka Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice: assay of enzyme activities by high-resolution gas chromatography;-mass spectrometry J. Lipid Res., March 1, 2000; 41(3): 442 - 451. [Abstract] [Full Text]
|
|
|
|
|
|
G. J. Schroepfer Jr. Oxysterols: Modulators of Cholesterol Metabolism and Other Processes Physiol Rev, January 1, 2000; 80(1): 361 - 554. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Kannenberg, P. Ellinghaus, G. Assmann, and U. Seedorf Aberrant Oxidation of the Cholesterol Side Chain in Bile Acid Synthesis of Sterol Carrier Protein-2/Sterol Carrier Protein-x Knockout Mice J. Biol. Chem., December 10, 1999; 274(50): 35455 - 35460. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K Shiga, R Fukuyama, S Kimura, K Nakajima, and S Fushiki Mutation of the sterol 27-hydroxylase gene (CYP27) results in truncation of mRNA expressed in leucocytes in a Japanese family with cerebrotendinous xanthomatosis J. Neurol. Neurosurg. Psychiatry, November 1, 1999; 67(5): 675 - 677. [Abstract] [Full Text]
|
|
|
|
|
|
J. Li-Hawkins, E. G. Lund, A. D. Bronson, and D. W. Russell Expression Cloning of an Oxysterol 7alpha -Hydroxylase Selective for 24-Hydroxycholesterol J. Biol. Chem., May 26, 2000; 275(22): 16543 - 16549. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Li-Hawkins, E. G. Lund, S. D. Turley, and D. W. Russell Disruption of the Oxysterol 7alpha -Hydroxylase Gene in Mice J. Biol. Chem., May 26, 2000; 275(22): 16536 - 16542. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Baes, S. Huyghe, P. Carmeliet, P. E. Declercq, D. Collen, G. P. Mannaerts, and P. P. Van Veldhoven Inactivation of the Peroxisomal Multifunctional Protein-2 in Mice Impedes the Degradation of Not Only 2-Methyl-branched Fatty Acids and Bile Acid Intermediates but Also of Very Long Chain Fatty Acids J. Biol. Chem., May 19, 2000; 275(21): 16329 - 16336. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Hosseinpour and K. Wikvall Porcine Microsomal Vitamin D3 25-Hydroxylase (CYP2D25). CATALYTIC PROPERTIES, TISSUE DISTRIBUTION, AND COMPARISON WITH HUMAN CYP2D6 J. Biol. Chem., October 27, 2000; 275(44): 34650 - 34655. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Repa, E. G. Lund, J. D. Horton, E. Leitersdorf, D. W. Russell, J. M. Dietschy, and S. D. Turley Disruption of the Sterol 27-Hydroxylase Gene in Mice Results in Hepatomegaly and Hypertriglyceridemia. REVERSAL BY CHOLIC ACID FEEDING J. Biol. Chem., December 8, 2000; 275(50): 39685 - 39692. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Rose, A. Allan, S. Gauldie, G. Stapleton, L. Dobbie, K. Dott, C. Martin, L. Wang, E. Hedlund, J. R. Seckl, et al. Neurosteroid Hydroxylase CYP7B. VIVID REPORTER ACTIVITY IN DENTATE GYRUS OF GENE-TARGETED MICE AND ABOLITION OF A WIDESPREAD PATHWAY OF STEROID AND OXYSTEROL HYDROXYLATION J. Biol. Chem., June 22, 2001; 276(26): 23937 - 23944. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Memon, A. H. Moser, J. K. Shigenaga, C. Grunfeld, and K. R. Feingold In Vivo and in Vitro Regulation of Sterol 27-Hydroxylase in the Liver during the Acute Phase Response. POTENTIAL ROLE OF HEPATOCYTE NUCLEAR FACTOR-1 J. Biol. Chem., August 3, 2001; 276(32): 30118 - 30126. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Honda, G. Salen, Y. Matsuzaki, A. K. Batta, G. Xu, E. Leitersdorf, G. S. Tint, S. K. Erickson, N. Tanaka, and S. Shefer Side Chain Hydroxylations in Bile Acid Biosynthesis Catalyzed by CYP3A Are Markedly Up-regulated in Cyp27-/- Mice but Not in Cerebrotendinous Xanthomatosis J. Biol. Chem., September 7, 2001; 276(37): 34579 - 34585. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Sehayek, S. Shefer, L. B. Nguyen, J. G. Ono, M. Merkel, and J. L. Breslow Apolipoprotein E regulates dietary cholesterol absorption and biliary cholesterol excretion: Studies in C57BL/6 apolipoprotein E knockout mice PNAS, March 28, 2000; 97(7): 3433 - 3437. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |