From the Gladstone Institute of Cardiovascular Disease,
Cardiovascular Research Institute, and ** Department of
Medicine, University of California, San Francisco, California
94141-9100 and the ¶ Department of Microbiology and Molecular
Genetics, Department of Medicine, and Molecular Biology Institute,
University of California, Los Angeles, California 90095
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ABSTRACT |
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ald, a recessive allele in AKR inbred
mice, is responsible for complete adrenocortical lipid depletion in
postpubertal males, which appears to be androgen dependent. Two recent
observations (adrenocortical lipid depletion in acyl-CoA:cholesterol
acyltransferase-deficient (Acact/
) mice and the mapping
of Acact to a region of chromosome 1 containing the
ald locus) prompted us to ask whether adrenocortical lipid
depletion in AKR mice results from an Acact mutation.
Refined genetic mapping of Acact and ald was
consistent with colocalization of these loci. Crossing
Acact
/
with AKR (ald/ald) mice yielded postpubertal male offspring characterized by adrenocortical lipid depletion, indicating that these loci are not complementational and are
therefore allelic. Immunoblotting of preputial gland homogenates demonstrated that AKR mice had an ACAT protein with a lower molecular mass than other mouse strains. Analysis of Acact cDNA
from AKR mice revealed a deletion of the first coding exon and two
missense mutations. Despite these coding sequence differences, the ACAT protein from the ald allele catalyzed cholesterol
esterification activity at levels similar to that of wild-type protein.
We speculate that the adrenocortical lipid depletion resulting from the
ald mutation is caused by an altered susceptibility of the
mutant protein to modifying factors, such as androgen production at
puberty, in an as yet undetermined manner.
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INTRODUCTION |
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About a dozen naturally occurring mutations that affect lipid metabolism in mice have been described (1). In most cases, the mutant genes have not yet been cloned. One such mutation, adrenocortical lipid depletion (ald),1 is expressed as a recessive trait in inbred AKR mice and was first described in 1955 by Arnesen (2, 3). The ald phenotype is characterized by spontaneous lipid depletion in the zona fasciculata at the time of sexual maturation (2, 4). The lipid depletion results from a loss of cholesterol esters, which are normally abundant in the adrenocortical cells, but not of free cholesterol or triacylglycerols. In male ald/ald mice, the lipid depletion is nearly total and appears to be androgen-dependent (5). Male ald/ald mice younger than 30 days have normal adrenocortical lipid content, but cholesterol ester stores are rapidly lost between 30 and 40 days, and the depletion is essentially complete by 50 days (5). In female mice, the lipid depletion is subtotal and variable (4). Prepubertal orchiectomy prevents the ald phenotype in male AKR mice, and the phenotype has been reversed by postpubertal orchiectomy (6) or by the administration of adrenocorticotropic hormone (2) or cortisone (7). The ald locus was mapped to chromosome 1 in 1976 (8). The DBA/2 strain is characterized by a similar adrenocortical lipid depletion phenotype, which appears to be due to a different genetic locus (6).
We have recently generated mice in which we have disrupted the gene
encoding acyl-CoA:cholesterol acyltransferase (ACAT) (9), an enzyme
responsible for catalyzing the formation of cholesterol esters from
free cholesterol and fatty acids (10-12). ACAT-deficient (Acact/
) mice have tissue-specific reductions in
cholesterol esters, including near-total depletion of cholesterol
esters in the adrenal cortex in both males and females (9). Coincident with these observations, we (13) and others (14) mapped the Acact gene to chromosome 1 near the ald locus.
These observations prompted us to ask whether ald results
from an Acact mutation.
In the current study, we examined whether ald and
Acact are allelic through genetic crosses of AKR and
Acact/
mice and by molecular analysis of the
Acact mRNA, protein, and enzyme activity levels in AKR
mice. The results indicate that ald is indeed due to a
naturally occurring mutant Acact allele but that the
ald protein is capable of catalyzing cholesterol
esterification normally. This finding suggests that the expression of
the adrenocortical cholesterol ester depletion phenotype in AKR mice is
caused by an altered susceptibility of the mutant protein to modifying
influences.
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MATERIALS AND METHODS |
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Mice--
All inbred strains including AKR were obtained from
The Jackson Laboratory (Bar Harbor, ME). Acact/
mice (9)
were of mixed C57BL/6 and 129/Sv genetic background and were genotyped with the polymerase chain reaction (PCR) as described (9). Mice were
housed in a pathogen-free barrier facility operating on a 12-h
light/12-h dark cycle and were fed rodent chow (Ralston Purina, St.
Louis, MO).
Genetic Mapping of ald and Acact-- Colocalization of ald and Acact was confirmed by typing microsatellite markers flanking a small region of chromosome 1 presumed to contain both loci. For Acact, a ((C57BL/6J × Mus spretus)F1 × C57BL/6J) interspecific backcross mapping panel (15) was typed, and for ald, a set of (AKXL)RI strains was typed. RI strain and progenitor control DNAs were purchased from The Jackson Laboratory. Genomic DNA was amplified with PCR primers for microsatellite markers (Research Genetics, Huntsville, AL) as described (16). Amplification products were separated on 5% polyacrylamide gels and detected by autoradiography. Variant alleles were scored, and the segregation patterns were entered into a Map Manager version 2.6.5 (17) data base containing either backcross (13) or RI strain data. The backcross panel has been typed for ~400 markers and the AKXL strains for 343 markers (17) distributed throughout the genome. Map positions were determined by minimizing the number of double and multiple recombination events. These mapping data have been deposited in the Mouse Genome Data Base (accession number MGD-JNUM-42073).
Tissue Lipid Analysis-- For neutral lipid staining, adrenal glands were perfusion fixed in 3% paraformaldehyde in Tyrode's solution, dissected from surrounding tissues, and frozen. Vibratome-cut sections were subsequently stained with Oil Red O (18). For chromatographic analysis, lipids were extracted from adrenal gland homogenates with the Bligh-Dyer method (19), and high-performance thin-layer chromatography (HPTLC) was performed as described (20).
Immunoblot Analysis-- Tissues were homogenized in 20 mM Tris, 150 mM NaCl, and 0.1% Triton X-100 (pH 7.4). An equal volume of homogenization buffer containing 10% SDS was then added to protein aliquots (final concentration, 5% SDS); the samples were heated to 65 °C for 20 min, and protein aliquots were size fractionated by SDS-polyacrylamide gel electrophoresis with 10% gels. The separated proteins were transferred to nylon membranes and immunoblotted with a rabbit antiserum generated against a glutathione S-transferase/mouse ACAT fusion protein containing the amino-terminal 120 residues of the mouse ACAT protein (21). The low density lipoprotein receptor-related protein was detected with a rabbit antiserum (22). Binding of the antibodies was detected with an enhanced chemiluminescence kit (Amersham Corp.).
RNA and cDNA Analysis--
RNA was prepared from tissues by
standard techniques (23). For Northern blots, 10-µg samples were
analyzed with a 32P-labeled 501-bp mouse ACAT cDNA
fragment (nucleotides 804-1304 (14)) or 28S primer (24) as
hybridization probes. cDNA was synthesized from RNA samples with a
first-strand cDNA kit (Stratagene, La Jolla, CA). 5-rapid
amplification of cDNA ends (RACE) experiments were performed with
an antisense primer (5
GAGAAGGTTGTGAGTGCACA 3
; nucleotides 1053-1072
(14)) and a 5
-RACE kit (Life Technologies, Inc.). For reverse
transcriptase-PCR, cDNA was amplified with sense (5
CTACCCTCCGCTCGCAG 3
; nucleotides 762-778 (14)) and antisense (5
GAGAAGGTTGTGAGTGCACA 3
; nucleotides 1053-1072 (14)) primers. PCR
conditions were 28 cycles, each of 30 s at 96 °C, 2 min at
55 °C, and 2 min at 72 °C. PCR-amplified DNA fragments were
sequenced directly with an automatic sequencer (Applied
Biosystems).
Cholesterol Esterification Assays-- Adrenal glands were homogenized in 0.1 M sucrose, 50 mM KCl, 40 mM KH2PO4, and 30 mM EDTA (pH 7.4) with a Potter-Elvehjem homogenizer. After removal of intact cells and nuclei by centrifugation of homogenates at 600 × g for 10 min, total membranes were isolated by ultracentrifugation (100,000 × g pellet). The rate of incorporation of [14C]oleoyl-CoA (Amersham Corp.) into cholesterol esters was assayed essentially as described by Erickson et al. (25). Reactions were performed at 37 °C for 5 min with 100-150 µg of protein homogenate and 25 µM oleoyl-CoA (specific activity, ~18 mCi/mmol). Exogenous cholesterol (20 nmol) was added to the reaction by preincubating membranes for 30 min at 37 °C with phosphatidylcholine:cholesterol (4:1 molar ratio) liposomes. In some experiments, exogenous cholesterol was provided as an acetone solution.
Transient Transfection of ACAT-deficient Chinese Hamster Ovary Cells-- ACAT-deficient Chinese hamster ovary (CHO)-(AC29) cells (provided by Dr. T. Y. Chang, Dartmouth University, Hanover, NH) were transfected with pcDNA3 mammalian expression vectors (Invitrogen, Carlsbad, CA) containing the wild-type human, wild-type mouse, or AKR mouse (ald) ACAT cDNAs. DNA vectors (1 µg) were transfected with LipofectAMINE (Life Technologies, Inc.) into equivalent amounts of cells (4 × 105) plated on 6-well dishes. Approximately 48 h after transfection, cells were washed twice with phosphate-buffered saline, harvested by scraping, and homogenized in the buffer described above using a syringe and 10 passes through a 25-gauge needle. Total membranes (excluding nuclei) were isolated, and immunoblotting and cholesterol esterification assays were performed as described above.
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RESULTS |
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We previously mapped Acact to a region of mouse
chromosome 1 (13) containing the ald locus (8). Because the
phenotype of Acact/
mice included cholesterol ester
depletion in the adrenal cortex of both male and female mice (9), we
performed refined mapping of Acact and ald with
microsatellite markers flanking the region containing both loci,
utilizing DNA from backcrossed or recombinant inbred strains.
Acact and ald colocalized within the proximal to
distal interval D1Mit346-At3 (Fig.
1). The segregation pattern of
Acact exhibited no recombination with D1Mit14 or
D1Mit33 among 62 or 55 backcross progeny, respectively.
ald was localized proximal to D1Mit33 but within
the 95% confidence interval for the location of Acact,
indicating that the Acact and ald loci were
indistinguishable as determined within the resolution of these genetic
markers.
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To determine whether Acact and ald were allelic,
we performed two genetic experiments designed to test whether the
ald locus would complement the Acact locus. We
crossed Acact
/
mice (mixed C57BL/6 and 129/Sv
background) with ald/ald (AKR) mice and assessed the adrenal neutral lipid content in Oil Red O-stained adrenal sections
of male postpubertal F1 offspring. Whereas
Acact+/+ and Acact+/
control male mice had
abundant neutral lipids in their adrenal cortex, male postpubertal
F1 offspring (carrying one ald and one
Acact
allele) exhibited marked adrenocortical lipid
depletion, similar to that in Acact
/
controls (Fig.
2A). In a separate experiment, we
crossed Acact+/
mice with ald/ald
mice and analyzed the adrenal gland cholesterol ester content of the
postpubertal male F1 offspring by HPTLC. All F1
offspring that carried an Acact
allele and an
ald allele had marked cholesterol ester depletion (Fig.
2B), similar to that observed in Acact
/
controls and less than that observed in ald/ald
controls. In contrast, control littermate F1 offspring
carrying an Acact+ allele and an ald allele had
cholesterol ester levels equivalent to or greater than that in
Acact+/
controls (Fig. 2B). The failure of the
ald allele to complement the Acact
allele in
both experiments indicated that the two loci were allelic.
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Immunoblotting of preputial gland homogenates from adult male mice of 10 inbred strains demonstrated that the Acact protein from AKR mice migrated at a lower molecular mass (~42 kDa) on SDS-PAGE than the ~45-kDa protein in the other mouse strains (Fig. 3A). Similarly, a protein of reduced molecular mass was observed in adrenal gland homogenates from male and female AKR mice and in ovary homogenates from female AKR mice (not shown). Quantitative analysis of the Acact protein in adrenal homogenates of AKR mice demonstrated that its levels in males and females were reduced to ~33 and ~46%, respectively, of the levels in male control (C57BL/6 × 129/Sv) mice (Fig. 3B).
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Northern blotting of RNA from the preputial glands of adult AKR mice
revealed that the Acact mRNA was less abundant and
slightly smaller than that of control mice (Fig.
4A). Similar findings were
observed in RNA samples from kidney and testes of AKR mice (data not
shown). Because the immunoblotting and Northern blotting experiments
suggested that the ald protein may be truncated, we examined
the cDNA sequences of the Acact mRNA and observed
several differences as compared with the wild-type Acact
cDNA (14). Initial 5-RACE experiments followed by cDNA
sequencing demonstrated that the ald cDNA contained a
118-bp deletion spanning part of the 5
untranslated region and the
initial coding sequences (cDNA nucleotides 790-897 (14)). To
further investigate this finding, we performed reverse
transcriptase-PCR experiments using primers located on either side of
the deleted region (Fig. 4B). Whereas a 312-bp wild-type
product was consistently amplified from cDNA of control mice, a
194-bp product (reflecting the 118-bp deletion) was amplified from all
tissues tested of both male and female AKR mice; the 194-bp product was
also observed at very low levels in the cDNA from the control mice.
DNA sequencing of this amplification product demonstrated that it
lacked the identical 118 bp identified in the 5
-RACE experiments. DNA
sequencing from C57BL/6 genomic DNA revealed that the 118 bp deleted in
the ald cDNA corresponded to an exon containing the AUG
translation initiation codon. In addition to the deleted sequences, the
ald cDNA contained two missense mutations: A
G at
cDNA nucleotide 1248, resulting in Ile
Val at amino acid 147;
and C
T at nucleotide 1422, resulting in His
Tyr at amino acid
205. Several other nucleotide differences were observed (A
G at
position 917, A
G at 971, and A
C at 1232) that did not result
in amino acid coding differences.
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To determine whether the protein product of the ald allele
could esterify cholesterol, we performed cholesterol esterification activity assays on adrenal membranes harvested from AKR mice and controls. The adrenal membranes from adult male AKR mice had activity levels (121 ± 11 pmol/mg protein/min) similar to those of
membranes from wild-type control (C57BL/6 × 129/Sv) male mice
(111 ± 17 pmol/mg protein/min) and much greater than those of
Acact/
controls (3.8 ± 0.5 pmol/mg protein/min)
(Fig. 5A). Membranes from female AKR mice exhibited ~3-4-fold greater activity (351 ± 171 pmol/mg protein/min) than those from male mice (consistent with the
higher cholesterol ester in adrenal glands of female mice (26)).
Because these assays were performed at Vmax,
with saturating concentrations of cholesterol substrate (20 nmol), we
hypothesized that the activity levels of the ald protein
might be altered at lower substrate concentrations owing to altered
affinity of the protein for cholesterol substrate. We therefore
analyzed activity levels of postpubertal male AKR adrenal membranes in
response to various concentrations of cholesterol. At each cholesterol
concentration tested, however, the cholesterol esterification activity
in membranes from AKR mice was equal to or greater than that of
wild-type control membranes (Fig. 5B).
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To confirm that the protein expressed by the ald allele could catalyze cholesterol esterification, we transiently expressed ald and wild-type Acact cDNAs in cultured ACAT-deficient (AC29) CHO cells (27) and measured cholesterol esterification activity in membrane homogenates. The transfection of the ald cDNA resulted in the expression of an Acact protein of lower molecular mass than that obtained with wild-type cDNAs, confirming the results of the immunoblot experiments in tissues from AKR mice. Membranes prepared from cells transfected with ald cDNA catalyzed cholesterol esterification at levels much greater than membranes of untransfected cells and similar to that of membranes from cells transfected with wild-type cDNA (Fig. 6). Normal cholesterol esterification activity was also observed in a pulse cholesterol esterification assay performed on intact transfected AC29 cells (data not shown).
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DISCUSSION |
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ald, a naturally occurring mutation in the AKR inbred mouse strain, is associated with cholesterol ester depletion in the adrenal cortex, which becomes manifest at the time of sexual maturation (3). In the current study, we provide genetic and molecular evidence indicating that ald is associated with a naturally occurring mutant Acact allele. The AKR strain carries mutations at the Acact locus that result in an amino-terminal deletion in the Acact protein and two amino acid changes compared with the wild-type protein. Despite these differences, the ACAT protein from the ald allele catalyzed cholesterol esterification at levels similar to that of the wild-type protein. This finding suggests that the adrenocortical cholesterol ester depletion phenotype due to the ald mutation results from an altered susceptibility of the mutant ald protein to modifying influences present in vivo.
Two lines of genetic evidence from our studies indicate that
ald and Acact are allelic. First, refined genetic
mapping demonstrated colocalization of the two loci to the same region
of chromosome 1. Second, a cross between Acact/
and AKR
(ald/ald) mice demonstrated a lack of
complementation between the ald and Acact
alleles with regard to adrenocortical lipid depletion in male
postpubertal mice. Male postpubertal F1 offspring from this
cross with an ald/
genotype were characterized by
cholesterol ester depletion in the adrenal cortex. If ald
and Acact were alleles of different loci, then this cross
would result in heterozygosity at each locus (i.e. Acact+/
and ald/+), and male postpubertal offspring would be
expected to have abundant cholesterol esters in the adrenal cortex at
levels similar those in Acact+/
mice or +/ald
mice. The fact that DNA from AKR mice could not correct the
adrenocortical lipid depletion caused by the Acact
allele
provides near-definitive evidence that the ald mutation
results from a mutant Acact allele.
The RNA and immunoblot analyses demonstrate clearly that the ald allele results in the production of a mutant Acact protein. Of 10 inbred strains surveyed, an Acact protein of lower molecular mass was found only in the AKR strain. The analysis of Acact cDNA from AKR mice demonstrated that the truncated Acact protein lacks the first coding exon containing the AUG translation initiation codon. Further experiments will be necessary to identify the genomic mutation that leads to the missing exon. The lack of this exon in the ald mRNA results in an amino-terminal deletion of at least the first 30 amino acids of the Acact protein. We hypothesize that translation of the Acact protein from the ald mRNA begins at the AUG codon at amino acid 34; this AUG is in the correct reading frame and is located in a Kozak consensus sequence for translation initiation (28). Supporting this hypothesis, the expression of the ald cDNA in cultured cells resulted in a truncated protein of identical molecular mass to that observed in tissues from AKR mice. The mutant protein was detected at lower levels than the wild-type Acact protein by immunoblotting, both in homogenates from male and female AKR mice and in cultured cells expressing equal amounts of wild-type or mutant Acact cDNAs. These immunoblot results may truly reflect lower levels of the ald protein, possibly due to a lower rate of translation originating from the alternate AUG start codon. However, it is also possible that the amino-terminal deletion in the ald protein resulted in altered reactivity of this truncated protein with the polyclonal antiserum (which recognizes epitopes at the amino terminus of ACAT) and that the immunoblotting data reflect this difference.
Despite the alterations, the ald protein catalyzed cholesterol esterification at levels equivalent to or greater than those of the wild-type Acact protein. This finding was observed both in adrenal tissues from adult male and female postpubertal AKR mice and in cultured cells expressing the mutant protein. Similar findings indicating that the ald protein can catalyze cholesterol esterification have been observed in pulse assays of cultured peritoneal macrophages from AKR mice.2 We observed normal or supranormal activity levels in assays carried out both at Vmax and at a range of cholesterol substrate concentrations. Thus, it is clear that the ald protein can catalyze cholesterol esterification, at least under the conditions utilized in our experiments. These data provide important structure-function information indicating that the Acact protein can still catalyze cholesterol esterification at normal levels despite an amino-terminal truncation (likely of 33 amino acids) and the amino acid changes at residues 147 and 205. Although this finding was somewhat surprising given the cholesterol ester depletion phenotype, it was not totally unexpected, inasmuch as the adrenal glands of AKR mice contain normal levels of cholesterol esters before sexual maturation. It is possible that the ald protein is associated with reduced ACAT activity in intact male AKR adrenal glands and that we were unable to reproduce the conditions or modifying factors that inhibit the activity in adrenal glands in our in vitro assays.
How does the ald allele contribute to cholesterol ester
depletion at the time of puberty? The answers to this question are largely speculative. It is clear that alterations of different proteins
involved in cholesterol metabolism can cause adrenocortical lipid
depletion. Phenotypes of adrenocortical lipid depletion have been
observed for knockouts of Acact (9) and Apoa1
(26) and in the naturally occurring ald mutations in AKR and
DBA/2 (6) mice. In contrast to the cholesterol ester depletion in both
male and female Acact/
and Apoa1
/
mice,
however, the ald phenotype in AKR and DBA/2 mice is
conditional and is manifested only in the presence of modifying
influences, such as androgen production at the time of puberty.
Supporting the hypothesis that adrenocortical lipid depletion can
result from the interactions of different factors, preliminary studies
in DBA/2 mice suggest that the primary defect is not due to the
Acact locus and that at least two loci are responsible for
the phenotype.3 In AKR mice, we
speculate that an increase in androgens affects or interacts with the
ability of the ald protein to esterify cholesterol or to
facilitate the storage of cholesterol esters. One possibility is that
the amino-terminal deletion in the ald protein affects protein-protein interactions or the localization of the ald
protein in intracellular compartments or in cell membranes and that
these alterations influence the susceptibility of the protein to
androgen-associated factors. Identifying factors that modulate the
adrenocortical lipid depletion phenotype could provide a better
understanding of cholesterol metabolism in adrenocortical cells and
intracellular cholesterol metabolic pathways.
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ACKNOWLEDGEMENTS |
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We thank Jim McGuire and Dale Newland for technical assistance, Dr. T. Y. Chang for the AC29 cell line, Dr. Joachim Herz for advice and the LRP antiserum, Drs. Joseph Goldstein and Ira Tabas for helpful discussions, Amy Corder and John Carroll for graphics, Stephen Ordway and Gary Howard for editorial support, and Angela Chen for manuscript preparation.
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FOOTNOTES |
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* This work was supported by the J. David Gladstone Institutes, American Heart Association, California Affiliate Grant-in-aid 95-239 (to R. V. F.), and National Institutes of Health Grants HL30568 and HL42488 (to A. J. L.).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.
§ Present address: Department of Genetics, Hadassah University Hospital, P. O. Box 12000, Jerusalem, Israel 91120.
Present address: Columbia University, College of Physicians & Surgeons, 630 W. 168th St., New York, NY 10032.
To whom correspondence should be addressed: Gladstone Institute
of Cardiovascular Disease, P. O. Box 419100, San Francisco, CA 94141-9100. Tel.: 415-695-3759; Fax: 415-285-5632; E-mail: bob_farese.gicd{at}quickmail.ucsf.edu.
1 The abbreviations used are: ald, adrenocortical lipid depletion; ACAT, acyl-CoA:cholesterol acyltransferase; Acact, acyl-CoA:cholesterol acyltransferase mouse gene; CHO, Chinese hamster ovary; HPTLC, high-performance thin-layer chromatography; PCR, polymerase chain reaction; bp, base pair(s); RACE, rapid amplification of cDNA ends.
2 I. Tabas, personal communication.
3 C. Welch, unpublished observations.
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REFERENCES |
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