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J Biol Chem, Vol. 273, Issue 41, 26755-26764, October 9, 1998
From the The synthesis of cholesterol
esters by acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) is
an important component of cellular cholesterol homeostasis. Cholesterol
ester formation also is hypothesized to be important in several
physiologic processes, including intestinal cholesterol absorption,
hepatic lipoprotein production, and macrophage foam cell formation in
atherosclerotic lesions. Mouse tissue expression studies and the
disruption of the mouse ACAT gene (Acact) have indicated
that more than one ACAT exists in mammals and specifically that another
enzyme is important in mouse liver and intestine. We now describe a
second mammalian ACAT enzyme, designated ACAT-2, that is 44% identical to the first cloned mouse ACAT (henceforth designated ACAT-1). Infection of H5 insect cells with an ACAT-2 recombinant baculovirus resulted in expression of a ~46-kDa protein in cell membranes that
was associated with high levels of cholesterol esterification activity.
Both ACAT-1 and ACAT-2 also catalyzed the esterification of the
3 The ability to synthesize sterol esters is fundamental to most
eukaryotic cells. Sterol esterification is thought to participate in
the maintenance of cell membrane sterols at levels optimal for normal
cell function. In mammalian cells, cholesterol is the predominant
cellular sterol, and cholesterol esterification is catalyzed by the
enzyme acyl-CoA:cholesterol acyltransferase (E.C. 2.3.1.26, ACAT)1 (reviewed in Refs.
1-3). Oxygenated sterols (oxysterols) and their esters also are found
in mammalian tissues (4). Although oxysterol esterification likely is
catalyzed by ACAT (5), a systematic analysis of the enzyme (5) and
substrates involved has not been done.
ACAT activity has been implicated in a number of physiologic processes
(1). In the small intestine, ACAT has been proposed to play a role in
cholesterol absorption by maintaining a free cholesterol diffusion
gradient across the enterocyte surface through the formation of
cholesterol esters intracellularly (6, 7). Cholesterol ester formation
by ACAT also has been hypothesized to be important for the assembly and
secretion of apolipoprotein B-containing lipoproteins in the intestine
and the liver (reviewed in Ref. 8). In the adrenal glands and other
steroidogenic tissues, ACAT synthesizes cholesterol esters that
accumulate in cytosolic droplets where they can serve as cholesterol
substrate stores for steroidogenesis. In macrophages, ACAT generates
intracellular cholesterol esters that are stored as cytosolic lipid
droplets, a characteristic feature of macrophage foam cells in
atherosclerotic lesions (9). Because of ACAT's apparently prominent
role in cholesterol metabolism, a number of ACAT inhibitors have been developed for use as anti-atherosclerosis agents (reviewed in Ref. 10).
Some of these act by inhibition of intestinal cholesterol absorption
(10) or hepatic lipoprotein synthesis and secretion (11, 12), and
others, as specific inhibitors of macrophage foam cell formation
(13).
Recent evidence has suggested that more than one ACAT exists in
mammals. A human ACAT cDNA was first identified from a macrophage cDNA library by Chang and co-workers (14). The disruption of the
mouse homolog of this ACAT gene (Acact) yielded viable,
ACAT-deficient (Acact In this study, we report the identification and cloning of a second
mammalian ACAT, designated ACAT-2. We have expressed the ACAT-2
cDNA in insect cells and characterized its sterol esterification activity in detail. In addition, the ability to express ACAT-1 and
ACAT-2 in the low-background insect cell system enabled us to compare
the two enzymes with respect to substrate preferences and response to
inhibitors. The characteristics of the mouse ACAT-2 gene product and
its tissue distribution support the hypothesis that ACAT-2 may
contribute to cholesterol esterification activity in the liver and
intestine.
Cloning of ACAT-2 cDNA--
An EST (accession number R10292)
was identified by BLAST searches
(http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast) of the human
EST data base with sequences from ACAT-1. Degenerate primers derived
from the EST sequences were subsequently used to amplify a partial
ACAT-2 cDNA from mouse liver cDNA. Sequences for the 5' and 3'
ends of the ACAT-2 cDNA were determined by 5'- and 3'-rapid
amplification of cDNA ends methodology using a mouse spleen
Marathon ReadyTM cDNA library
(CLONTECH, Palo Alto, CA). ACAT-2 sequences have been deposited in GenBank (accession number AF078751).
Generation of Recombinant Baculoviruses--
Mouse ACAT-2 and
human ACAT-1 baculovirus transfer vectors were constructed by inserting
ACAT-2 coding sequences (amplified from mouse liver cDNA) or ACAT-1
coding sequences (amplified from HepG2 cell cDNA) downstream of the
polyhedrin promoter of the pVL1392 vector (PharMingen, San Diego, CA).
Vectors were co-transfected with viral baculogold DNA into Sf9
insect cells, according to the manufacturer's protocol (PharMingen
Kit), to generate recombinant baculoviruses. Viruses were
plaque-purified and high titers of recombinant baculoviruses were
generated by two rounds of amplification in Sf9 cells. Cells
were cultured at 27 °C in Grace's medium (Life Technologies,
Gaithersburg, MD), supplemented with 10% fetal bovine serum.
Expression Studies--
For protein expression studies, H5
insect cells were cultured in optimized serum-free Express-Five medium
(Life Technologies), supplemented with 20 mM
L-glutamine. Cells were plated on day 0 at 8.5 × 106 cells per 100-mm dish and infected on day 1 with
high-titer baculovirus stocks at an optimal multiplicity of infection
that was determined empirically. On day 3 (48 h after infection), cells
were collected by low-speed centrifugation and washed twice with
phosphate-buffered saline. Cell pellets were resuspended in 0.1 M sucrose, 0.05 M KCl, 0.04 M
KH2PO4, 0.03 M EDTA (pH 7.2) and
homogenized by 10 passages through a 27-gauge needle. Cell homogenates
were fractionated by centrifugation at 100,000 × g for
1 h at 4 °C. The cell membrane fraction (pellet) was
resuspended in the isolation buffer, and aliquots were frozen
( Metabolic Labeling--
For metabolic labeling, H5 insect cells
were plated on day 0 at 2.9 × 106 cells per 60-mm
dish and infected at day 1 with high-titer baculoviruses at empirically
determined multiplicity of infectioins. On day 3 (48 h after
infection), cells were washed and incubated in methionine-free and
cysteine-free medium (SF900 II, Life Technologies) for 1 h. The
medium was then replaced by 3 ml of methionine-free and cysteine-free medium containing 715 µCi of [35S]methionine and
[35S]cysteine (Amersham Pro-Mix, Amersham) and the cells
were incubated for 2 h. Cells were washed twice with
phosphate-buffered saline, collected by low-speed centrifugation,
resuspended in 500 µl of lysis buffer (50 mM Tris-HCl,
150 mM NaCl, 5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride (PMSF), 1% Triton X-100 (pH 7.4)), and
sonicated. Cell lysate samples (100 µg of protein) were analyzed by
10% SDS-PAGE. Gels were fixed in 30% acetic acid, washed in 25%
ethanol and 8% acetic acid, dried, and exposed to x-ray film.
ACAT Activity Assays--
The rate of incorporation of
[14C]oleoyl-CoA (Amersham) into cholesterol esters was
assayed essentially as described by Erickson et al. (21).
Reactions were performed at 37 °C for 5 min with 100 µg of cell
membrane protein and 25 µM oleoyl-CoA (specific activity = 18 µCi/µmol). Exogenous cholesterol (20 nmol) was
added as phosphatidylcholine (PC):cholesterol (4:1 molar ratio)
liposomes to measure apparent Vmax activities
(22).
Determination of Fatty Acyl-CoA Specificity--
ACAT activity
was assayed essentially as described (22), except that unlabeled fatty
acyl-CoAs and [14C]cholesterol (51.3 mCi/mmol, NEN Life
Science Products, Boston, MA) provided as cholesterol:egg PC liposomes
(molar ratio = 0.2) were used. Parallel assays were performed with
[14C]oleoyl-CoA (57 mCi/mmol, Amersham) and unlabeled
cholesterol provided as PC liposomes (molar ratio = 0.2). The
relative competition of fatty acyl-CoAs for cholesterol esterification
was assessed using [14C]oleoyl-CoA diluted to ~20,000
dpm/nmol with unlabeled fatty acyl-CoAs (~1:6, mol:mol dilution) and
unlabeled cholesterol:PC liposomes (molar ratio = 0.7). The
abilities of [14C]oleoyl-CoA and
[14C]palmitoyl-CoA (55.0 mCi/mmol, Amersham) to catalyze
cholesterol esterification were assessed directly. Fatty acyl-CoAs, egg
PC, and cholesterol were from Sigma. Briefly, membrane proteins (25 µg) were incubated in 0.25 M sucrose, 1 mM
EDTA, 100 mM Tris-HCl (pH 7.5) containing 400 µg of
bovine serum albumin and cholesterol:PC liposomes (final volume = 0.2 ml). The reaction was started by the addition of 5 nmol of fatty
acyl-CoA and carried out for 2 min at 37 °C. The reaction was
terminated by the addition of CHCl3:methanol (2:1, v:v),
and the products analyzed as described (21). All assays were in the
linear range with respect to assay time and protein concentration.
Determination of Acyl Acceptor Substrate Specificity--
The
ACAT assay described above was used, except that acceptors other than
cholesterol were added to the incubations as acceptor:PC liposomes
(molar ratio = ~0.2) or, in the case of ethanol, added directly.
The acceptors included ethanol, retinol, tocopherol, Inhibitor Studies--
Cholesterol esterification activity was
assayed as described above (using [14C]oleoyl-CoA and
cholesterol:PC liposomes (molar ratio = 0.2)) after a 5-min
preincubation of the membranes at 37 °C with 1 mM PMSF,
0.1 mM para-hydroxymercuribenzoate (PHMB), or
0.1 mM progesterone. Inhibitors were purchased from
Sigma.
ACAT-2, A Second Mammalian Acyl-CoA:Cholesterol
Acyltransferase
ITS CLONING, EXPRESSION, AND CHARACTERIZATION*
§,§,,
,,
,, and¶||
Gladstone Institute of Cardiovascular
Disease, § Cardiovascular Research Institute, and
¶ Department of Medicine, University of California, San Francisco,
California 94141, the Veterans Administration Medical Center,
San Francisco, California 94121, the ** Department of Microbiology and
Molecular Genetics, Department of Medicine, and Molecular Biology
Institute, University of California, Los Angeles, California 90095, the
§§ Department of Chemistry, Dartmouth College,
Hanover, New Hampshire 03755, and ¶¶ Parke-Davis
Pharmaceutical Research, Ann Arbor, Michigan 48105
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-hydroxyl group of a variety of oxysterols. Cholesterol esterification activities for ACAT-1 and ACAT-2 exhibited different IC50 values when assayed in the presence of several
ACAT-specific inhibitors, demonstrating that ACAT inhibitors can
selectively target specific forms of ACAT. ACAT-2 was expressed
primarily in mouse liver and small intestine, supporting the hypothesis that ACAT-2 contributes to cholesterol esterification in these tissues.
The mouse ACAT-2 gene (Acact2) maps to chromosome 15 in a
region containing a quantitative trait locus influencing plasma
cholesterol levels. The identification and cloning of ACAT-2 will
facilitate molecular approaches to understanding the role of ACAT
enzymes in mammalian biology.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
/) mice
that were characterized by tissue-specific reductions in cholesterol
esters (15). Cholesterol ester stores were markedly reduced in adrenal
cortices and cultured peritoneal macrophages; however, substantial
levels of ACAT activity were present in
Acact/ livers, and intestinal cholesterol
absorption was normal, indicating that another ACAT was active in these
tissues (15). Studies examining the tissue distribution of
Acact mRNA expression also supported the hypothesis that
more than one ACAT exists (16), as did previous biochemical (17) and
ACAT inhibitor (18) studies showing differences between liver and
aorta/macrophage ACAT activities. Taken together, these data led us to
hypothesize that a second ACAT contributes to cholesterol
esterification activity in the liver and small intestine. A precedent
for eukaryotic organisms having more than one sterol esterification
enzyme was established for Saccharomyces cerevisiae, which
have two genes encoding sterol esterification enzymes, ARE1
and ARE2 (19, 20). Although the reason for yeast having two
genes is unclear, the disruption of both was necessary to render this
organism deficient in sterol esterification (19, 20).
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
80 °C) for activity assays. Protein concentrations were
determined using a DC Protein Assay kit (Bio-Rad).
-sitosterol,
lanosterol, vitamins D1 and D2, diacylglycerol, and a number of oxysterols (25-hydroxy-, 27-hydroxy-, 7
-hydroxy-, 7-hydroxy, 7-keto-, 24(S)-hydroxy-,
24(R)-hydroxy-, 24(S),25-epoxy-, and
24(R),25-epoxycholesterols). The sterols were assessed at three different concentrations (4, 8, and 16 µg) at a constant sterol:PC molar ratio (~0.2). Acceptors were from Sigma or Steraloids (Wilton, NH). 24(S)-Hydroxy- and
24(R)-hydroxycholesterol (23, 24),
24(S),25-epoxy-, and 24(R),25-epoxycholesterols
were prepared as described (24-26). The reaction products were
extracted and separated by thin-layer chromatography (21), and the
plates exposed to x-ray film for 4 days to visualize the radioactive spots. Fatty acyl ester standards were used to identify the migration positions of the products. The spots were cut out and counted in a
liquid scintillation counter (21).
20 °C. Membrane proteins (100 µg for ACAT-1 and 50 µg for
ACAT-2) were preincubated with cholesterol:PC liposomes and different
inhibitors (final Me2SO concentration = 2.5%) for 30 min on ice. After equilibrating for 5 min at 37 °C, the reaction was
started by adding [14C]oleoyl-CoA. Assays were performed
otherwise essentially as described above.
ACAT-2 Expression in Mouse and Human Tissues-- For RT-PCR analysis, total RNA was prepared from fresh mouse tissues, human hepatoma cells (HepG2), human small intestine, or human fibroblasts using Trizol (Life Technologies), and cDNA was synthesized using random hexamer primers and Superscript reverse transcriptase (Life Technologies). For mouse ACAT-2, a 531-base pair fragment was amplified using sense (5'-ACTGTGCCTGGGATCTTTTGTGTC-3') and antisense (5'-CTCGCGGGGTGGCCATGCTGGGAGTG-3') primers. For human ACAT-2, a 742-base pair fragment was amplified using sense (5'-TCTTCTATCCCGTCATGCTG-3') and antisense (5'-GGTCCACATCAGCACGTTCC-3') primers. Amplification of glyceraldehyde-3-phosphate dehydrogenase cDNA (an internal control) was as described (27).
For Northern blots, total RNA was extracted from tissues using Trizol (Life Technologies) and 10-µg aliquots were analyzed. Blots were hybridized with a 32P-labeled 1.5-kb DNA fragment containing the mouse ACAT-2 coding sequences and re-probed with coding sequences for glyceraldehyde-3-phosphate dehydrogenase as an internal standard.Mapping of Mouse Acact2 by Linkage Analysis-- Linkage analysis was performed using a panel of 67 progeny derived from a ((C57BL/6J × Mus spretus)F1 X C57BL/6J) interspecific backcross (28). This backcross panel has been typed for more than 400 loci distributed throughout the genome (29). Briefly, parental strain DNAs were screened for restriction fragment-length variants following digestion of genomic DNA with a panel of 10 restriction enzymes and hybridization with a fluorescein-labeled (Gene Images random prime labeling module, Amersham), 1.3-kb human Acact2 cDNA fragment. Filters were washed under conditions of high stringency (0.1 × SSC/0.1% SDS, 60 °C, 20 min). Acact2 hybridization products were detected by chemiluminescence (Gene Images CDP-Star detection module, Amersham). Linkage to previously typed chromosomal markers was detected using Map Manager v2.6.5, and loci were ordered by minimizing the number of recombination events between Acact2 and the markers (30). The data have been deposited in the Mouse Genome Data base under accession number MGD-J:45730 (http://www.infomatics.jax.org).
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RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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A human EST cDNA (accession R10292) that was highly similar to the C-terminal region of ACAT-1 was identified from BLAST data base searches. By using the EST sequence, a full-length cDNA containing the entire coding sequence was subsequently isolated from mouse liver and designated ACAT-2. The mouse ACAT-2 cDNA is predicted to encode a 525-amino acid protein that is 44% identical to mouse ACAT-1 (31) (Fig. 1A). The ACAT-2 protein has multiple hydrophobic domains, and its hydrophobicity plot is strikingly similar to that for ACAT-1 (Fig. 1B). The analysis of mouse ACAT-2 sequences with a transmembrane prediction program (http://ulrec3.unil.ch/software/TMPRED_form.html) indicated that ACAT-2 may have eight transmembrane-spanning regions (amino acid residues 125-143, 154-181, 192-220, 265-285, 304-326, 351-369, 439-459, and 475-496). Using the same program, mouse ACAT-1 was predicted to have seven or eight transmembrane regions. The alignment of the mouse ACAT-2 protein sequence with those of other ACAT gene family members (Fig. 2) reveals many conserved residues, especially in the C termini of these proteins. Each family member has a potential tyrosine phosphorylation site and a conserved serine residue (amino acid 248 of ACAT-2) that has been implicated in the catalytic activity of hamster ACAT-1 (32).
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To determine if ACAT-2 was capable of catalyzing sterol esterification, we expressed its cDNA in H5 insect cells using a baculovirus expression system. Insect cells were chosen for these expression studies because they have very low levels of cellular cholesterol and lack significant cholesterol esterification activity (33). Metabolic labeling of cellular proteins in insect cells 48 h after infection demonstrated the expression of a ~46-kDa protein (Fig. 3A). The apparent molecular mass of ACAT-2 on SDS-polyacrylamide gels was smaller than that predicted from the amino acid sequence (60.7 kDa), a finding similar to that reported for ACAT-1 (14). For ACAT-1, it has been suggested that the apparent size of the protein is less than predicted due to high amounts of SDS binding (33). Cell membrane preparations from insect cells infected with the ACAT-2 baculovirus were characterized by high levels of cholesterol esterification activity (Fig. 3B). The amount of ACAT activity present in ACAT-2-infected cells was consistently 10-fold higher than that in cells infected with virus containing ACAT-1. However, metabolic labeling studies (Fig. 3A) indicated that considerably more ACAT-2 protein may have been expressed than ACAT-1. Membrane cholesterol esterification activity was dependent upon the amount of ACAT-2 protein expressed, as demonstrated by a time course of ACAT-2 virus infection (Fig. 4).
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The ability to express ACAT-1 or ACAT-2 independently in insect cells
enabled us to examine several biochemical properties of the enzymes.
The esterification of cholesterol by both ACAT-1 and ACAT-2 was
dependent on the presence of a fatty acyl-CoA substrate; in its
absence, cholesterol esterification activity greater than background
was not observed (data not shown). Of the fatty acyl-CoAs tested,
ACAT-1 showed a slight preference for oleoyl, while that for ACAT-2 was
palmitoyl 
linoleoyl > oleoyl > arachidonyl (Table I). The ability of unlabeled fatty
acyl-CoAs to compete with [14C]oleoyl-CoA for
incorporation into cholesterol esters by ACAT-1 or ACAT-2 was also
examined. For ACAT-1, palmitoyl, linoleoyl, and to a lesser extent,
arachidonyl, competed with oleoyl for incorporation into cholesterol
esters (Table II). For ACAT-2, both
linoleoyl and palmitoyl competed with oleoyl for incorporation into
cholesterol esters, but arachidonyl did not. When cholesterol esterification was measured using [14C]oleoyl-CoA or
[14C]palmitoyl-CoA directly, ACAT-1 had 58% higher
activity with oleoyl-CoA compared with palmitoyl-CoA, while ACAT-2
showed a negligible difference (16%) (Table
III).
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Fatty acyl acceptor specificities also were examined for the expressed
proteins. Esterification activity was not detected for either ACAT-1 or
ACAT-2 with ethanol, retinol, tocopherol, -sitosterol, lanosterol,
vitamins D1 and D2, or diacylglycerol as
acceptors (not shown). However, in addition to esterifying cholesterol,
both ACAT-1 and ACAT-2 esterified the 3-hydroxyl group of a variety
of oxysterols. All oxysterols analyzed exhibited greater esterification
capability than cholesterol itself, with the exception of
7-hydroxycholesterol for ACAT-1 (Fig.
5A) and 25-hydroxycholesterol
for ACAT-2 (Fig. 5B). For ACAT-1, the highest levels of
incorporation into sterol esters (more than 5-fold greater than for
cholesterol) were for 24(R)-hydroxycholesterol,
7-hydroxycholesterol, and 27-hydroxycholesterol. For ACAT-2,
24(R)-hydroxycholesterol, 27-hydroxycholesterol, and
7-hydroxycholesterol were esterified at rates 2-4-fold higher than
for cholesterol.
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We also examined the effects of added oxysterols on membrane
cholesterol esterification, using the small but significant mass of
endogenous membrane cholesterol as a substrate. For ACAT-1, cholesterol
esterification was increased approximately 2.5-fold by
27-hydroxycholesterol and 24(S),25-epoxycholesterol, but it was decreased substantially by 7-hydroxycholesterol,
25-hydroxycholesterol, and 24(S)-hydroxy-cholesterol
(Fig. 6A). For ACAT-2, none of
the oxysterols increased cholesterol esterification; instead, they either had little effect or decreased it by 40-60% (Fig.
6B).
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The effects of nonspecific (PMSF, PHMB, and progesterone) and specific inhibitors of ACAT were examined next. PMSF, which inhibits enzymes utilizing serine in their active site, inhibited both ACAT-1 and ACAT-2 by 20-40% (Fig. 7). PHMB, which acts on enzymes that require sulfhydryl groups for activity, inhibited ACAT-1 to a greater extent than ACAT-2 (~90% and ~55%, respectively), and progesterone, a compound known to inhibit ACAT (34), inhibited both enzymes by ~70% (Fig. 7). Membranes from ACAT-1 and ACAT-2 virus-infected cells were also assayed in the presence of ACAT-specific inhibitors PD 132301-2, CI-976, and CI-1011. PD 132301-2 inhibited both ACAT-1 and ACAT-2 to similar degrees (IC50 < 1 µM) (Fig. 8A). In contrast, CI-976 exhibited some selectivity for the expressed enzymes. ACAT-1 activity was suppressed by CI-976 with an apparent IC50 of ~5 µM, whereas ACAT-2 activity was more sensitive with an apparent IC50 of < 1 µM (Fig. 8B). A significant difference in selectivity was also observed for CI-1011, which also inhibited ACAT-2 (IC50 ~2.5 µM) more than ACAT-1 (IC50 > 10 µM) (Fig. 8C).
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5% in all cases. The inhibitors
were incubated with membranes for 5 min at 37 °C prior to start of
the assay. Final concentrations were: PMSF, 1 mM; PHMB, 0.1 mM; and progesterone, 0.1 mM. The
cholesterol:phospholipid liposome molar ratio was 0.2. Control values
were 172 pmol of cholesterol ester/mg of protein/min for ACAT-1 and
1078 pmol of cholesterol ester/mg of protein/min for ACAT-2.
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ACAT-2 mRNA was detected in a variety of mouse tissues by RT-PCR with the highest levels of expression in the liver, small intestine, and embryo liver (Fig. 9A). Northern analysis of mouse tissues demonstrated a ~2-kb ACAT-2 mRNA in the liver and small intestine (Fig. 9B). To determine if ACAT-2 was also expressed in the liver and intestine in humans, we performed RT-PCR experiments on cDNA from HepG2 cells (a human hepatoma cell line), human intestine, and human fibroblasts. ACAT-2 mRNA was detected in HepG2 cells and small intestine, but not in fibroblasts (Fig. 9C). In a separate experiment, we also detected ACAT-2 mRNA in cDNA from human liver (not shown).
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The mouse ACAT-2 gene (Acact2) was localized to mouse chromosome 15 by linkage analysis from an interspecific backcross (Fig. 10). The hybridization of BamHI-digested DNA of the parental strains with the Acact2 cDNA probe resulted in the detection of single fragments in DNA from each (C57BL/6J = 10 kb and Mus spretus = 8.8 kb) and both fragments in DNA from the F1 progeny. The segregation pattern of the M. spretus allele among the backcrossed mice revealed linkage to markers on chromosome 15; linkage was not detected with markers on any other chromosome. No recombination was observed between Acact2 and the microsatellite marker D15Mit16 (0/65 mice). The following gene order was obtained (distance ± S.E., in centiMorgans): centromere-D15Mit31-(3.6 ± 2.5 centiMorgans)-Ppara-(9.2 ± 3.6)-D15Ucla3, Pou6f1-(1.5 ± 1.5)-Acact2, D15Mit16. A human EST for Acact2 (accession R10292) has been mapped to a homologous region on human chromosome 12q13.3-q15 (http://www.ncbi.nlm.nih. gov/XREFdb).
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DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Prior studies, including the disruption of the mouse ACAT-1 gene (Acact) (15) and tissue expression studies of Acact (16), indicated that more than one ACAT exists in mammals. In the current study, we report the cloning and expression of a second mammalian ACAT, ACAT-2. The expression of the ACAT-2 cDNA in insect cells, which normally lack ACAT activity, resulted in high levels of fatty acyl CoA-dependent cholesterol esterification in isolated cell membranes, thereby establishing that this cDNA encodes a sterol esterification enzyme.
The predicted protein sequence for mouse ACAT-2 shows similarities to that for mouse ACAT-1 and other members of the sterol acyltransferase gene family. This family of proteins are most similar in their C termini, suggesting that this region contains domains important for enzyme catalysis. The motif MKX(H/Y)SF in the mid-region of the proteins is conserved in all family members; the serine residue in this motif is necessary for catalytic activity in the hamster ACAT-1 ortholog (32). A tyrosine residue located within a tyrosine phosphorylation consensus motif also is found in most family members. The hydrophobicity analyses for mouse ACAT-1 and ACAT-2 suggest that the proteins may have seven or eight transmembrane regions, consistent with their being intrinsic membrane proteins.
The expression of ACAT-1 or ACAT-2 independently in insect cell membranes made it possible to characterize the two activities separately with respect to substrate specificities and response to inhibitors. The level of cholesterol esterification specific activity for ACAT-1 was similar to that reported previously in insect cell expression studies (33). ACAT-2-containing membranes catalyzed cholesterol esterification at a consistently higher rate than that observed for ACAT-1; however, ACAT-2 protein expression levels appeared higher, which could account for this difference. In preliminary studies, ACAT-2 activity was not saturable with cholesterol delivered in liposomes (up to 275 µM), whereas ACAT-1 activity was saturable at ~200 µM and even inhibited at higher cholesterol concentrations (not shown).
In addition to esterifying cholesterol, both ACAT-1 and ACAT-2 showed
considerable activity with oxysterol substrates. Of note, ACAT-1
exhibited higher levels of sterol esterification for nearly all of the
oxysterols tested than it did for cholesterol. ACAT-1 is expressed
highly in macrophages (16), where oxysterols are found in abundance
(35-37). Because oxysterols can be toxic to cells (38-40), a
potential role for ACAT-1 in macrophages may be to convert free
oxysterols to oxysterol esters as part of a detoxification mechanism.
The ability of ACAT-2 to esterify both cholesterol and oxysterols would
be expected for an intestinal ACAT activity because dietary oxysterols
derived from oxidized cholesterol are absorbed in both animals (41-43)
and humans (44). Oxysterols also are esterified by liver microsomes
(5). Of note, two oxysterols involved in bile acid synthesis,
7-hydroxycholesterol and 27-hydroxycholesterol, were esterified by
both ACAT enzymes, suggesting a potential role for ACAT in regulating
bile acid metabolism. Recently, specific oxysterols, in particular
24(S),25-epoxycholesterol and
24(S)-hydroxycholesterol, were shown to be ligands for
nuclear transcription factors LXR
and LXR
(45, 46). LXR has been implicated in the sterol-mediated
regulation of cholesterol-7-hydroxylase expression (45). The finding
that these oxysterols are esterified by ACAT-1 and ACAT-2 raises the
possibility that ACAT activity may modulate the effects of oxysterols
on LXR-responsive genes.
The effects of oxysterols on cholesterol esterification activity were mixed for ACAT-1 in this expression system. Both 27-hydroxycholesterol and 24(S),25-epoxycholesterol stimulated cholesterol esterification, consistent with the possibility that ACAT-1 may be allosterically regulated by oxysterols (33, 47). In contrast, a number of oxysterols were inhibitory, in particular 25-hydroxycholesterol. The reason for this result, which differs from findings reported previously (33), is unclear, but could relate to differences in ACAT assay methods. In contrast, none of the added oxysterols stimulated ACAT-2 activity, suggesting that ACAT-2 may not have an allosteric binding site for the up-regulation of cholesterol esterification by specific oxysterols. For both ACAT-1 and ACAT-2, the inhibition of cholesterol esterification by oxysterols suggests that, in some instances, oxysterols may compete with cholesterol for the active site. Of interest in this regard is that oxysterols are reported to inhibit cholesterol absorption (43).
The fatty acyl-CoA specificity for ACAT-1 was broad with some preference for oleoyl-CoA, similar to results reported previously for ACAT-1 expressed in yeast cells deficient in sterol esterification (48). ACAT-2 exhibited a preference for linoleoyl-CoA or palmitoyl-CoA over oleoyl-CoA and had the least activity with arachidonyl-CoA. The lower activity with oleoyl-CoA was unexpected because ACAT-2 is expressed predominantly in the liver and small intestine and oleoyl-CoA was reported to be the preferred substrate for hepatic and intestinal ACAT activities (49, 50).
The finding that ACAT-1 and ACAT-2 activities responded differently to ACAT inhibitors may be of interest with regard to development of drugs designed specifically to target ACAT in selected tissues. Responses of ACAT-1 and ACAT-2 to ACAT inhibitors differed with the biochemical class. PD 132301-2, a urea-based compound whose use is associated with adrenal gland toxicity in animals (51, 52), markedly inhibited both enzymes. The IC50 data for this compound are similar to those reported previously (53). CI-976, a fatty acid amide (13), and CI-1011, an acyl sulfamate (54), have been used successfully without adrenal gland toxicity in animals. Both compounds demonstrated selective inhibition of ACAT-2 compared with ACAT-1. For CI-976, this result was surprising inasmuch as this drug has been used in animal studies to inhibit macrophage ACAT selectively without affecting plasma lipid levels (13). CI-1011, a potent hypocholesterolemic agent (54) currently under investigation in human trials, also exhibited a high degree of selectivity toward ACAT-2. Data from human subjects administered CI-1011 at 500 mg/day indicate that the maximal drug concentration ranges from ~7 to 13 µM, depending on the fat content of the diet (55), suggesting that at these concentrations, both ACAT-1 and ACAT-2 would be inhibited significantly. These studies demonstrate that the ability to express the two enzymes independently may provide a useful screen for designing inhibitors with narrow specificity profiles and low toxicity. It is important to realize, however, that the results in both the inhibitor and substrate preference studies reflect data obtained from enzymes expressed in insect cells. The findings therefore may not necessarily reflect what occurs in mammalian cells, where potential differences in post-translational modifications of the enzymes or differences in the membrane lipid environment may occur.
Previous studies (15, 16) indicated that ACAT-1 likely is not the major cholesterol esterification enzyme in mouse liver or intestine. Although it remains to be determined whether ACAT-2 is responsible for the bulk of the activity in these tissues, several findings make it an attractive candidate. First, ACAT-2 is expressed predominantly in the liver and the small intestine. Its mRNA was detected specifically in mouse liver and small intestine by Northern analysis and in human intestinal samples by RT-PCR analysis. ACAT-2 mRNA also was detected in HepG2 cells, a human hepatoma cell line that expresses high levels of ACAT activity (56). In agreement with our findings, Rudel and co-workers2 have detected ACAT-2 mRNA expression in the liver and intestine of primates by Northern blotting. The mouse ACAT-2 gene mapping data also are consistent with a possible role for ACAT-2 in intestinal cholesterol absorption or lipoprotein production by the liver or intestine. A quantitative trait locus influencing plasma low density lipoprotein and very low density lipoprotein cholesterol levels in response to high-fat feeding has been mapped to a region on mouse chromosome 15 from crosses of NZB/B1NJ and SM/J mice (57). The mapping of Acact2 to this region makes it an attractive candidate gene for this locus.
It remains to be determined why mammals or yeast (19, 20) have a need for more than one sterol esterification enzyme. These experiments comparing ACAT-1 and ACAT-2 suggest that evolutionary conservation of the two enzymes may relate either to different substrate preferences or different patterns of tissue expression. Another hypothesis is that the two enzymes have fundamentally different functions within cells. For example, it is possible that cholesterol esters generated by ACAT-1 may be preferentially directed toward intracellular cytosolic droplets for storage, whereas cholesterol esters generated by ACAT-2 may be directed to secretion from the cell by entering the lipoprotein assembly process in tissues like the liver and intestine. This hypothesis is supported by the observation that ACAT-1-deficient mice lacked cholesterol ester droplets in adrenocortical cells and macrophages, but had no apparent defects in lipoprotein production or cholesterol absorption (15). To answer these and other questions related to the biology of ACAT enzymes, the identification and characterization of an ACAT-2 cDNA provides a valuable tool.
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ACKNOWLEDGEMENTS |
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We thank Parke-Davis Pharmaceuticals for providing PD 132301-2, CI-976, and CI-1011. We also thank Dr. Vardiella Meiner for assistance during the early phase of these studies, Dr. Jan Borén for assistance with metabolic labeling experiments, Yu-Rong Xia for assistance with the mouse gene-mapping experiments, John Carroll and Niele Shea for graphics, Gary Howard and Stephen Ordway for editorial assistance, Angela Chen and Maggie Chow for assistance with manuscript preparation, and Drs. Stephen Young, Tom Innerarity, and Steve Smith for comments on the manuscript.
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Note Added in Proof |
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Anderson et al. (58) and Oelkers et al. (59) report the identification of ACAT-2 in primates and humans, respectively, in this issue.
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FOOTNOTES |
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* This work was supported by the J. David Gladstone Institutes, National Institutes of Health Grants HL57170 (to R. V. F), HL52069 (to S. K. E. and T. A. S.), and HL42488 (to A. J. L.), an American Heart Association Postdoctoral Fellowship Award (to S. C.), and a Merit Award from the Department of Veterans Affairs (to S. K. E.).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 nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF078751.
Present address: Columbia University, College of Physicians & Surgeons, 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.: 415-826-7500; Fax: 415-285-5632; E-mail: bfarese{at}gladstone.ucsf.edu.
The abbreviations used are: ACAT, acyl-CoA:cholesterol acyltransferase; Acact, mouse acyl-CoA:cholesterol acyltransferase geneEST, expressed sequence tagHepG2, human hepatoma cellsPC, phosphatidylcholinePCR, polymerase chain reactionPHMB, para-hydroxymercuribenzoatePMSF, phenylmethylsulfonyl fluorideRT, reverse transcriptasekb, kilobase(s)PAGE, polyacrylamide gel electrophoreis.
2 L. Rudel, personal communication.
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Abstract
Introduction
Materials & Methods
Results
Discussion
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 R. G. Lee, K. L. Kelley, J. K. Sawyer, R. V. Farese Jr, J. S. Parks, and L. L. Rudel Plasma Cholesteryl Esters Provided by Lecithin:Cholesterol Acyltransferase and Acyl-Coenzyme A:Cholesterol Acyltransferase 2 Have Opposite Atherosclerotic Potential Circ. Res., November 12, 2004; 95(10): 998 - 1004. [Abstract] [Full Text] [PDF]
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 W. R Hiatt, E. Klepack, M. Nehler, J. G Regensteiner, J. Blue, J. Imus, and M. H Criqui The effect of inhibition of acyl coenzyme A-cholesterol acyltransferase (ACAT) on exercise performance in patients with peripheral arterial disease Vascular Medicine, November 1, 2004; 9(4): 271 - 277. [Abstract] [PDF]
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J. J. Liang, P. Oelkers, C. Guo, P.-C. Chu, J. L. Dixon, H. N. Ginsberg, and S. L. Sturley Overexpression of Human Diacylglycerol Acyltransferase 1, Acyl-CoA:Cholesterol Acyltransferase 1, or Acyl-CoA:Cholesterol Acyltransferase 2 Stimulates Secretion of Apolipoprotein B-containing Lipoproteins in McA-RH7777 Cells J. Biol. Chem., October 22, 2004; 279(43): 44938 - 44944. [Abstract] [Full Text] [PDF]
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P. Parini, M. Davis, A. T. Lada, S. K. Erickson, T. L. Wright, U. Gustafsson, S. Sahlin, C. Einarsson, M. Eriksson, B. Angelin, et al. ACAT2 Is Localized to Hepatocytes and Is the Major Cholesterol-Esterifying Enzyme in Human Liver Circulation, October 5, 2004; 110(14): 2017 - 2023. [Abstract] [Full Text] [PDF]
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M. Hori, M. Satoh, K. Furukawa, Y.-i. Sakamoto, H. Hakamata, Y. Komohara, M. Takeya, Y. Sasaki, A. Miyazaki, and S. Horiuchi Acyl-Coenzyme A:Cholesterol Acyltransferase-2 (ACAT-2) Is Responsible for Elevated Intestinal ACAT Activity in Diabetic Rats Arterioscler. Thromb. Vasc. Biol., September 1, 2004; 24(9): 1689 - 1695. [Abstract] [Full Text] [PDF]
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N.D. Vaziri and K.H. Liang Acyl-Coenzyme A:Cholesterol Acyltransferase Inhibition Ameliorates Proteinuria, Hyperlipidemia, Lecithin-Cholesterol Acyltransferase, SRB-1, and Low-Denisty Lipoprotein Receptor Deficiencies in Nephrotic Syndrome Circulation, July 27, 2004; 110(4): 419 - 425. [Abstract] [Full Text] [PDF]
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D. Sorger, K. Athenstaedt, C. Hrastnik, and G. Daum A Yeast Strain Lacking Lipid Particles Bears a Defect in Ergosterol Formation J. Biol. Chem., July 23, 2004; 279(30): 31190 - 31196. [Abstract] [Full Text] [PDF]
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T. M. Lewin, N. M. J. Schwerbrock, D. P. Lee, and R. A. Coleman Identification of a New Glycerol-3-phosphate Acyltransferase Isoenzyme, mtGPAT2, in Mitochondria J. Biol. Chem., April 2, 2004; 279(14): 13488 - 13495. [Abstract] [Full Text] [PDF]
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J. L. Smith, K. Rangaraj, R. Simpson, D. J. Maclean, L. K. Nathanson, K. A. Stuart, S. P. Scott, G. A. Ramm, and J. de Jersey Quantitative analysis of the expression of ACAT genes in human tissues by real-time PCR2 J. Lipid Res., April 1, 2004; 45(4): 686 - 696. [Abstract] [Full Text] [PDF]
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A. T. Lada, M. Davis, C. Kent, J. Chapman, H. Tomoda, S. Omura, and L. L. Rudel Identification of ACAT1- and ACAT2-specific inhibitors using a novel, cell-based fluorescence assay: individual ACAT uniqueness J. Lipid Res., February 1, 2004; 45(2): 378 - 386. [Abstract] [Full Text] [PDF]
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 I. Namatame, H. Tomoda, S. Ishibashi, and S. Omura Antiatherogenic activity of fungal beauveriolides, inhibitors of lipid droplet accumulation in macrophages PNAS, January 20, 2004; 101(3): 737 - 742. [Abstract] [Full Text] [PDF]
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 S. Lin, X. Lu, C. C.Y. Chang, and T.-Y. Chang Human Acyl-Coenzyme A:Cholesterol Acyltransferase Expressed in Chinese Hamster Ovary Cells: Membrane Topology and Active Site Location Mol. Biol. Cell, June 1, 2003; 14(6): 2447 - 2460. [Abstract] [Full Text] [PDF]
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Y. Zhang, C. Yu, J. Liu, T. A. Spencer, C. C. Y. Chang, and T.-Y. Chang Cholesterol Is Superior to 7-Ketocholesterol or 7alpha -Hydroxycholesterol as an Allosteric Activator for Acyl-coenzyme A:Cholesterol Acyltransferase 1 J. Biol. Chem., March 21, 2003; 278(13): 11642 - 11647. [Abstract] [Full Text] [PDF]
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E. L. Willner, B. Tow, K. K. Buhman, M. Wilson, D. A. Sanan, L. L. Rudel, and R. V. Farese Jr. Deficiency of acyl CoA:cholesterol acyltransferase 2 prevents atherosclerosis in apolipoprotein E-deficient mice PNAS, February 4, 2003; 100(3): 1262 - 1267. [Abstract] [Full Text] [PDF]
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H. Chao, M. Zhou, A. McIntosh, F. Schroeder, and A. B. Kier ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT J. Lipid Res., January 1, 2003; 44(1): 72 - 83. [Abstract] [Full Text] [PDF]
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K. Liang and N. D. Vaziri Upregulation of acyl-CoA:cholesterol acyltransferase in chronic renal failure Am J Physiol Endocrinol Metab, October 1, 2002; 283(4): E676 - E681. [Abstract] [Full Text] [PDF]
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C. Xie, L. A. Woollett, S. D. Turley, and J. M. Dietschy Fatty acids differentially regulate hepatic cholesteryl ester formation and incorporation into lipoproteins in the liver of the mouse J. Lipid Res., September 1, 2002; 43(9): 1508 - 1519. [Abstract] [Full Text] [PDF]
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L. L. Rudel, M. Davis, J. Sawyer, R. Shah, and J. Wallace Primates Highly Responsive to Dietary Cholesterol Up-regulate Hepatic ACAT2, and Less Responsive Primates Do Not J. Biol. Chem., August 23, 2002; 277(35): 31401 - 31406. [Abstract] [Full Text] [PDF]
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P. Oelkers, D. Cromley, M. Padamsee, J. T. Billheimer, and S. L. Sturley The DGA1 Gene Determines a Second Triglyceride Synthetic Pathway in Yeast J. Biol. Chem., March 8, 2002; 277(11): 8877 - 8881. [Abstract] [Full Text] [PDF]
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J. W. Furbee Jr., O. Francone, and J. S. Parks In vivo contribution of LCAT to apolipoprotein B lipoprotein cholesteryl esters in LDL receptor and apolipoprotein E knockout mice J. Lipid Res., March 1, 2002; 43(3): 428 - 437. [Abstract] [Full Text] [PDF]
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 H.H. HOBBS, G.A. GRAF, L. YU, K.R. WILUND, and J.C. COHEN Genetic Defenses against Hypercholesterolemia Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 499 - 506. [Abstract] [PDF]
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T. Y. Chang, C. C. Y. Chang, X. Lu, and S. Lin Catalysis of ACAT may be completed within the plane of the membrane: a working hypothesis J. Lipid Res., December 1, 2001; 42(12): 1933 - 1938. [Abstract] [Full Text] [PDF]
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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]
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 W. M. Pandak, C. Schwarz, P. B. Hylemon, D. Mallonee, K. Valerie, D. M. Heuman, R. A. Fisher, K. Redford, and Z. R. Vlahcevic Effects of CYP7A1 overexpression on cholesterol and bile acid homeostasis Am J Physiol Gastrointest Liver Physiol, October 1, 2001; 281(4): G878 - G889. [Abstract] [Full Text] [PDF]
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H. C. Chen Molecular Mechanisms of Sterol Absorption J. Nutr., October 1, 2001; 131(10): 2603 - 2605. [Abstract] [Full Text] [PDF]
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 K. Jensen-Pergakes, Z. Guo, M. Giattina, S. L. Sturley, and M. Bard Transcriptional Regulation of the Two Sterol Esterification Genes in the Yeast Saccharomyces cerevisiae J. Bacteriol., September 1, 2001; 183(17): 4950 - 4957. [Abstract] [Full Text] [PDF]
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Z. Guo, D. Cromley, J. T. Billheimer, and S. L. Sturley Identification of potential substrate-binding sites in yeast and human acyl-CoA sterol acyltransferases by mutagenesis of conserved sequences J. Lipid Res., August 1, 2001; 42(8): 1282 - 1291. [Abstract] [Full Text] [PDF]
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 C. K. Garcia, G. Mues, Y. Liao, T. Hyatt, N. Patil, J. C. Cohen, and H. H. Hobbs Sequence Diversity in Genes of Lipid Metabolism Genome Res., June 1, 2001; 11(6): 1043 - 1052. [Abstract] [Full Text] [PDF]
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L. J. Wilcox, N. M. Borradaile, L. E. de Dreu, and M. W. Huff Secretion of hepatocyte apoB is inhibited by the flavonoids, naringenin and hesperetin, via reduced activity and expression of ACAT2 and MTP J. Lipid Res., May 1, 2001; 42(5): 725 - 734. [Abstract] [Full Text]
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Z. Zhang, D. Li, D. E. Blanchard, S. R. Lear, S. K. Erickson, and T. A. Spencer Key regulatory oxysterols in liver: analysis as {{Delta}}4-3-ketone derivatives by HPLC and response to physiological perturbations J. Lipid Res., April 1, 2001; 42(4): 649 - 658. [Abstract] [Full Text]
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K. K. Maung, A. Miyazaki, H. Nomiyama, C. C. Y. Chang, T.-Y. Chang, and S. Horiuchi Induction of acyl-coenzyme A:cholesterol acyltransferase-1 by 1,25-dihydroxyvitamin D3 or 9-cis-retinoic acid in undifferentiated THP-1 cells J. Lipid Res., February 1, 2001; 42(2): 181 - 187. [Abstract] [Full Text]
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V. W. Bloks, T. Plösch, H. van Goor, H. Roelofsen, J. Baller, R. Havinga, H. J. Verkade, A. van Tol, P. L. M. Jansen, and F. Kuipers Hyperlipidemia and atherosclerosis associated with liver disease in ferrochelatase-deficient mice J. Lipid Res., January 1, 2001; 42(1): 41 - 50. [Abstract] [Full Text]
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 A. Ruiz, M. H. Kuehn, J. L. Andorf, E. Stone, G. S. Hageman, and D. Bok Genomic Organization and Mutation Analysis of the Gene Encoding Lecithin Retinol Acyltransferase in Human Retinal Pigment Epithelium Invest. Ophthalmol. Vis. Sci., January 1, 2001; 42(1): 31 - 37. [Abstract] [Full Text]
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R. G. Lee, M. C. Willingham, M. A. Davis, K. A. Skinner, and L. L. Rudel Differential expression of ACAT1 and ACAT2 among cells within liver, intestine, kidney, and adrenal of nonhuman primates J. Lipid Res., December 1, 2000; 41(12): 1991 - 2001. [Abstract] [Full Text]
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C. W. Joyce, G. S. Shelness, M. A. Davis, R. G. Lee, K. Skinner, R. A. Anderson, and L. L. Rudel ACAT1 and ACAT2 Membrane Topology Segregates a Serine Residue Essential for Activity to Opposite Sides of the Endoplasmic Reticulum Membrane Mol. Biol. Cell, November 1, 2000; 11(11): 3675 - 3687. [Abstract] [Full Text]
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C. G. Panousis and S. H. Zuckerman Regulation of cholesterol distribution in macrophage-derived foam cells by interferon-{gamma} J. Lipid Res., January 1, 2000; 41(1): 75 - 83. [Abstract] [Full Text]
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J. Kusunoki, K. Aragane, T. Kitamine, H. Kozono, K. Kano, K. Fujinami, K. Kojima, T. Chiwata, and Y. Sekine Postprandial Hyperlipidemia in Streptozotocin-Induced Diabetic Rats Is Due to Abnormal Increase in Intestinal Acyl Coenzyme A:Cholesterol Acyltransferase Activity Arterioscler. Thromb. Vasc. Biol., January 1, 2000; 20(1): 171 - 178. [Abstract] [Full Text] [PDF]
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 N. Sakashita, A. Miyazaki, M. Takeya, S. Horiuchi, C. C. Y. Chang, T.-Y. Chang, and K. Takahashi Localization of Human Acyl-Coenzyme A:Cholesterol Acyltransferase-1 (ACAT-1) in Macrophages and in Various Tissues Am. J. Pathol., January 1, 2000; 156(1): 227 - 236. [Abstract] [Full Text] [PDF]
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C. Yu, J. Chen, S. Lin, J. Liu, C. C. Y. Chang, and T.-Y. Chang Human Acyl-CoA:Cholesterol Acyltransferase-1 Is a Homotetrameric Enzyme in Intact Cells and in Vitro J. Biol. Chem., December 17, 1999; 274(51): 36139 - 36145. [Abstract] [Full Text] [PDF]
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F. Lammert, D. Q-H. Wang, B. Paigen, and M. C. Carey Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: integrated activities of hepatic lipid regulatory enzymes J. Lipid Res., November 1, 1999; 40(11): 2080 - 2090. [Abstract] [Full Text]
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S. K. Cheema and L. B. Agellon Metabolism of Cholesterol Is Altered in the Liver of C3H Mice Fed Fats Enriched with Different C-18 Fatty Acids J. Nutr., September 1, 1999; 129(9): 1718 - 1724. [Abstract] [Full Text]
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S. Lin, D. Cheng, M.-S. Liu, J. Chen, and T.-Y. Chang Human Acyl-CoA:Cholesterol Acyltransferase-1 in the Endoplasmic Reticulum Contains Seven Transmembrane Domains J. Biol. Chem., August 13, 1999; 274(33): 23276 - 23285. [Abstract] [Full Text] [PDF]
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J. R. Burnett, L. J. Wilcox, D. E. Telford, S. J. Kleinstiver, P. H. R. Barrett, R. S. Newton, and M. W. Huff Inhibition of ACAT by avasimibe decreases both VLDL and LDL apolipoprotein B production in miniature pigs J. Lipid Res., July 1, 1999; 40(7): 1317 - 1328. [Abstract] [Full Text]
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R. L. Hamilton, A. Moorehouse, S. R. Lear, J. S. Wong, and S. K. Erickson A rapid calcium precipitation method of recovering large amounts of highly pure hepatocyte rough endoplasmic reticulum J. Lipid Res., June 1, 1999; 40(6): 1140 - 1147. [Abstract] [Full Text]
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D. Warnecke, R. Erdmann, A. Fahl, B. Hube, F. Muller, T. Zank, U. Zahringer, and E. Heinz Cloning and Functional Expression of UGT Genes Encoding Sterol Glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum J. Biol. Chem., May 7, 1999; 274(19): 13048 - 13059. [Abstract] [Full Text] [PDF]
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C. D. Jolley, J. M. Dietschy, and S. D. Turley Genetic differences in cholesterol absorption in 129/Sv and C57BL/6 mice: effect on cholesterol responsiveness Am J Physiol Gastrointest Liver Physiol, May 1, 1999; 276(5): G1117 - G1124. [Abstract] [Full Text] [PDF]
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B.-L. Li, X.-L. Li, Z.-J. Duan, O. Lee, S. Lin, Z.-M. Ma, C. C. Y. Chang, X.-Y. Yang, J. P. Park, T. K. Mohandas, et al. Human Acyl-CoA:Cholesterol Acyltransferase-1 (ACAT-1) Gene Organization and Evidence That the 4.3-Kilobase ACAT-1 mRNA Is Produced from Two Different Chromosomes J. Biol. Chem., April 16, 1999; 274(16): 11060 - 11071. [Abstract] [Full Text] [PDF]
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R. L. Walzem and R. L. Hamilton Introduction J. Nutr., February 1, 1999; 129(2): 449 - 449. [Full Text]
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B. A. Janowski, M. J. Grogan, S. A. Jones, G. B. Wisely, S. A. Kliewer, E. J. Corey, and D. J. Mangelsdorf Structural requirements of ligands for the oxysterol liver X receptors LXRalpha and LXRbeta PNAS, January 5, 1999; 96(1): 266 - 271. [Abstract] [Full Text] [PDF]
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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]
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H. Yagyu, T. Kitamine, J.-i. Osuga, R.-i. Tozawa, Z. Chen, Y. Kaji, T. Oka, S. Perrey, Y. Tamura, K. Ohashi, et al. Absence of ACAT-1 Attenuates Atherosclerosis but Causes Dry Eye and Cutaneous Xanthomatosis in Mice with Congenital Hyperlipidemia J. Biol. Chem., July 7, 2000; 275(28): 21324 - 21330. [Abstract] [Full Text] [PDF]
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C. C. Y. Chang, N. Sakashita, K. Ornvold, O. Lee, E. T. Chang, R. Dong, S. Lin, C.-Y. G. Lee, S. C. Strom, R. Kashyap, et al. Immunological Quantitation and Localization of ACAT-1 and ACAT-2 in Human Liver and Small Intestine J. Biol. Chem., September 1, 2000; 275(36): 28083 - 28092. [Abstract] [Full Text] [PDF]
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D. K. Spady, M. N. Willard, and R. S. Meidell Role of Acyl-Coenzyme A:Cholesterol Acyltransferase-1 in the Control of Hepatic Very Low Density Lipoprotein Secretion and Low Density Lipoprotein Receptor Expression in the Mouse and Hamster J. Biol. Chem., August 25, 2000; 275(35): 27005 - 27012. [Abstract] [Full Text] [PDF]
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J.-B. Yang, Z.-J. Duan, W. Yao, O. Lee, L. Yang, X.-Y. Yang, X. Sun, C. C. Y. Chang, T.-Y. Chang, and B.-L. Li Synergistic Transcriptional Activation of Human Acyl-coenzyme A: Cholesterol Acyltransterase-1 Gene by Interferon-gamma and All-trans-Retinoic Acid THP-1 Cells J. Biol. Chem., June 8, 2001; 276(24): 20989 - 20998. [Abstract] [Full Text] [PDF]
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S. Sonda, L.-M. Ting, S. Novak, K. Kim, J. J. Maher, R. V. Farese Jr., and J. D. Ernst Cholesterol Esterification by Host and Parasite Is Essential for Optimal Proliferation of Toxoplasma gondii J. Biol. Chem., September 7, 2001; 276(37): 34434 - 34440. [Abstract] [Full Text] [PDF]
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K. K. Buhman, H. C. Chen, and R. V. Farese Jr. The Enzymes of Neutral Lipid Synthesis J. Biol. Chem., October 26, 2001; 276(44): 40369 - 40372. [Full Text] [PDF]
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