THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 275, No. 28, Issue of July 14, pp. 21324 –21330, 2000
Printed in U.S.A.

Absence of ACAT-1 Attenuates Atherosclerosis but Causes Dry Eye
and Cutaneous Xanthomatosis in Mice with Congenital
Hyperlipidemia*
Received for publication, March 26, 2000, and in revised form, April 17, 2000
Published, JBC Papers in Press, April 20, 2000, DOI 10.1074/jbc.M002541200

Hiroaki Yagyu‡§, Tetsuya Kitamine‡§, Jun-ichi Osuga‡, Ryu-ichi Tozawa‡, Zhong Chen‡,Yuichi Kaji¶, Teruaki Oka储, Stéphane Perrey‡, Yoshiaki Tamura‡, Ken Ohashi‡,
Hiroaki Okazaki‡, Naoya Yahagi‡, Futoshi Shionoiri‡, Yoko Iizuka‡, Kenji Harada‡,
Hitoshi Shimano‡, Hidetoshi Yamashita¶**, Takanari Gotoda‡, Nobuhiro Yamada‡ ‡‡,and Shun Ishibashi‡ §§From the Departments of ‡Metabolic Diseases, ¶Ophthalmology, and 储Pathology, Faculty of Medicine, University of Tokyo,
Hongo, Tokyo 113-8655, Japan

Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes esterification of cellular cholesterol. To investigate the role of ACAT-1 in atherosclerosis, we have generated ACAT-1 null (ACAT-1ⴚ/ⴚ) mice. ACAT activities
were present in the liver and intestine but were completely absent in adrenal, testes, ovaries, and peritoneal
macrophages in our ACAT-1ⴚ/ⴚ mice. The ACAT-1ⴚ/ⴚ
mice had decreased openings of the eyes because of
atrophy of the meibomian glands, a modified form of
sebaceous glands normally expressing high ACAT activities. This phenotype is similar to dry eye syndrome inhumans. To determine the role of ACAT-1 in atherogenesis, we crossed the ACAT-1ⴚ/ⴚ mice with mice lacking
apolipoprotein (apo) E or the low density lipoprotein
receptor (LDLR), hyperlipidemic models susceptible toatherosclerosis. High fat feeding resulted in extensive
cutaneous xanthomatosis with loss of hair in both
ACAT-1ⴚ/ⴚ:apo Eⴚ/ⴚ and ACAT-1ⴚ/ⴚ:LDLRⴚ/ⴚ mice.
Free cholesterol content was significantly increased intheir skin. Aortic fatty streak lesion size as well as cholesteryl ester content were moderately reduced in both
double mutant mice compared with their respective
controls. These results indicate that the local inhibitionof ACAT activity in tissue macrophages is protective
against cholesteryl ester accumulation but causes cutaneous xanthomatosis in mice that lack apo E or LDLR.

Esterification of intracellular cholesterol is catalyzed by a

* This work was supported by a grant-in-aid for scientific researchfrom the Ministry of Education, Science and Culture, the Promotion of
Fundamental Studies in Health Science of The Organization for Pharmaceutical Safety and Research, and Health Sciences Research grants(Research on Human Genome and Gene Therapy) from the Ministry of
Health and Welfare. The costs of publication of this article were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
** Present address: Dept. of Ophthalmology, Yamagata University
School of Medicine, 2-2-2 Iidanishi, Yamagata, Yamagata 990-9585,
Japan.
‡‡ Present address: Metabolism, Endocrinology, and Atherosclerosis,
Inst. of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai,
Tsukuba, Ibaraki 305-8575, Japan.
§§ To whom correspondence should be addressed: Dept. of Metabolic
Diseases, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-8655, Japan. Tel.: 81-3-3815-5411 (ext. 33113);
Fax: 81-3-5802-2955; E-mail: ishibash-tky@umin.ac.jp.

microsomal enzyme, acyl-CoA:cholesterol acyltransferase(ACAT; EC 2.3.1.26) (1, 2).1 In the liver and small intestine,cholesterol is esterified by ACAT and incorporated into apolipoprotein (apo) B-containing lipoproteins, which are subsequently secreted into circulation. After lipolytic conversion,these lipoproteins are taken up by the cells of various organs,where their cholesteryl esters (CE) are hydrolyzed in lysosomes
to liberate free cholesterol (FC), which is re-esterified by ACAT
again for storage in cytoplasmic lipid droplets.
Two ACAT genes have been identified in mammals (3– 6).
ACAT-1, which was originally cloned from a macrophage cDNA
library (3), is ubiquitously expressed. In particular, it is highly
expressed in preputial glands, steroidogenic organs, sebaceous
glands, and macrophages (7–9). However, its physiologicalroles in each organ are yet to be determined. ACAT-1 is also
expressed in macrophage-derived foam cells in human atherosclerotic lesions (10), where it is thought to mediate foam cell
formation, an initial event of atherosclerosis. Therefore, it isplausible that inhibition of ACAT in the arterial wall cells
exerts a protective effect against atherosclerosis. In contrast,
ACAT-2 is expressed only in the liver and small intestine andis conceivably involved in lipoprotein assembly and secretion(4 – 6). Consistently, ACAT activities were eliminated from themacrophages and adrenal glands but not from the liver in
ACAT-1 knockout mice reported by Meiner et al. (11, 12).
To further explore the other unknown functions of ACAT-1
and to determine whether the elimination of ACAT-1 has ananti-atherogenic effect, we have generated ACAT-1 knockout
mice by homologous recombination with a targeting vector thatis designed to delete the first membrane spanning region plus
a serine 268 that is crucial for the catalytic activity of ACAT-1(13) and crossed these animals with hyperlipidemic mutant
mice, apo E-deficient (apo E⫺/⫺) (14), and the low density
lipoprotein (LDL) receptor-deficient (LDLR⫺/⫺) mice (15),which are susceptible to atherosclerosis.
MATERIALS AND METHODS

General Methods—Standard molecular biology techniques were used(16). Before sacrifice, mice were anesthetized with pentobarbital. Sickmice were euthanized. The current experiments were performed in

1
The abbreviations used are: ACAT, Acyl-CoA:cholesterol acyltransferase; apo, apolipoprotein; LDL, low density lipoprotein; LDLR, LDL
receptor; HPLC, high performance liquid chromatography; TC, totalcholesterol; TG, triglycerides; FC, free cholesterol; CE, cholesteryl esters; HDL-C, cholesterol contents in high density lipoprotein fraction;
kb, kilobase(s).

21324This paper is available on line at http://www.jbc.org

Eye, Skin, and Vascular Lesions in Hyperlipemic ACAT-1KO Miceaccord with institutional guidelines for animal experiments at the University of Tokyo.
Diets—Three diets were used: (i) a normal chow diet (MF diet from
Oriental Yeast Co., Tokyo) that contained 5.6% (w/w) fat with 0.09%(w/w) cholesterol; (ii) a high fat diet A: MF diet containing 0.15% (w/w)
cholesterol and 15% (w/w) butter (essentially similar to Western style
diet (17)); (iii) a high fat diet B: MF diet containing 1.25% (w/w)
cholesterol, 15% (w/w) cocoa butter, 0.5% (w/w) cholic acid essentially as
described by Paigen et al. (18, 19). Preliminary experiments have shownthat feeding with high fat diet B made many of the apo E⫺/⫺ mice sickprobably because of extreme hyperlipidemia irrespective of the presence of the ACAT-1 gene. On the other hand, LDLR⫺/⫺ mice developed
only a minimal lesions in 8 weeks when fed with high fat diet A.
Accordingly, two different high fat diets were used to analyze ACAT1⫺/⫺:apo E⫺/⫺ or ACAT-1⫺/⫺:LDLR⫺/⫺ double knockout mice.
Generation of ACAT-1 Knockout Mice—A fragment (1623 base pairs)of mouse ACAT-1 cDNA was amplified by polymerase chain reaction
using primers that were designed based on reported sequences of human ACAT-1 cDNA (sense primer, 5⬘-ATGGTGGGTGAAGAGAAGATGTCTCTAAGAA-3⬘, and antisense primer, 5⬘-AAACACGTAACGACAAGTCCAGGAACGTGG-3⬘) (3) and was subcloned into pCRII(Invitrogen). After labeling with 32P using the Megaprime DNA labelingsystem (Amersham Pharmacia Biotech) and [␣-32P]dCTP (Amersham
Pharmacia Biotech), the cDNA fragment was used as a probe to screen
a 129/Sv mouse genomic library, which had been constructed in
Lambda Dash II (Stratagene) as described previously (20, 21). One
genomic clone coding the 5⬘ side of ACAT-1 cDNA was sequenced. A
1-kb fragment in intron 5 was amplified by polymerase chain reaction
using the following primers: sense primer, 5⬘-CGCTCGAGTTCAGTCCTCATTAATGAG-3⬘ and antisense primer, 5⬘-CGCTCGAGGTAAATTGTTCTGATGTGG-3⬘, and used as the short arm (22). Another 13-kb
genomic clone that contained a fragment of the ACAT gene spanning
intron 11 to exon 16 was directly used as the long arm. A replacementtype targeting vector was constructed by ligating the short and long arms
into the XhoI and NotI sites, respectively, of the vector pPolIIshortneobpA-HSVTK as described previously (15, 21) (see Fig. 1A). A 4-kb pieceof the gene that contained exons 6 –10, which encoded the first membrane
spanning domain (23) and serine 268 that is crucial for the enzyme
activity (13), was replaced by a neomycin-resistant cassette. Theoretically,
homologous recombination would produce a totally inactive enzyme.
After digestion with SalI, the vector was electroporated into JH1embryonic stem cells. Targeted clones that had been selected in thepresence of G418 and 1-(2-deoxy, 2-fluoro-␤-D-arabinofuranosyl)-5 iodouracil were identified by polymerase chain reaction using the following primers: 5⬘-TCTGGAGAAAAATGCAGGCTTCTTAC-3⬘ and 5⬘GATTGGGAAGACAATAGCAGGCATGC-3⬘ (see Fig. 1A). Homologous
recombination was verified by Southern blot analysis after digestion
with EcoRI using a fragment that was upstream of the short arm as aprobe (probe B; see Fig. 1A). Targeted embryonic stem clones were
injected into the C57BL/6J blastocysts, yielding four lines of chimericmice that transmitted the disrupted allele through the germline. All
experiments reported here were performed with 129/Sv-C57BL/6J hybrid descendants (F1 and subsequent generations) of these animals. For
genotyping, tail DNA was digested with BamHI and subjected to Southern blot analyses using probe A (see Fig. 1A).
Breeding Experiments—ACAT-1⫹/⫺ mice were cross-bred to apoE⫺/⫺ or LDLR⫺/⫺ mice to produce mice that were heterozygous for the
disrupted alleles of both ACAT-1 and either apo E or the LDL receptor
loci. The intercross of these animals was performed to produce ACAT1⫹/⫺:apo E⫺/⫺ and ACAT-1⫹/⫺:LDLR⫺/⫺. Brother-sister mating of
ACAT-1⫹/⫺:apo E⫺/⫺ or ACAT-1⫹/⫺:LDLR⫺/⫺ mice was performed
to produce a set of apo E-deficient mice (ACAT-1⫹/⫹:apo E⫺/⫺, ACAT1⫹/⫺:apo E⫺/⫺, and ACAT-1⫺/⫺:apo E⫺/⫺) or a set of the LDL receptor-deficient mice (ACAT-1⫹/⫹:LDLR⫺/⫺, ACAT-1⫹/⫺:LDLR⫺/⫺, andACAT-1⫺/⫺:LDLR⫺/⫺), respectively. Littermates were used as controls. Therefore, genetic background of these animals were mixed: 50%
C57BL/6J and 50% 129/Sv.
Preparation of Peritoneal Macrophages—Peritoneal macrophages
were prepared as described previously (17).
Northern Blot Analyses—Total RNA was isolated from cultured peritoneal macrophages by using TRIZOL reagent (Life Technologies, Inc.).
10 ␮g of total RNA was subjected to electrophoresis in 1% agarose gel
containing formamide and transferred to a nylon filter (Hybond N, Amersham Pharmacia Biotech). cDNA probes (the 1623-base pair coding
region of ACAT-1 and ␤-actin) were radiolabeled with [␥-32P]dCTP. After
prehybridization for 2 h, blots were hybridized in RapidhybR buffer (Amersham Pharmacia Biotech) with indicated probes for 1 h at 65 °C.
Immunoblot Analyses—cDNA fragment encoding N-terminal 12521325

amino acids of ACAT-1 was amplified by polymerase chain reaction
using primers (sense primer, 5⬘-GAGCATATGTCACTAAGAAACCGGCTGTCA-3⬘; antisense primer, 5⬘-TTCGGATCCTTAGTCTAAAAGAGACTGCCT-3⬘) and cDNA pool made from mouse peritoneal macrophages and subcloned into the expression vector PW6A (24). Aftertransformation, Escherichia coli, BL21(DE3), were grown in LB medium in the presence of isopropyl-␤-D-thiogalactopyranoside. The cells
were disrupted by sonication and centrifuged to remove cellular debris.
The recombinant ACAT-1 peptide was purified by chromatographyusing SP Sepharose FF column followed by Superdex 75 columns.
ACAT-1⫺/⫺ mice were immunized with the purified recombinant
ACAT-1 peptide. Standard technique was used to generate a monoclonal antibody (25).
Peritoneal macrophages (1 ⫻ 107 cells) were resuspended in 100 ␮l ofbuffer A (50 mM Tris-HCl, pH 7.8, 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride) containing 5% SDS. After an overnight incubation at
4 °C, 50 ␮g of protein was subjected to SDS/polyacrylamide gel electrophoresis. After transfer to a nitrocellulose membrane, immunoblot was
performed using an ECL kit (Amersham Pharmacia Biotech) with antimouse IgG (Amersham Pharmacia Biotech) as the secondary antibody.
Plasma Lipoprotein Analyses—After a 16-h fast, blood was collectedfrom the retro-orbital venous plexus into tubes containing EDTA.
Plasma levels of total cholesterol (TC) and triglycerides (TG) were
determined enzymatically using kits (Determiner TC555 and Determiner
TG555, Kyowa Medex). Lipoproteins were fractionated by high performance chromatography (HPLC), and the cholesterol contents in high density lipoprotein fraction (HDL-C) were determined as described (17).
Tissue and Cellular Lipids—Lipids were extracted from tissues by
the method of Folch et al. (26). TC and FC were determined by fluorometric microassay according to a modified method of Heider and Boyett(27) with the exception that 0.01% (v/v) Triton X-100 was used insteadof Carbowax-600.
Assay of ACAT Activity—Tissue was homogenized in buffer B (0.25 M
sucrose, 1 mM EDTA, 2 ␮g/ml leupeptin, and 50 mM Tris-HCl, pH 7.0)
and centrifuged at 107,000 ⫻ g for 60 min at 4 °C. The pellets were
resuspended and used for the assay. ACAT activity in microsomes orcell homogenates was determined by the rate of incorporation of [14C]
oleoyl-CoA into the CE fraction according to Billheimer et al. with minor
modifications (28, 29).
Histology—Tissues were fixed with neutral-buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin. The aortic
cross-sectional lesion area was evaluated according to a modified
method of Paigen et al. (17, 30). In brief, the hearts were perfused withsaline containing 4% (w/v) formalin and fixed for more than 48 h in thesame solution. The basal half of the hearts was embedded in Tissue-Tek
OCT compound (Miles, Inc.), and the serial sections were captured
using Cryostat microtome (6-␮m thick) as described (31). Four sections,each separated by 60 ␮m, were used to evaluate the lesions: two at theend of the aortic sinus and two at the junctional site of sinus andascending aorta. The sections were stained with Oil Red O and counterstained with hematoxylin.
Statistics—Data are represented as the means ⫾ S.D. Student’s t test
and analysis of variance were used to compare the mean values between
two and three groups, respectively.
RESULTS

Tissue-specific Role of ACAT-1—A replacement-type vector
was used to generate chimeric mice (Fig. 1A). Heterozygous
ACAT-1 knockout mice (ACAT-1⫹/⫺) born by these chimera,
which were viable and fertile, were intercrossed to obtain homozygous ACAT-1 knockout mice (ACAT-1⫺/⫺). Wild type,
heterozygotes, and homozygotes were born in accordance with
Mendelian fashion (⫹/⫹:⫹/⫺:⫺/⫺ ⫽ 17:40:19, ␹2 ⫽ 0.16; p ⫽0.92) (Fig. 1B).
To determine whether the expression of the ACAT-1 gene
was ablated, we performed Northern blot analyses (Fig. 1C).
Although three major mRNA transcripts (9.0, 4.6, and 3.5 kb)
were detectable in wild-type macrophages, adrenal glands, ovaries, and testes, no bands were detectable in these organs from
ACAT-1⫺/⫺ mice. Approximately one-half of the amounts ofthe three transcripts were expressed by the cells from ACAT1⫹/⫺ mice. Consistent with the Northern blot results, immunoblot analyses revealed no proteins that were immunoreactivewith an antibody raised against recombinant mouse ACAT-1 in
the peritoneal macrophages (Fig. 1D), adrenal glands, ovaries,

21326

Eye, Skin, and Vascular Lesions in Hyperlipemic ACAT-1KO Mice

FIG. 1. Targeted disruption of the ACAT-1 gene. A, a map of the ACAT-1 locus, together with the sequence replacement gene-targeting
vector and the targeted ACAT-1 allele. The gene targeting event replaced a 4-kb fragment containing the first transmembrane domain and aconserved serine with a cassette containing a neomycin-resistance gene (neor) driven by the RNA polymerase II promoter. Two copies of herpes
simplex virus thymidine kinase (HSV-TK) expression cassette were used as a negative selection marker. Gene-targeting events were verified bySouthern blot analyses using EcoRI and probe B for embryonic stem cell DNA and BamHI and probe A for tail DNA. B, Southern blot of BamHI
fragment in the targeted allele is 7 kb versus 10 kb for the wild-type allele when hybridized with probe A. DNA was isolated from tails of offspringof heterozygous matings. C, Northern blot analyses. 10 ␮g of total RNA was isolated from peritoneal macrophages in culture and subjected to
Northern blot analyses. Two probes were used: cDNA fragment containing the entire coding region of ACAT-1 (1623 base pairs) and ␤-actin as areference. Three major transcripts are indicated. D, immnunoblot analyses. Peritoneal macrophages were isolated, and 50 ␮g of cellular protein
was subjected to SDS/polyacrylamide gel electrophoresis. After transfer to a nitrocellulose membrane, immunoblot was performed using an ECL
kit with the antibody against a recombinant peptide encoding N-terminal 125 amino acids of ACAT-1 and anti-mouse IgG as the secondary
antibody.

and testes. Therefore, it is reasonable to conclude that our
ACAT-1⫺/⫺ mice were virtually null for ACAT-1.
Table I compares ACAT activities in microsome fractions
prepared from the liver, small intestine, adrenal glands, testes,and ovaries, between the wild-type and ACAT-1⫺/⫺ mice. In
ACAT-1⫺/⫺ mice, ACAT activity was barely detectable in theadrenal glands, testes, and ovaries that normally express substantial ACAT activity in the wild-type mice. In contrast tothese steroidogenic organs, the liver and small intestine fromthe ACAT-1⫺/⫺ mice expressed ACAT activities similar to
those from wild-type mice.
Atrophy of Meibomian Gland—ACAT-1⫺/⫺ mice weregrossly normal except that they had narrow eye fissures andlipid-depleted adrenal glands. Abnormal facial expression wasnoted around the weaning age (3– 4 weeks) in almost all ACAT1⫺/⫺ mice (Fig. 2). Pathological examination of the eye balls
and adnexa revealed that ACAT-1⫺/⫺ mice had atrophic acinar
cells in the meibomian glands (Fig. 2). Upon bio-microscopic
examination, fine punctate stripping of the corneal epithelium
was observed. No other abnormalities were found in conjunctiva,
cornea, retina, or Harderian glands. Lipids in adrenal cortex
were depleted in ACAT-1⫺/⫺ mice (data not shown).
We measured FC and CE contents in steroidogenic organs
whose ACAT activities were markedly reduced in ACAT-1⫺/⫺
mice and meibomian glands that exhibited remarkable acinar
cell shrinkage in ACAT-1⫺/⫺ mice (Fig. 3). In wild-type mice,
most of cholesterol was stored in its esterified form in adrenal
glands, ovaries, and meibomian glands. CE contents of theseorgans were markedly reduced to a base-line level in ACAT1⫺/⫺ mice. Interestingly, FC content was increased only in the
meibomian glands of ACAT-1⫺/⫺ mice. There were virtually no

TABLE I
Microsomal ACAT activities in various organs of wild-type and
ACAT-1⫺/⫺ miceMicrosome fractions were prepared from the indicated organs ofwild-type (⫹/⫹) and ACAT-1⫺/⫺ (⫺/⫺) mice aged 3 months. ACAT
activities were measured as described under “Materials and Methods.”
TissueACATnACAT activity
pmol/mg/min

Liver
Small intestine
AdrenalTestis
Ovary

⫹/⫹
⫺/⫺
⫹/⫹
⫺/⫺
⫹/⫹
⫺/⫺
⫹/⫹
⫺/⫺
⫹/⫹
⫺/⫺

3
3
2
2
3a
3a
1
1
2a
2a

156 ⫾ 20139 ⫾ 36
111
221
286
⬍5
192
⬍5
2330
23

a
The assay was performed on pooled samples from the indicatednumber of animals. Data represent the means ⫾ S.D.

differences in CE content of testes between wild-type and
ACAT-1⫺/⫺ mice.
Massive Cutaneous Xanthomatosis and Alopecia in ACAT1⫺/⫺ Mice Lacking apo E or the LDL Receptor—To determine
whether hyperlipidemia affects the phenotypes of ACAT-1⫺/⫺
mice, we generated mice lacking both ACAT-1 and either apo E(ACAT-1⫺/⫺:apo E⫺/⫺) or LDL receptor (ACAT-1⫺/⫺:LDLR⫺/⫺) by cross-breeding and used their littermates ascontrols. Although the inactivation of the ACAT-1 gene did nothave a significant effect on the lipoprotein profiles in apo E⫺/⫺
mice, it slightly increased intermediate density lipoprotein/
LDL cholesterol levels in LDLR⫺/⫺ mice (Table II). TheseEye, Skin, and Vascular Lesions in Hyperlipemic ACAT-1KO Mice

21327

FIG. 2. Facial appearance and histology of meibomian glands of wildtype (ⴙ/ⴙ) and ACAT-1ⴚ/ⴚ (ⴚ/ⴚ)
mice. Facial appearance (A, ⫹/⫹; B, ⫺/⫺)and histology of meibomian glands (C,⫹/⫹; D, ⫺/⫺) are shown. Tarsus was prepared from eyelids and used for histological examination (hematoxylin and eosin
staining). The mice were females aged 5months. Note the narrow eye fissures andatrophic meibomian glands in ACAT1⫺/⫺ mice.

FIG. 3. FC and CE contents of adrenal glands, testes, ovaries
and meibomian glands. Open bars represent FC, and closed bars
represent CE. Lipids were extracted by Folch’s method and quantitatedby the fluorometric assay. Three mice aged 5 months were used. *, p ⬍0.01; **, p ⬍ 0.001 versus ⫹/⫹. Significant reduction in CE was noted in
the adrenals, ovaries, and meibomian glands. FC was increased only inthe meibomian glands of ACAT-1⫺/⫺ mice.

hyperlipidemic mice lacking the ACAT gene developed skinlesions upon aging (Fig. 4). Even on a normal chow diet, about
20% of ACAT-1⫺/⫺:apo E⫺/⫺ mice developed skin lesions by theage of 6 months (Fig. 4C). Typically, hair was lost, and skinbecame rough and thick. In contrast, ACAT-1⫺/⫺:LDLR⫺/⫺mice did not develop obvious skin lesions, at least by the age of 6months, as long as they were maintained on a normal chow diet.
When ACAT-1⫺/⫺:apo E⫺/⫺ and ACAT-1⫺/⫺:LDLR⫺/⫺mice were fed high fat diets A and B, respectively, both micedeveloped more pronounced hypercholesterolemia (Table II)
and more extensive skin lesions (Fig. 4D). Compared with
ACAT-1⫹/⫹:apo E⫺/⫺ mice, ACAT-1⫺/⫺:apo E⫺/⫺ mice had a
42% decrease in very LDL/intermediate density lipoproteincholesterol levels. There was no difference in the plasma lipoprotein profiles between ACAT-1⫹/⫹:LDLR⫺/⫺ and
ACAT-1⫺/⫺:LDLR⫺/⫺ mice, whereas very LDL/intermediate density lipoprotein/LDL-cholesterol levels were increasedin ACAT-1⫹/⫺:LDLR⫺/⫺ mice by 22%.
Microscopic examination revealed extensive infiltration ofmacrophages throughout the entire dermal layer of ACAT-1⫺/⫺:
apoE⫺/⫺ and ACAT-1⫺/⫺:LDLR⫺/⫺ mice fed the high fat
diets (Fig. 5, C and D). Many macrophages exhibited foamy
appearance with multiple nuclei similar to the Touton typegiant cells (Fig. 5, E and F). The thickened dermis was also
filled with necrotic acellular areas containing cholesterol clefts(Fig. 5). The number of hair follicles was remarkably reduced.
It is noteworthy that subcutaneous adipose tissues, which werenormally present in mice with ACAT-1 gene (Fig. 5, A and B),
were not discernible in the hyperlipidemic ACAT-1⫺/⫺ mice.
Measurements of FC and CE contents of the xathomatous skins
showed that FC content was specifically increased by 5.5-foldin ACAT-1⫺/⫺:apoE⫺/⫺ (20.0 ⫾ 14.8 versus 3.7 ⫾ 1.5 ␮g/mg
tissue; n ⫽ 3) and 9.7-fold in ACAT-1⫺/⫺:LDLR⫺/⫺ mice
(31.0 ⫾ 19.6 versus 3.2 ⫾ 1.1 ␮g/mg tissue; p ⬍ 0.05, n ⫽ 3).
Moderate Suppression of Diet-induced Atherosclerosis in
ACAT-1⫺/⫺ Mice Lacking apo E or the LDL Receptor—After
feeding the sets of apo E and the LDL receptor-deficient micewith high fat diets A and B, respectively, for 8 weeks, atherosclerotic lesion size was evaluated at the aortic roots (Fig. 6).
ACAT-1⫺/⫺:apo E⫺/⫺ mice developed a 33% smaller lesionarea than ACAT-1⫹/⫹:apo E⫺/⫺ mice. ACAT-1⫺/⫺:LDLR⫺/⫺mice had a 53% reduction in lesion area compared with ACAT1⫹/⫹:LDLR⫺/⫺ mice. There were no significant differences in
the lesion size between males and females in each genotype.
With regard to the aortic CE contents, 3-fold and 2-fold decreases were observed in ACAT-1⫺/⫺:apo E⫺/⫺ mice and
ACAT-1⫺/⫺:LDLR⫺/⫺ mice, respectively, as compared with
their ACAT-1⫹/⫹ controls (Fig. 7). In contrast to cutaneous
xanthomas, aortic FC contents were not increased.
DISCUSSION

In the present study, we have established ACAT-1 null miceand shown that ACAT-1 is involved in meibomian gland function, xanthoma formation, and atherosclerosis development in
hyperlipidemic mouse models. Previously, Meiner et al. (11)disrupted the ACAT-1 gene in mice. They reported that their
ACAT-1 knockout mice showed significant decreases in cholesterol esterification in embryonic fibroblasts and adrenal membranes. Although these ACAT-1⫺/⫺ mice had adrenal lipid
depletion and decreased CE content in peritoneal macrophages, the liver retained significant cholesterol esterification
activity, suggesting that cholesterol esterification involves
more than one form of esterification enzyme. In addition to21328Eye, Skin, and Vascular Lesions in Hyperlipemic ACAT-1KO Mice

TABLE II
Effects of the high fat diet feeding on the plasma lipid levels of ACAT-1⫺/⫺ mice lacking either apo E or LDL receptor
At the age of 3 months, apo E-deficient mice were fed high fat diet A, and LDL receptor-deficient mice were fed high fat diet B. Blood was taken
before and after feeding for 8 weeks. HDL-C was determined by HPLC using pooled plasma. TC, TG, and HDL-C are in mg/dl. Data represent themeans ⫾ S.D.
Genotype

Apo E⫺/⫺LDLR⫺/⫺ab

Diet

ACAT-1⫹/⫹10 (5, 5)

ACAT-1⫹/⫺
10 (4, 6)

ACAT-1⫺/⫺9 (5, 4)

Normal diet

TC
TG
HDL-C

777 ⫾ 236
133 ⫾ 329837 ⫾ 392
134 ⫾ 329573 ⫾ 163142 ⫾ 479High fat diet A

TC
TG
HDL-C

1471 ⫾ 472
181 ⫾ 86111202 ⫾ 572
152 ⫾ 528857 ⫾ 157a
156 ⫾ 521010 (5, 5)

9 (4, 5)8 (3, 5)

Normal diet

TC
TG
HDL-C

254 ⫾ 42191 ⫾ 70149

292 ⫾ 48203 ⫾ 63136

308 ⫾ 45b
279 ⫾ 107b
168

High fat diet B

TC
TG
HDL-C

1918 ⫾ 453
141 ⫾ 89142334 ⫾ 308b
145 ⫾ 79162045 ⫾ 297
140 ⫾ 4420p ⬍ 0.01 versus ACAT-1⫹/⫹ mice by analysis of variance.
p ⬍ 0.05 versus ACAT-1⫹/⫹.

FIG. 4. Cutaneous xanthomatosis
and alopecia in ACAT-1-deficient
mice that also lacked either apo E or
the LDL receptor. No apparent skin lesions were observed in ACAT-1⫹/⫹:apoE⫺/⫺ (A) and ACAT-1⫹/⫹:LDLR⫺/⫺mice (B). ACAT-1⫺/⫺:apoE⫺/⫺ (C) and
ACAT-1⫺/⫺:LDLR⫺/⫺ mice (D) developed generalized alopecia with scaly
thickened skin. ACAT-1⫹/⫹:apo E⫺/⫺and ACAT-1⫺/⫺:apo E⫺/⫺ mice were
7-month-old males fed a normal chow
diet. ACAT-1⫹/⫹:LDLR⫺/⫺ and ACAT1⫺/⫺:LDLR⫺/⫺ mice were 5-month-old
females fed the high fat diet B for 8
weeks.

confirming Meiner’s original observations, we found that cholesterol esterification in the epithelium of small intestine is not
mediated by ACAT-1. This is in agreement with the observations that wild-type small intestine expresses negligible
amounts of ACAT-1 (7, 8) and that human intestinal ACAT
activity is largely resistant to immunodepletion with an antibody against ACAT-1 (32). Recently, three groups independently reported that a second gene product (ACAT-2) is exclusively expressed in the small intestine and liver in bothnonhuman primates and mice (4 – 6). Therefore, ACAT-2 appears to mediate the formation of the CE that is used forlipoprotein assembly and secretion.
Unexpectedly, we noticed that our ACAT-1⫺/⫺ mice hadaltered facial appearance mainly because of narrow eye fissures (Fig. 2). This phenotype was noticeable as early as 3
weeks old and had almost complete penetrance. It can result
from either palpebral edema, brepharoptosis, corneal diseases,
impaired sight, reduction in retro-orbital tissue mass, etc. We
found no abnormalities except for corneal erosion probably
because of atrophic meibomian glands (Fig. 2). Therefore, dysfunction of meibomian glands may account for the narrow eye
fissures. Consistently, ACAT-1 is highly expressed in meibo-

mian glands (data not shown). The meibomian dysfunction may
result from reduced amounts of CE, increases in FC, or both(Fig. 3). Meibomian gland dysfunction may cause a decrease in
the superficial lipid layer of tear film, thereby increasing theevaporation of aqueous component of the tear. This condition issimilar to dry eye syndrome in humans (33). In this context, it
is interesting to note that meibomian glands are modified sebaceous glands which also express high levels of ACAT-1 (8). In
addition, we observed atrophic sebaceous glands in ACAT1⫺/⫺ mice, but the penetrance of this phenotype was not complete. Atrophy of sebaceous glands has been reported in cynomolgus monkeys treated with an ACAT inhibitor, PD 132301-2(34). Clinically, meibomian and sebaceous glands are commonly affected together; keratoconjunctivitis is frequently associated with sebaceous gland diseases such as seborrhea sicca(35). Further studies will be needed to determine whether
abnormal ACAT-1 underlies these diseases.
Jong et al. (36) have recently described cutaneous abnormalities including atrophy of both sebaceous and meibomian
glands in transgenic mice overexpressing apo CI. Because FC is
increased in the skin of the apo CI transgenic mice, the overexpressed apo CI may inhibit ACAT activities in the glands,

Eye, Skin, and Vascular Lesions in Hyperlipemic ACAT-1KO Mice

21329

FIG. 6. Effects of the inactivation of the ACAT-1 gene on atherosclerotic plaque size. 3-month-old mice were used for experiments of high fat feeding. Apo E⫺/⫺ mice were fed high fat diet A for 8
weeks, and LDLR⫺/⫺ mice were fed high fat diet B for 8 weeks. 10
ACAT-1⫹/⫹:apo E⫺/⫺ (5 males and 5 females), 10 ACAT-1⫹/⫺:apoE⫺/⫺ (4 males and 6 females), and 9 ACAT-1⫺/⫺:apo E⫺/⫺ mice (5males and 4 females) were fed high fat diet A for 8 weeks. Ten ACAT1⫹/⫹:LDLR⫺/⫺ (5 males and 5 females), 9 ACAT-1⫹/⫺:LDLR⫺/⫺ (4males and 5 females), and 8 ACAT-1⫺/⫺:LDLR⫺/⫺ mice (3 males and5 females) were fed high fat diet B for 8 weeks. Plasma lipoprotein
profiles are shown in Table II. After feeding with the high fat diets for
8 weeks, cross-sectional lesion areas were evaluated in the aortic rootsof the mice. †, p ⬍ 0.05; *, p ⬍ 0.01 by analysis of variance.

FIG. 5. Histopathology of cutaneous lesions developed in
ACAT-1ⴚ/ⴚ:apo Eⴚ/ⴚ and ACAT-1ⴚ/ⴚ:LDLRⴚ/ⴚ mice fed thehigh fat diets. 3-month-old mice were used for experiments of high fat
feeding. ACAT-1⫹/⫹:apo E⫺/⫺ and ACAT-1⫺/⫺:apo E⫺/⫺ mice were fed
high fat diet A for 8 weeks; ACAT-1⫹/⫹:LDLR⫺/⫺ and ACAT-1⫺/⫺:LDLR⫺/⫺ mice were fed high fat diet B for 8 weeks. ACAT-1⫹/⫹:apoE⫺/⫺ (A) and ACAT-1⫹/⫹:LDLR⫺/⫺ mice (B) had normal epidermaland dermal structure with substantial subcutaneous adipose tissue. No
foam cells were observed. In ACAT-1⫺/⫺:apo E⫺/⫺ (C and D) and
ACAT-1⫺/⫺:LDLR⫺/⫺ mice (D and F), the dermal layers were extensively infiltrated with fat-laden macrophage (foam cells). Acellular necrotic areas with cholesterol clefts were present. There were a reduced
number of hair follicles. Note that subcutanous adipose tissue was notdiscernible. High magnification of the dermal layer of ACAT-1⫺/⫺:apoE⫺/⫺ (E) and ACAT-1⫺/⫺:LDLR⫺/⫺ mice (F) is shown. Many macrophages exhibited multinucleated giant cell appearance with eosinophilic cytoplasms.

thereby producing phenotypes similar to those of our ACAT1⫺/⫺ mice. Phenotypic similarity to the mutant mouse asebia(ab) was discussed in their paper (37). Recently, Zheng et al.
(38) have reported that the stearoyl-CoA desaturates 1 (Scd1)
gene is disrupted in the ab mice. Because Scd1 catalyzes theformation of monounsaturated fatty acids, it is tempting to
speculate that cholesterol esterified with monusaturated fatty
acids is required for normal meibomian function. Further studies are needed to clarify this intriguing issue.
The most outstanding phenotype observed in this study was
cutaneous xanthomatosis with generalized alopecia in the double mutant mice (Figs. 4 and 5). Hair loss may be secondary to
the severe xanthomatosis; however, the LDL receptor knockout
mice fed an atherogenic diet develop massive xanthomatosis
but not alopecia (19). Therefore, xanthomatosis and alopecia
may be independent of each other. Here again, the alopecia

FIG. 7. Cholesterol content of the aorta. 3-month-old mice wereused for experiments of high fat feeding. Apo E⫺/⫺ mice were fed highfat diet A for 8 weeks, and LDLR⫺/⫺ mice were fed high fat diet B for
8 weeks. Aortas were prepared from the same animals that were used
for the evaluation of atherosclerotic lesion sizes as shown in Fig. 6.
Attached connective tissue and fat were removed from the aorta asmuch as possible. Lipids were extracted, and TC and FC content wasmeasured. CE content was obtained by subtracting FC from TC. Resultsfor FC and CE are shown in the upper and lower panels, respectively.
There were significant decreases in the CE content of the aortas frommice lacking ACAT-1 as compared with those from mice with ACAT-1.
†, p ⬍ 0.05; *, p ⬍ 0.01; **, p ⬍ 0.001 by analysis of variance.

21330Eye, Skin, and Vascular Lesions in Hyperlipemic ACAT-1KO Micedeveloped in apo CI transgenic mice may be relevant; apo CI
transgenics have atrophic sebaceous glands, infiltration of inflammatory cells, and a hairless coat (36). Because ACAT-1 ishighly expressed in sebaceous glands (8), it is reasonable toassume that ACAT-1 deficiency causes atrophy of these glands
as it does in meibomian glands resulting in reduced sebaceous
outflow of sebum onto the skin. Indeed, some, but not all,sebaceous glands showed atrophic changes in ACAT-1⫺/⫺ mice(data not shown). There was massive accumulation of FC in the
cutaneous xanthomas, as evidenced histologically by the presence of cholesterol clefts. Probably, plasma lipoproteins aretaken up by macrophages in which their CE are hydrolyzed but
are not re-esterified because of the absence of ACAT-1. This
may lead to an unlimited increase in cellular FC, which may
eventually result in cell death (39). The presence of giant
multinucleated cells indicates the inflammatory nature of thelesions. At present, we are unaware of a human disease which
parallels this skin lesion. Common pathogenesis may underlie
certain types of non-X histiocytosis such as xanthoma disseminatum or necrobiotic xanthogranuloma (40). Absence of subcutaneous fats may reflect the wasting nature of the disease.
Alternatively, the adipose tissue may be destroyed by infiltrating inflammatory cells.
The effects of ACAT-1 deficiency on atherosclerosis are interesting. Several ACAT inhibitors reduced plasma lipids bysuppressing lipoprotein production in the liver and intestine(41). In addition to the hypolipemic effects, these compounds
are expected to reduce foam cell formation in atherosclerotic
lesions, thereby inhibiting the progression of atherosclerosis insitu (42). As expected, CE accumulation was markedly reduced
by ACAT-1 disruption in the settings of both apo E and the LDL
receptor deficiency (Fig. 7). However, the effects on the atherosclerotic lesion size appeared more moderate compared withthose on CE contents. Because Oil-Red-O stains only neutral
lipids, the staining could potentially underestimate the lesion
size in the animals that lacked CE formation. In the setting of
apo E deficiency, the mean cross-sectional lesion size was reduced by 30%. This anti-atherogenic effect may be accounted
for simply by the reduction of the plasma TC levels, because theplasma TC levels were reduced by 40% in ACAT-1⫺/⫺:
apoE⫺/⫺ mice compared with in ACAT-1⫹/⫹:apoE⫺/⫺ mice.
In the setting of the LDL receptor deficiency, a similar degree of suppression in atherosclerotic lesions was observed
despite the fact that the plasma TC levels were not reduced. In
this particular model, it is reasonable to speculate that local
inhibition of ACAT activity results in reduction in atherosclerosis. Therefore, it may be important to take background clinical condition into account, when treating atherosclerosis with
ACAT inhibitors.
During the preparation of this manuscript, Accad et al. (43)published a report on atherosclerosis and cutaneous xanthomatosis of mice lacking both ACAT-1 and either apo E or the
LDL receptor. Their observations are essentially similar toours; however, they did not provide quantitative results of thelesion size and CE contents of the aortas. Instead, they performed more detailed histological analyses of the lesions andfound paucity of macrophage-derived foam cells in the lesions
from mice lacking ACAT-1.
In conclusion, the current study reveals that ACAT-1 isrequired for normal meibomian function. Inhibition of ACAT-1
is useful to prevent the CE accumulation in atherosclerotic
lesions that develop in the hyperlipidemic state. However, exacerbation of xanthomas is a potential complication of therapy
with ACAT inhibitors.
Acknowledgment—We thank A. H. Hasty for critical reading of themanuscript.

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