Increased cholesterol biosynthesis andhypercholesterolemia in mice overexpressing squalene
synthase in the liver
Hiroaki Okazaki,1,* Fumiko Tazoe,1,§§ Sachiko Okazaki,* Naoyuki Isoo,* Kazuhisa Tsukamoto,*
Motohiro Sekiya,* Naoya Yahagi,* Yoko Iizuka,* Ken Ohashi,* Tetsuya Kitamine,*
Ryu-ichi Tozawa,* Toshihiro Inaba,§§ Hiroaki Yagyu,§§ Mitsuyo Okazaki,§ Hitoshi Shimano,**Norihito Shibata,†† Hiroyuki Arai,†† Ryo-zo Nagai,† Takashi Kadowaki,* Jun-ichi Osuga,* andShun Ishibashi2,*,§§

Abstract Squalene synthase (SS) is the first committed
enzyme for cholesterol biosynthesis, located at a branch
point in the mevalonate pathway. To examine the role of SS
in the overall cholesterol metabolism, we transiently overexpressed mouse SS in the livers of mice using adenovirusmediated gene transfer. Overexpression of SS increased denovo cholesterol biosynthesis with increased 3-hydroxy-3methyglutaryl-CoA (HMG-CoA) reductase activity, in spite ofthe downregulation of its own mRNA expression. Furthermore, overexpression of SS increased plasma concentrationsof LDL, irrespective of the presence of functional LDL receptor (LDLR). Thus, the hypercholesterolemia is primarily
caused by increased hepatic production of cholesterol-rich
VLDL, as demonstrated by the increases in plasma cholesterol levels after intravenous injection of Triton WR1339.
mRNA expression of LDLR was decreased, suggesting that
defective LDL clearance contributed to the development ofhypercholesterolemia. Curiously, the liver was enlarged, witha larger number of Ki-67-positive cells. These results demonstrate that transient upregulation of SS stimulates cholesterol biosynthesis as well as lipoprotein production, providing
the first in vivo evidence that SS plays a regulatory role incholesterol metabolism through modulation of HMG-CoA
reductase activity and cholesterol biosynthesis.—Okazaki, H.,
F. Tazoe, S. Okazaki, N. Isoo, K. Tsukamoto, M. Sekiya, N.
Yahagi, Y. Iizuka, K. Ohashi, T. Kitamine, R-i. Tozawa, T.
Inaba, H. Yagyu, M. Okazaki, H. Shimano, N. Shibata, H. Arai,
R-z. Nagai, T. Kadowaki, J-i. Osuga, and S. Ishibashi. Increasedcholesterol biosynthesis and hypercholesterolemia in miceoverexpressing squalene synthase in the liver. J. Lipid Res.
2006. 47: 1950–1958.

Manuscript received 19 May 2006 and in revised form 31 May 2006.
Published, JLR Papers in Press, June 1, 2006.
DOI 10.1194/jlr.M600224-JLR200

Supplementary key words lipoprotein . adenovirus . hyperlipoproteinemia . 3-hydroxy-3-methyglutaryl-CoA reductase . farnesyl
diphosphate . mevalonate . hepatomegaly . cell proliferation .
feedback regulation

Cholesterol biosynthesis is subject to tight regulation
by a multivalent feedback mechanism at both the transcriptional and the posttranscriptional level (1). The transcriptional regulation is mediated through the action ofsterol-regulatory element binding proteins (SREBPs), membrane-bound transcription factors that enhance transcription of genes encoding cholesterol biosynthetic enzymes
and the LDL receptor (LDLR) (2). The translational regulation of 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) reductase (EC1.1.1.34), the rate-limiting enzyme in cholesterol
biosynthesis, is mediated by nonsterol mevalonate-derived
isoprenoids, which act by an undefined mechanism (3). Its
degradation is regulated by both sterols and nonsterolend products of mevalonate metabolism (4, 5). The sterolregulated degradation of HMG-CoA reductase is mediatedAbbreviations: Ad-SS, recombinant adenovirus carrying SS cDNA
under the control of cytomegalovirus promoter; apoB, apolipoprotein
B; E, embryonic day; ER, endoplasmic reticulum; LDLR, LDL receptor;
m.o.i., multiplicity of infection; SREBP, sterol-regulatory element binding protein; SS, squalene synthase; TC total cholesterol; TG, triglyceride; TUNEL, terminal deoxyribonucleotidyl transferase-mediateddUTP nick end labeling.
1
H. Okazaki and F. Tazoe contributed equally to this work.
2
To whom correspondence should be addressed.
e-mail: ishibash@jichi.ac.jp
Copyright D 2006 by the American Society for Biochemistry and Molecular Biology, Inc.

1950

Journal of Lipid Research

Volume 47, 2006

This article is available online at http://www.jlr.org

Downloaded from www.jlr.org at Univ of Tokyo Medical Library on August 23, 2006

Departments of Metabolic Diseases* and Cardiovascular Diseases,† Graduate School of Medicine, Universityof Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan; Laboratory of Chemistry, College of Liberal Arts andScience,§ Tokyo Medical and Dental University, Chiba 272-0827, Japan; Metabolism, Endocrinology andAtherosclerosis, Institute of Clinical Medicine,** University of Tsukuba, Ibaraki 305-8575, Japan; Department
of Health Chemistry, Graduate School of Pharmaceutical Sciences,†† University of Tokyo 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033 Japan; Division of Endocrinology and Metabolism, Department of Medicine,§§Jichi Medical University, Tochigi 329-0498, Japan

MATERIALS AND METHODS
Construction of recombinant adenoviruses
Recombinant adenovirus that carried murine SS cDNA under
the control of the cytomegalovirus promoter, designated as AdSS, was constructed using the cDNA cloned by RT-PCR frommouse liver as described previously (20, 21). The recombinantadenoviruses were expanded in HEK293 cells and purified bycesium chloride ultracentrifugation. The purified viruses werestored in 10% (v/v) glycerol in PBS at 2808C. In our preparations,
1 multiplicity of infection (m.o.i.) corresponded to 25 particlesof adenovirus per cell, and cells were infected at 300 m.o.i.

Animal studies
C57BL/6 male mice and LDLR knockout mice (22) that had
been backcrossed into the C57BL/6 background were purchasedfrom Charles River Japan, Inc. (Yokohama, Japan) and JacksonLaboratory (Bar Harbor, ME), respectively. Mice were maintained and cared for according to the regulations of the Animal
Care Committees of the University of Tokyo and Jichi Medical
University. Mice were caged separately, with 12 h light/darkcycles, and were given free access to standard chow diet containing 0.075% cholesterol (MF; Oriental Yeast Co., Ltd., Osaka,
Japan). Mice were injected intravenously with 1.5 3 1011 particles (6 3 109 plaque-forming units) of Ad-LacZ or Ad-SS. Eachgroup contained six mice. Seven days after virus injection, food
was withdrawn 4 h before collection of blood samples fromthe retroorbital plexus of anesthetized animals. Tissues were immediately collected, snap frozen in liquid nitrogen, and stored
at 2808C.

Real-time RT-PCR
Total RNA was prepared from livers with TRIzol reagent (LifeTechnologies, Inc.). Two micrograms of total RNA was reverse
transcribed (Taq Man Reverse Transcription Reagents; Applied
Biosystems, Foster City, CA), and synthesized cDNA was quantified using Taq Man quantitative PCR analysis of each gene with the
ABI Prism 7700 sequence detection system (Applied Biosystems)
according to the manufacture’s protocol. The specific primer pairs
for SS, HMG-CoA reductase, FAS, LDLR, apolipoprotein B(apoB), CYP7A, and SREBP-1a were designed as previously described (23); the others were purchased from Applied Biosystems.
The relative amounts of all mRNAs were calculated by using thecomparative 2-DDCT method (User Bulletin, Applied Biosystems).
b-actin mRNA was used as the invariant control.

Western blot analysis
Livers were homogenized in a buffer containing 15 mM
nicotinamide, 2 mM MgCl2, and 100 mM potassium phosphate,
pH 7.4, and centrifuged at 10,000 g for 20 min at 48C. The
supernatants were centrifuged at 105,000 g for 1 h at 48C, andthe resultant pellets, a microsome fraction, were washed, resuspended in the same buffer, and stored in aliquots at 2808C.
The supernatant was used for Western blot analysis as described
previously (21), using an anti-SS antibody that was raised by immunizing rabbits with glutathione S-transferase fusion protein
containing amino acid sequence 174–339 of mouse SS. For
Western blot of HMG-CoA reductase, we prepared and solubilized the liver membrane fraction exactly as described by Severet al. (7) and used antibodies from Upstate Cell Signaling Solutions (Charlottesville, VA) and from Dr. Y. K. Ho at Universityof Texas Southwestern Medical Center at Dallas.

Adenovirus-mediated gene transfer of squalene synthase

1951

Downloaded from www.jlr.org at Univ of Tokyo Medical Library on August 23, 2006

by the binding of a pair of endoplasmic reticulum (ER)retention proteins called Insig-1 and Insig-2 to the sterolsensing domain in membrane-spanning segments of HMGCoA reductase (6, 7).
Inhibition of cholesterol biosynthesis in the liver byusing inhibitors of HMG-CoA reductase, collectively calledstatins, is a widely used strategy for preventing heart attacks by lowering plasma cholesterol. However, statinshave limited effects (8), partly because the deficiency ofmevalonate-derived products following the inhibition of
HMG-CoA reductase activity leads to a compensatory increase in HMG-CoA reductase protein, invoking the needfor higher doses of statins (9), and to occasional severe adverse effects, such as rhabdomyolysis (10). Therefore, exploration of another target for the inhibition of cholesterol
biosynthesis is needed.
Squalene synthase (SS, or farnesyl-diphosphate:farnesyldiphosphate farnesyltransferase, EC2.5.1.21) catalyzes the
reductive head-to-head condensation of two molecules offarnesyl diphosphate to form squalene, the first specificintermediate in the cholesterol biosynthetic pathway (11,12). SS is unique among other enzymes in cholesterol biosynthesis in that, in addition to the sterol-mediated transcriptional control through the action of SREBPs (13),expression of the SS gene is regulated by lipopolysaccharide, which potently inhibits SS, whereas it stimulates
HMG-CoA reductase expression (14). SS is an attractive target for cholesterol-lowering therapy, because the inhibition of this step theoretically may not perturb the nonsterol
pathway. Indeed, several potent SS inhibitors successfully
lower plasma cholesterol levels without the potential limitations reported for statins (15–17).
To elucidate the role of SS in cholesterol metabolism,
we have generated SS knockout mice. SS knockout mice(SS 2/2) were lethal between embryonic day (E) 9.5 and E
12.5 and exhibited severe retardation of development
and defective neural tube closure, indicating that SS isessential for development, particularly of the central nervous system (18). Although SS 1/2 mice expressed only 50%
SS activities in the liver compared with SS 1/1 mice, theirplasma lipoprotein profiles and responses to dietary challenges were indistinguishable from those of SS 1/1 mice.
These observations closely resemble those seen in HMGCoA reductase knockout mice: HMG-CoA reductase2/2 mice
were embryonic lethal, and HMG-CoA reductase1/2 mice
showed no significant changes in lipoprotein profiles (19).
However, the role of SS in cholesterol metabolism in adultliver remains largely unknown.
To better understand the role of SS in overall cholesterol metabolism in vivo in adult liver, we have usedadenovirus-mediated gene delivery to overexpress SS in
the liver and have examined changes in hepatic cholesterol biosynthesis and plasma lipoprotein profiles in vivo.
We show here that increased SS activity in liver leads
to increased cholesterol synthesis, as well as secretion ofcholesterol-rich lipoproteins, providing the first in vivo
evidence that SS plays a regulatory role in the development of hypercholesterolemia.

SS activity assay
Liver microsomal protein was obtained as described above,and stored in aliquots at 2808C. SS activities were measured
according to a modified method of Cohen et al. (18, 24). In brief,
the microsome fractions (z20 mg) were incubated in 50 ml of abuffer containing 20 mM [1-3H]farnesyl pyrophosphate (25 mCi/
mmol; NEN Life Science Products, Inc.), 1 mM NADPH, 5 mM
MgCl2, 6 mM glutathione, and 100 mM potassium phosphate,
pH 7.4, at 378C for 15 min. The reaction was terminated by theaddition of 150 ml chloroform-methanol (1:2; v/v) containing
0.2% unlabeled squalene. After 50 ml of chloroform and 50 mlof 3 M NaOH were added, the reaction mixtures were vortexed
and centrifuged. The infranatant organic phase was used for thedetermination of the radioactivities in the squalene produced.

Plasma lipids and lipoprotein analyses

Histology
Tissues were fixed with neutral-buffered formalin, embedded
in paraffin, and stained with hematoxylin and eosin. Ki-67 staining was performed using rat ant-mouse Ki-67 antibody (DAKO;Glostrup, Denmark). Apoptosis was detected by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining using the in situ Apoptosis Detection Kit(TaKaRa, Japan) according to the instruction manual. The number of cells positive for staining with Ki-67 or TUNEL was counted
in each field of representative slides from three independent experiments at a magnification of 3100.

Tissue and cellular lipids
Lipids were extracted from tissues by the method of Folch,Lees, and Sloane Stanley (26). TC was determined by fluorometric microassay according to a modified method of Heider andBoyett (27) with the exception that 0.01% (v/v) Triton X-100 wasused instead of Carbowax-600. TG was determined enzymaticallyusing a kit (Triglyceride-G, WAKO Pure Chemicals).

HMG-CoA reductase activity assay
Liver microsomal proteins were prepared as described above.
HMG-CoA reductase activities were measured essentially as described previously (9). Briefly, the microsome fractions (50 mg)
were incubated in 20 ml of a buffer containing 110 mM DL[3-14C]HMG-CoA (4.5 mCi/mmol; NEN Life Science Products,Inc.), 5 mM NADPH, 10 mM EDTA, 10 mM dithiothreitol, and100 mM potassium phosphate, pH 7.4, at 378C for 30 min. The
reaction was terminated by the addition of 10 ml of 2 N HCl andincubated for another 30 min at 378C to lactonize the mevalonate
formed. The [14C]mevalonate was isolated by TLC and counted
using [3H]mevalonate as an internal standard. HMG-CoA reductase activity is expressed as picomoles of [14C]mevalonate formed
per minute per mg of protein.

Measurement of cellular cholesterol synthesis in vitro
Cholesterol biosynthesis in vitro was determined essentially as
described previously (29, 30). Briefly, McARH7777 cells (American Type Culture Collection) were grown in DMEM containing
10% FBS and 10% fetal horse serum and were plated on day 0 at adensity of 2.5 3 104 cells per square centimeter into 24-wellcollagen-coated plates (IWAKI). On day 1, transfection of recombinant adenovirus (Ad-LacZ or Ad-SS) was performed at theindicated m.o.i. On day 3, [2-14C]acetate (51 mCi/mmol; NEN
Life Science Products, Inc.) was added to a final concentration of
10 mCi per well. After incubation for 5 h, the medium was removed and centrifuged. The cells were harvested by scraping into
100 ml of 0.1 N sodium hydroxide, followed by 100 ml of water.
Radiolabeled lipids were saponified by 15% KOH, extracted bypetroleum ether. The content of 14C-labeled squalene, 14Clabeled cholesterol, and 14C-labeled fatty acids in the cells and in
the medium was quantified by TLC and scintillation counting.
The cellular protein was measured by the BCA Protein Assay Kit(Pierce Chemical Co., Rockford, IL).

Assay of microsomal ACAT activity
Liver was homogenized in buffer A (0.25 M sucrose, 1 mM
EDTA, 2 mg/ml leupeptin, and 50 mM Tris-HCl, pH 7.0) andcentrifuged at 100,000 g for 45 min at 48C. The pellets were resuspended and used for the assay. ACAT activity in microsomes
was determined by the rate of incorporation of [1-14C]oleoyl-CoA(53.5 mCi/mmol; NEN Life Science Products, Inc.) into thecholesteryl ester fraction according to Yagyu et al. (25).

VLDL secretion rate
Secretion rates of TG or cholesterol in vivo were estimated by
the intravenous administration of Triton WR1339 as described
previously (31). Plasma levels of TG or cholesterol were measured at 1, 2, 3, and 4 h after the treatment. Aliquots of plasma
were subjected to sequential ultracentrifugation to separate lipoprotein fractions using S100AT3 angle rotor for himac CS120GXL(HITACHI). In brief, 60 ml of plasma was mixed with 60 ml of saline and centrifuged at 188,000 g for 3 h at 168C. Sixty microliters
was aspirated from the bottom by Hamilton syringe and mixed
with a KBr solution (d 5 1.12) and centrifuged at 188,000 g for 5 h
at 168C. The remaining top fraction was used as the VLDL
fraction. Sixty microliters was aspirated as described above andused as the HDL fraction, and the remaining top fraction was usedas the IDL/LDL fraction.

Statistical analyses
All values are given as mean 6 SE, and differences between
groups were evaluated with Student’s t-test or ANOVA, unlessotherwise stated. All calculations were performed with STAT
view, version 5.0, for Macintosh (SAS Institute).

Measurement of hepatic cholesterol synthesis in vivo
Seven days after administration of adenovirus to mice, cholesterol synthesis in the liver was estimated during the mid lightcycle as previously described (18, 19, 28). In brief, animals were
given food and water ad libitum and injected intraperitoneally

1952

Journal of Lipid Research

Volume 47, 2006

RESULTS
Intravenous injection of Ad-SS elicited an 11-fold increase in mRNA expression of mouse SS in the liver as

Downloaded from www.jlr.org at Univ of Tokyo Medical Library on August 23, 2006

Plasma levels of total cholesterol (TC), triglycerides (TGs),and FFAs were determined enzymatically using kits: Determiner
TC555 (Kyowa Medex), Triglyceride-G, and NEFA-C (WAKO
Pure Chemicals). Lipoproteins were fractionated by HPLC as
described (25), and the cholesterol contents in each lipoprotein
fraction were determined.

with 37 kBq/kg body weight of [2-14C]acetate (NEN Life Science
Products, Inc.). After 1 h, animals were euthanized and the liver
was removed. Two portions of the liver (200–300 mg/each) were
saponified, and the digitonin-precipitable sterols were isolated
for the measurement of radioactivities. The results were expressed as 14C dpm/100 mg of wet weight of liver/h.

TABLE 1. Relative amounts of mRNAs in the livers
Gene

HMG-CoA
reductase
Squalene synthase
Squalene epoxydase
Lanosterol 14ademethylase
SREBP-1aFatty acid synthase
ACAT2
ApoB
LDLR
CYP7A
ABCA1

PBS

Ad-LacZ

Ad-SS

1.00 6 0.14

1.19 6 0.08

0.72 6 0.13b

1.00 6 0.40
1.00 6 0.18
1.00 6 0.23

1.24 6 0.21
1.26 6 0.28a
1.34 6 0.23

13.43 6 2.20b
0.35 6 0.03b
0.41 6 0.07b

1.00 6 0.09
1.00 6 0.07
1.00 6 0.10
1.00 6 0.17
1.00 6 0.05
1.00 6 0.37
1.00 6 0.08

0.91 6 0.08a
0.53 6 0.06a
1.61 6 0.24a
1.08 6 0.06
0.90 6 0.04
6.75 6 2.01a
0.79 6 0.03a

0.54 6 0.06b
0.46 6 0.04
0.49 6 0.04b
0.55 6 0.05b
0.47 6 0.04b3.40 6 0.49
0.52 6 0.03b

compared with Ad-LacZ (Table 1). In parallel, mouse SS
protein was markedly expressed in liver of Ad-SS-injected
mice (Fig. 1A). Hepatic SS activities were also remarkably
increased in the liver, by 52-fold (Fig. 1B).
To determine whether the overexpressed SS accompanied increased cholesterol synthesis, we measured theamounts of cholesterol synthesized from acetyl-CoA in theliver (Fig. 2A). Although the use of acetic acid as a substrate for cholesterol biosynthesis poses several limitations
to the interpretation of the results (32), we presume thatoverexpression of SS is unlikely to affect other potential
confounding factors such as transmembrane permeabilityof acetic acid and/or differential dilution by the intracellular pool of acetyl-CoA. Hepatic cholesterol synthesis wasincreased 2-fold. Given that HMG-CoA reductase is theFig. 2. Cholesterol synthesis, HMG-CoA reductase, and ACAT
activities in the liver. Seven days after intravenous injection of
adenovirus into C57BL/6 mice (n 5 6), rates of hepatic cholesterolsynthesis (A) and HMG-CoA reductase (B) and ACAT activities (C)in the livers were measured. *, P , 0.05 and **, P , 0.0001 vs. AdLacZ. Error bars represent standard deviation.

Fig. 1. Expression of squalene synthase (SS). Seven days after
intravenous injection of adenovirus into C57BL/6 mice (n 5 6),livers were excised and used for Western blot analysis of SS (A) andfor measurements of SS activities (B). *, P , 0.0001 vs. Ad-LacZ.
Three representative samples were used for Western blot analysis.
Error bars represent standard deviation.

rate-limiting enzyme of cholesterol synthesis, we measured
the activities of HMG-CoA reductase in the microsome
fraction of the liver (Fig. 2B). There was a 3.6-fold increasein the HMG-CoA reductase activities in the Ad-SS-injected
mice compared with Ad-LacZ-injected mice. To determine whether the increase in the HMG-CoA reductase
activities was associated with induction of the mRNA expression, we performed RT-PCR of HMG-CoA reductase
along with other enzymes in the cholesterol synthetic
pathway (Table 1). The hepatic mRNA level of HMGCoA reductase was rather decreased, by 39%, in the AdSS-treated mice, indicating that the increased enzymatic
activity was at posttranscriptional level. More notably, therelative mRNA expression of enzymes catalyzing the steps
downstream of HMG-CoA reductase, such as squalene
epoxidase and lanosterol 14a-demethylase, was significantly suppressed in the Ad-SS-injected mice by 72% and69%, respectively. To estimate the protein expression of
HMG-CoA reductase in the liver membrane fraction, we
performed Western blot analysis. Although we tried to
Adenovirus-mediated gene transfer of squalene synthase

1953

Downloaded from www.jlr.org at Univ of Tokyo Medical Library on August 23, 2006

Seven days after intravenous injection of adenovirus into C57BL/
6 mice (n 5 4), total RNA was isolated from the livers and used forreal-time PCR analysis. Values are indicated as means 6 SE. HMGCoA reductase, 3-hydroxy-3-methyglutaryl-CoA reductase; SREBP,
sterol-regulatory element binding protein; ACAT, acyl-CoA:cholesterolacyltransferase; apoB, apolipoprotein B; LDLR, LDL receptor; CYP7A,
cholesterol 7a-hydroxylase; ABCA1, ATP binding cassette transporter A1.
a
P , 0.05 vs. PBS by ANOVA and Fisher’s protected least significant difference (PLSD) test.
b
P , 0.05 vs. Ad-LacZ by ANOVA and Fisher’s PLSD.

TABLE 2. Lipid contents of the livers

Body weight (g)Liver weight (g)TC (mg/g wet tissue)
TG (mg/g wet tissue)

Ad-LacZ

Ad-SS

24.1 6 0.8
1.465 6 0.61
1.101 6 0.038
3.672 6 0.228

25.1 6 0.6
2.003 6 0.059a
1.310 6 0.132
2.387 6 0.310

Seven days after intravenous injection of adenovirus into C57BL/
6 mice (n 5 6), lipids were extracted from the livers by the methodof Folch, Lees, and Sloane Stanley (26). TC and TG were measured
enzymaticaly. TC, total cholesterol; TG, triglyceride. Values are indicated as means 6 SD.
a
P , 0.0001 vs. Ad-LacZ.

1954

Journal of Lipid Research

Volume 47, 2006

Fig. 3. Histology of the liver. Seven days after intravenous injection of adenovirus into C57BL/6 mice, liver samples from miceoverexpressing Ad-LacZ (A, C, E) or Ad-SS (B, D, F) were fixed inneutral-buffered formalin and embedded in paraffin. A, B: Tissue
sections were stained with hematoxylin and eosin (HE). C, D: Ki-67
staining was performed using rat anti-mouse Ki-67 antibody. Kipositive cells are brown. E, F: Apoptosis was detected by terminal
deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining using the in situ Apoptosis Detection Kit.
TUNEL-positive cells are brown.

cholesterol/protein ratio of VLDL from Ad-SS-injected
mice was 1.6-fold higher than that of VLDL from Ad-LacZinjected mice at 4 h after the injection of Triton WR1339(Table 4). Cholesterol in the IDL/LDL fraction did notincrease at 4 h after the injection of Triton WR1339 (AdLacZ, from 10.5 6 0.8 mg/dl to 9.8 6 0.7 mg/dl; Ad-SS,
from 30.3 6 3.6 mg/dl to 28.4 6 2.7 mg/dl). Thus, thecontribution of increased secretion of LDL to the development of hypercholesterolemia was negligible. Consistent
with the production of cholesterol-rich VLDL, ACAT activities in the liver microsome were significantly increased,by 81% (Fig. 2C). However, mRNA expression levels of
ACAT-2 or apoB in Ad-SS-injected mice were rather suppressed (Table 1).
It is conceivable that increased cholesterol synthesis
suppresses the expression of LDLR, the major pathway for
LDL removal, and thereby delays the plasma clearance of
LDL. To explore this possibility, we measured relative
mRNA expression of LDLR in the liver. It was decreased by
48% in the Ad-SS-injected mice compared with the AdLacZ-injected mice (Table 1).
Cholesterol could be eliminated from the liver as bile
acids. The mRNA level of cholesterol 7a-hydroxylase, therate-limiting enzyme for bile acid synthesis, however, was
not increased (Fig. 1). Cholesterol could be secreted into

Downloaded from www.jlr.org at Univ of Tokyo Medical Library on August 23, 2006

detect HMG-CoA reductase protein expression by using
two different antibodies, we failed to detect a 97 kDa bandthat reportedly corresponds to HMG-CoA reductase (datanot shown).
To determine whether the increased cholesterol synthesis in the liver overexpressing SS affected hepatic cholesterol contents, we measured lipid contents in the liver(Table 2). The weight of the liver was increased by 37%
in Ad-SS-injected mice compared with Ad-LacZ-injected
mice. Cholesterol contents per weight of wet tissue were
not different between the Ad-LacZ- and Ad-SS-injected
mice, whereas TG contents per weight of wet tissue weredecreased by 35% in Ad-SS-injected mice. Thus, cholesterol content in the whole liver was increased in proportion to the increase in liver weight. To determine thereasons for the hepatomegaly in Ad-SS-injected mice, we
performed histology (Fig. 3). Hematoxylin and eosin staining showed negligible infiltration of inflammatory cells;
there were no differences between the Ad-LacZ- and AdSS-injected mice. The number of Ki-67-positive cells, which
are correlated with cell proliferation activity (33), wassignificantly increased in the livers of Ad-SS-injected micecompared with Ad-LacZ-injected mice (17.7 6 2.3 vs. 0 6
0 cells/field; P , 0.01), whereas the number of TUNELpositive cells was not different between the Ad-LacZ- and
Ad-SS-injected mice. These results indicate that overexpression of SS stimulates cell proliferation of hepatocytes,
thereby leading to hepatomegaly.
Plasma lipoprotein profiles were determined 7 days
after the injection of the virus into wild-type mice (Fig. 4A
and Table 3). Plasma cholesterol levels were significantly
increased, by 66%, in Ad-SS-injected mice compared with
Ad-LacZ-injected mice, whereas there were no significant
differences in plasma levels of either TG or FFA. HPLC
lipoprotein analyses revealed that LDL- and HDL-cholesterol levels were increased by 1.9- and 1.3-fold, respectively.
To determine how the hepatic overexpression of SS led
to hypercholesterolemia, we determined hepatic VLDL
secretion rates (Fig. 5). Rates of increases in plasma cholesterol levels after the injection of Triton WR1339 weresignificantly increased in Ad-SS-injected mice comparedwith Ad-LacZ-injected mice (Fig. 5B), whereas those inplasma TG levels were not different between the two groups(Fig. 5A). These results indicate that the increase in plasma
LDL-cholesterol levels resulted primarily from the secretion of cholesterol-rich VLDL from the liver. Indeed, thecirculation directly from the liver via ABCA1, for example.
However, mRNA expression of ABCA1 was decreased by
34% (Table 1).
Secretion of cholesterol-rich VLDL as a major cause of
hypercholesterolemia was confirmed by an experiment inwhich Ad-SS was injected into LDLR knockout mice. In theabsence of LDLR, plasma cholesterol levels are determined primarily by the VLDL production rate, because thedifference in the LDL clearance is negligible. TC levels
were increased by 1.3-fold (Table 3). HPLC lipoprotein
analyses revealed that cholesterol was increased mainly inthe non-HDL fraction (Fig. 4B, Table 3).
To further verify the notion that the overexpressionof SS is associated with increased cholesterol synthesis and
TABLE 3. Changes in plasma lipids and lipoproteins in wild-type or
LDLR knockout miceMice

Wild-typeLDLRKO

Lipids and Lipoproteins

Ad-LacZ

Ad-SS

TC (mg/dl)
TG (mg/dl)
FFA (mM)
VLDL (mg/dl)
LDL (mg/dl)
HDL (mg/dl)
TC (mg/dl)
TG (mg/dl)
FFA (mM)
VLDL (mg/dl)
LDL (mg/dl)
HDL (mg/dl)

83.1 6 3.9
50.7 6 1.9
684.2 6 101.5
3.50 6 0.39
15.68 6 1.61
63.25 6 2.81
260.5 6 9.6
111.3 6 6.6
421.6 6 20.0
53.4
184.5
93.8

137.6 6 4.4a
44.2 6 2.3
583.8 6 56.4
4.77 6 0.40
29.58 6 3.62a
84.07 6 0.83a
334.7 6 22.3a
117.8 6 6.2
383.0 6 23.9
71.6
213.3
104.0

Seven days after intravenous injection of adenovirus into wildtype C57BL/6 mice (n = 6) or LDL receptor knockout (LDLRKO) mice(n 5 6), blood was collected after a 4 h fast. Plasma was subjected to
HPLC lipoprotein analyses individually. Pooled plasma was used toobtain lipoprotein data for LDLRKO mice. Values are indicated asmeans 6 SD.
a
P , 0.05 vs. Ad-LacZ.

Fig. 5. Hepatic VLDL secretion. Seven days after intravenous
injection of PBS (n 5 4), Ad-LacZ (n 5 6), or Ad-SS (n 5 6) into
C57BL/6 mice, VLDL secretion rates were determined by the increments of plasma levels of triglyceride (A) or cholesterol (B)after the intravenous injection of Triton WR1339. VLDL-cholesterol secretion rate in Ad-SS-injected mice was larger than that in
Ad-LacZ-injected mice. *, P , 0.05 vs. Ad-LacZ by 2-way ANOVA
and Fisher’s protected least significant difference test. Dotted lines
with open squares denote Ad-LacZ; solid lines with closed diamonds denote PBS; solid lines with closed squares denote Ad-SS.

VLDL secretion, we performed in vitro experiments using
McARH7777 cells (Fig. 6). Cultured McARH7777 cells
were infected with either Ad-LacZ or Ad-SS. After 48 h,[14C]acetate was added to the culture, and the cells wereincubated for 5 h. Radiolabeled lipids were extracted fromthe cells and medium, and the radioactivities incorporated into the sterols and fatty acids (unesterified plus esterified) were determined after they were resolved by TLC.
As expected, Ad-SS-infected cells synthesized larger
amounts of cholesterol than did the control cells infectedwith Ad-LacZ. Synthesis of fatty acids was also increased.
Furthermore, secretion of cholesterol and fatty acids into
the culture medium was also stimulated in Ad-SS-infected
cells more than in Ad-LacZ-infected cells.

DISCUSSION
In the present study, we showed that overexpression of
SS in the liver causes increased cholesterol biosynthesis
Adenovirus-mediated gene transfer of squalene synthase

1955

Downloaded from www.jlr.org at Univ of Tokyo Medical Library on August 23, 2006

Fig. 4. Plasma lipoprotein profiles. Seven days after intravenous
injection of adenovirus into C57BL/6 mice (A) (n 5 6) or LDL
receptor (LDLR) knockout mice (B) (n 5 6), blood was collected
after a 4 h fast. Pooled plasma was subjected to HPLC lipoprotein
analyses. WT, wild type; LDLRKO, LDLR knockout.

TABLE 4. Lipid contents in VLDL and IDL/LDL before and after intravenous injection of Triton WR1339
Lipid

h

PBS

Ad-LacZ

Ad-SS

lg/mg protein
Triglyceride
VLDL
IDL/LDL
Cholesterol
VLDL
IDL/LDL04
04

33.7 6 2.5
2,755 6 559
27.2 6 2.6
79.9 6 11.8

31.2 6 4.7
2,311 6 101
38.3 6 7.7
78.1 6 12.8

30.7 6 1.8
2,457 6 167
38.3 6 3.7
110 6 1704
04

26.2 6 1.6
225 6 2940.7 6 6.3
81.4 6 9.5

23.0 6 1.7
176 6 1032.9 6 4.5
48.6 6 5.7

30.3 6 1.9a
277 6 17a77.5 6 7.9a
156 6 17aSeven days after intravenous injection of PBS (n 5 4), Ad-LacZ (n 5 6), or Ad-SS (n 5 6) into wild-type
C57BL/6 mice, Triton WR1339 was injected intravenously. Blood was collected before and 4 h after the injection.
Plasma was subjected to sequential analytical ultracentrifugation. Concentrations of protein, TG, and cholesterol
were determined. Values are indicated as means 6 SE.
a
P , 0.05 vs. Ad-LacZ by ANOVA and Fisher’s PLSD.

Fig. 6. Lipid synthesis and secretion in McARH7777 cells.
McARH7777 cells were transfected with recombinant adenovirus[Ad-LacZ (open bars) and Ad-SS (closed bars)], and after 48 h,[14C]acetate was added to a final concentration of 28.6 mCi/ml.
After 5 h of incubation, the media (B) were removed and the cells(A) were harvested. Radiolabeled sterols and fatty acids were extracted and resolved by TLC, and the content of 14C-labeled steroland fatty acids in cells and media were measured. *, P , 0.05 and**, P , 0.01 vs. Ad-LacZ. Chol, cholesterol. Error bars represent
standard deviation.

1956

Journal of Lipid Research

Volume 47, 2006

expression. 2) Secretion of cholesterol-rich VLDL and possibly downregulation of LDLR are responsible for thedevelopment of hypercholesterolemia. 3) Increased cholesterol synthesis is associated with hepatomegaly due toincreased proliferation of hepatocytes. 4) Genes of enzymes in the cholesterol synthetic pathway, including the
enzymes downstream of SS, such as squalene epoxidase
and lanosterol 14a-demethylase, are downregulated.
The mRNA expression of enzymes in the cholesterol
biosynthetic pathway, such as HMG-CoA reductase, squalene epoxidase, and lanosterol 14a-demethylase, was downregulated (Table 1). The downregulation of these genes
might be mediated through a negative feedback regulation by the end product, cholesterol, which was overproduced by the liver (Fig. 2). The downregulation of thegenes for squalene epoxidase and lanosterol 14a-demethylase suggests that there are no other rate-limiting steps inthe cholesterol biosynthetic pathway past SS. Interestingly,cholesterol biosynthesis was increased in spite of the overall downregulation of genes in the cholesterol biosynthetic
pathway. This puzzling phenomenon might be explainedby an increase in HMG-CoA reductase activity, the ratelimiting enzyme in cholesterol biosynthesis. The inductionof HMG-CoA reductase activity should be through its posttranscriptional regulations; both translation and degradation of HMG-CoA reductase protein are subject to feedback
regulation by nonsterol mevalonate metabolites, as mentioned above. In the state of overwhelmingly increased activity of SS, most of the mevalonate metabolites may beutilized to produce squalene, leaving little substrates for
nonsterol pathways such as geranyl diphosphate and farnesyl diphosphate. Under this condition, it is expected thattranslation of HMG-CoA reductase is increased and that its
proteosomal ER degradation is suppressed, because farnesol (35, 36) and/or granylgeraniol (6) stimulate degradation of this enzyme. To verify this possibility, we needto examine the protein stability of HMG-CoA reductase andthe cellular contents of isoprenoids. Unfortunately, we
failed to detect HMG-CoA reductase protein in the liver
membrane fractions by Western blot analysis, although wefollowed the established protocol and used two different

Downloaded from www.jlr.org at Univ of Tokyo Medical Library on August 23, 2006

and hypercholesterolemia, which is the first in vivo evidence that SS plays a regulatory role in cholesterol metabolism, and is a mirror image of previous observations byothers that SS inhibitors possess cholesterol-lowering activity primarily through inhibition of hepatic VLDL production (16, 17, 34). Other new findings in the current
paper are as follows: 1) HMG-CoA reductase activity is
upregulated in spite of downregulation of its own mRNA

the liver into the bile in compensation for the increased
synthesis, although the expression of genes responsible forthis pathway, such as cholesterol 7a-hydroxylase, was not
significantly changed (Table 1). A final but most interesting possibility is that newly synthesized cholesterol may
be utilized for the plasma membranes of dividing cells.
The liver was heavier, with an increased number of cells
positive for Ki-67, after infection with Ad-SS (Table 2,
Fig. 3), indicating that the hepatocytes were in a hyperproliferative state. Although the precise mechanisms are
currently unknown, changes in the expression of Ras andRas-related proteins (46) or in the cholesterol content ofcaveolae (47) may be intriguing possibilities.
The present study shows that selective upregulation of
SS may lead to hypercholesterolemia. Mutations in the SSgene that confer stimulation of enzymatic activity may underlie some forms of hypercholesterolemia. In combination with liver-specific knockout of SS, which is being
generated in this lab, manipulation of hepatic SS activity
should provide the basis for understanding the complex
regulatory mechanisms in the cholesterol biosynthetic
pathway and for developing novel therapeutic modalities
for hyperlipoproteinemia.
We thank Drs. Y. K. Ho, Michael S. Brown, and Joseph L.
Goldstein for the generous gift of the antibody against HMGCoA reductase, Prof. Yusuke Furukawa for discussion, and Mika
Hayashi for technical assistance. This work was supported in partby a grant-in-aid for scientific research from the Ministry of Education, Science and Culture.

REFERENCES
1. Goldstein, J. L., and M. S. Brown. 1990. Regulation of the mevalonate pathway. Nature. 343: 425–430.
2. Brown, M. S., J. Ye, R. B. Rawson, and J. L. Goldstein. 2000.
Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans. Cell. 100: 391–398.
3. Nakanishi, M., J. L. Goldstein, and M. S. Brown. 1988. Multivalent
control of 3-hydroxy-3-methylglutaryl coenzyme A reductase.
Mevalonate-derived product inhibits translation of mRNA and accelerates degradation of enzyme. J. Biol. Chem. 263: 8929–8937.
4. Roitelman, J., and R. D. Simoni. 1992. Distinct sterol and nonsterol
signals for the regulated degradation of 3-hydroxy-3-methylglutarylCoA reductase. J. Biol. Chem. 267: 25264–25273.
5. Inoue, S., and R. D. Simoni. 1992. 3-Hydroxy-3-methylglutarylcoenzyme A reductase and T cell receptor alpha subunit are differentially degraded in the endoplasmic reticulum. J. Biol. Chem. 267:
9080–9086.
6. Sever, N., B. L. Song, D. Yabe, J. L. Goldstein, M. S. Brown, and R. A.
DeBose-Boyd. 2003. Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol. J. Biol. Chem. 278: 52479–52490.
7. Sever, N., T. Yang, M. S. Brown, J. L. Goldstein, and R. A. DeBose-Boyd.
2003. Accelerated degradation of HMG CoA reductase mediated bybinding of insig-1 to its sterol-sensing domain. Mol. Cell. 11: 25–33.
8. Grundy, S. M., J. I. Cleeman, C. N. Merz, H. B. Brewer, Jr., L. T.
Clark, D. B. Hunninghake, R. C. Pasternak, S. C. Smith, Jr., and N. J.
Stone. 2004. Implications of recent clinical trials for the National
Cholesterol Education Program Adult Treatment Panel III guidelines. Arterioscler. Thromb. Vasc. Biol. 24: e149–e161.
9. Kita, T., M. S. Brown, and J. L. Goldstein. 1980. Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in liversof mice treated with mevinolin, a competitive inhibitor of the reductase. J. Clin. Invest. 66: 1094–1100.

Adenovirus-mediated gene transfer of squalene synthase

1957

Downloaded from www.jlr.org at Univ of Tokyo Medical Library on August 23, 2006

antibodies against HMG-CoA reductase. Our standard dietcontaining 0.075% of cholesterol might suppress the HMGCoA reductase protein to a level below the sensitivity (37).
A similar deficiency of mevalonate metabolites may
underlie the marked upregulation of HMG-CoA reductase
in cells from mevalonate kinase-deficient patients, where
HMG-CoA reductase activity is increased by 6-fold despite
the absence of the increases in its expression level (38).
In addition to the substrate pool size of isoprenoids in thecholesterol biosynthetic pathway, flux to the isoprenoid
pathway might be regulated by a separate isoprenoid transport network that selectively extracts some of these compounds and distributes them to other synthetic enzymes.
There are two potential explanations for the hypercholesterolemia in mice infected with Ad-SS: i) secretion ofcholesterol-rich VLDL and ii) defective plasma clearanceof LDL. Involvement of the secretion of cholesterol-rich
VLDL is supported by the following findings: 1) LDLcholesterol was increased even in the LDLR knockout
mice in which the LDL clearance was maximally suppressed (Fig. 4B). 2) Cholesterol-rich VLDL was accumulated in the plasma of mice after the intravenous injectionof Triton WR1339 (Fig. 5B). 3) Hepatic ACAT activity,
which is tightly associated with VLDL secretion (39–41),
was increased. Supporting this, an in vitro study using
McARH7777 cells showed that the secretion of cholesteroland fatty acids into the medium was increased when thecells overexpressed SS (Fig. 6). In this regard, it is interesting to note that some SS inhibitors, but not statins, inhibit VLDL secretion from the liver (17, 34, 42). These
considerations suggest the intriguing possibility that
nonsterol mevalonate metabolites, for example, farnesol,or newly synthesized cholesterol regulate VLDL secretion(42); this awaits further investigation. Defective LDL clearance may also contribute to the development of hypercholesterolemia, because the mRNA expression of the
LDLR gene is downregulated (Table 1). The downregulation of the LDLR gene might result from the feedback
regulation by the increased cholesterol biosynthesis. Consistently, SS inhibitors stimulate the LDLR activity (16, 17).
It is of note that cholesterol contents per weight of wet
tissue did not significantly increase in the liver despite theincreased cholesterol synthesis (Table 2). This resistanceto cholesterol accumulation was not observed in transgenic mice overexpressing SREBP-1a, -1c, or -2 under thecontrol of the phosphoenolpyruvate carboxykinase promoter in which cholesterol synthesis is stimulated by theoverexpression of the active form of SREBPs and which exhibit massive accumulation of cholesterol in the liver (43–45). One explanation for this may be that the degree of
the increase in cholesterol synthesis was not large enoughto achieve a detectable buildup of cholesterol in the liver,possibly because the time span (7 days) was not longenough. An increased cholesterol flux into the lipoprotein
assembly without an increased hepatic uptake may be
another explanation. SREBP may promote the transcription of genes that participate in lipid droplet formation,rather than genes involved in VLDL secretion. A third
possibility is that cholesterol is efficiently eliminated out of

1958

Journal of Lipid Research

Volume 47, 2006

SREBP-1a mice unmasks hyperlipidemia resulting from productionof lipid-rich VLDL. J. Clin. Invest. 103: 1067–1076.
30. Shibata, N., M. Arita, Y. Misaki, N. Dohmae, K. Takio, T. Ono, K.
Inoue, and H. Arai. 2001. Supernatant protein factor, which stimulates the conversion of squalene to lanosterol, is a cytosolic squalene transfer protein and enhances cholesterol biosynthesis. Proc.
Natl. Acad. Sci. USA. 98: 2244–2249.
31. Aalto-Setala, K., E. A. Fisher, X. Chen, T. Chajek-Shaul, T. Hayek,
R. Zechner, A. Walsh, R. Ramakrishnan, H. N. Ginsberg, and J. L.
Breslow. 1992. Mechanism of hypertriglyceridemia in human apolipoprotein (apo) CIII transgenic mice. Diminished very low density lipoprotein fractional catabolic rate associated with increased
apo CIII and reduced apo E on the particles. J. Clin. Invest. 90:
1889–1900.
32. Dietschy, J. M., and D. K. Spady. 1984. Measurement of rates ofcholesterol synthesis using tritiated water. J. Lipid Res. 25: 1469–1476.
33. Gerdes, J., U. Schwab, H. Lemke, and H. Stein. 1983. Production ofa mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int. J. Cancer. 31: 13–20.
34. Ugawa, T., H. Kakuta, H. Moritani, O. Inagaki, and H. Shikama.
2003. YM-53601, a novel squalene synthase inhibitor, suppresses
lipogenic biosynthesis and lipid secretion in rodents. Br. J. Pharmacol. 139: 140–146.
35. Correll, C. C., L. Ng, and P. A. Edwards. 1994. Identification of
farnesol as the non-sterol derivative of mevalonic acid required
for the accelerated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. J. Biol. Chem. 269: 17390–17393.
36. Meigs, T. E., and R. D. Simoni. 1997. Farnesol as a regulator of
HMG-CoA reductase degradation: characterization and role offarnesyl pyrophosphatase. Arch. Biochem. Biophys. 345: 1–9.
37. Engelking, L. J., G. Liang, R. E. Hammer, K. Takaishi, H. Kuriyama,
B. M. Evers, W. P. Li, J. D. Horton, J. L. Goldstein, and M. S. Brown.
2005. Schoenheimer effect explained—feedback regulation of cholesterol synthesis in mice mediated by Insig proteins. J. Clin. Invest.
115: 2489–2498.
38. Houten, S. M., M. S. Schneiders, R. J. Wanders, and H. R.
Waterham. 2003. Regulation of isoprenoid/cholesterol biosynthesis in cells from mevalonate kinase-deficient patients. J. Biol. Chem.
278: 5736–5743.
39. Huff, M. W., D. E. Telford, P. H. Barrett, J. T. Billheimer, and P. J.
Gillies. 1994. Inhibition of hepatic ACAT decreases ApoB secretionin miniature pigs fed a cholesterol-free diet. Arterioscler. Thromb. 14:
1498–1508.
40. Musanti, R., L. Giorgini, P. P. Lovisolo, A. Pirillo, A. Chiari, and G.
Ghiselli. 1996. Inhibition of acyl-CoA: cholesterol acyltransferase
decreases apolipoprotein B-100-containing lipoprotein secretionfrom HepG2 cells. J. Lipid Res. 37: 1–14.
41. Spady, D. K., M. N. Willard, and R. S. Meidell. 2000. Role of acylcoenzyme 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. 275:
27005–27012.
42. Hiyoshi, H., M. Yanagimachi, M. Ito, N. Yasuda, T. Okada, H. Ikuta,
D. Shinmyo, K. Tanaka, N. Kurusu, I. Yoshida, et al. 2003. Squalene
synthase inhibitors suppress triglyceride biosynthesis through thefarnesol pathway in rat hepatocytes. J. Lipid Res. 44: 128–135.
43. Shimano, H., J. D. Horton, R. E. Hammer, I. Shimomura, M. S.
Brown, and J. L. Goldstein. 1996. Overproduction of cholesteroland fatty acids causes massive liver enlargement in transgenic miceexpressing truncated SREBP-1a. J. Clin. Invest. 98: 1575–1584.
44. Shimano, H., J. D. Horton, I. Shimomura, R. E. Hammer, M. S.
Brown, and J. L. Goldstein. 1997. Isoform 1c of sterol regulatory
element binding protein is less active than isoform 1a in livers oftransgenic mice and in cultured cells. J. Clin. Invest. 99: 846–854.
45. Horton, J. D., I. Shimomura, M. S. Brown, R. E. Hammer, J. L.
Goldstein, and H. Shimano. 1998. Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose
tissue of transgenic mice overproducing sterol regulatory elementbinding protein-2. J. Clin. Invest. 101: 2331–2339.
46. Holstein, S. A., C. L. Wohlford-Lenane, and R. J. Hohl. 2002.
Consequences of mevalonate depletion. Differential transcriptional, translational, and post-translational up-regulation of Ras,
Rap1a, RhoA, and RhoB. J. Biol. Chem. 277: 10678–10682.
47. Razani, B., J. A. Engelman, X. B. Wang, W. Schubert, X. L. Zhang,
C. B. Marks, F. Macaluso, R. G. Russell, M. Li, R. G. Pestell, et al. 2001.
Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. J. Biol. Chem. 276: 38121–38138.

Downloaded from www.jlr.org at Univ of Tokyo Medical Library on August 23, 2006

10. Thompson, P. D., P. Clarkson, and R. H. Karas. 2003. Statin-associated myopathy. J. Am. Med. Assoc. 289: 1681–1690.
11. Shechter, I., E. Klinger, M. L. Rucker, R. G. Engstrom, J. A. Spirito,
M. A. Islam, B. R. Boettcher, and D. B. Weinstein. 1992. Solubilization, purification, and characterization of a truncated form of
rat hepatic squalene synthetase. J. Biol. Chem. 267: 8628–8635.
12. McKenzie, T. L., G. Jiang, J. R. Straubhaar, D. G. Conrad, and I.
Shechter. 1992. Molecular cloning, expression, and characterization of the cDNA for the rat hepatic squalene synthase. J. Biol. Chem.
267: 21368–21374.
13. Guan, G., P. H. Dai, T. F. Osborne, J. B. Kim, and I. Shechter.
1997. Multiple sequence elements are involved in the transcriptional regulation of the human squalene synthase gene. J. Biol.
Chem. 272: 10295–10302.
14. Memon, R. A., I. Shechter, A. H. Moser, J. K. Shigenaga, C.
Grunfeld, and K. R. Feingold. 1997. Endotoxin, tumor necrosis
factor, and interleukin-1 decrease hepatic squalene synthase activity, protein, and mRNA levels in Syrian hamsters. J. Lipid Res. 38:
1620–1629.
15. Baxter, A., B. J. Fitzgerald, J. L. Hutson, A. D. McCarthy, J. M.
Motteram, B. C. Ross, M. Sapra, M. A. Snowden, N. S. Watson, R. J.
Williams, et al. 1992. Squalestatin 1, a potent inhibitor of squalene
synthase, which lowers serum cholesterol in vivo. J. Biol. Chem. 267:
11705–11708.
16. Hiyoshi, H., M. Yanagimachi, M. Ito, I. Ohtsuka, I. Yoshida, T.
Saeki, and H. Tanaka. 2000. Effect of ER-27856, a novel squalene
synthase inhibitor, on plasma cholesterol in rhesus monkeys: comparison with 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors.
J. Lipid Res. 41: 1136–1144.
17. Nishimoto, T., Y. Amano, R. Tozawa, E. Ishikawa, Y. Imura, H.
Yukimasa, and Y. Sugiyama. 2003. Lipid-lowering properties of
TAK-475, a squalene synthase inhibitor, in vivo and in vitro. Br. J.
Pharmacol. 139: 911–918.
18. Tozawa, R., S. Ishibashi, J. Osuga, H. Yagyu, T. Oka, Z. Chen, K.
Ohashi, S. Perrey, F. Shionoiri, N. Yahagi, et al. 1999. Embryoniclethality and defective neural tube closure in mice lacking squalene
synthase. J. Biol. Chem. 274: 30843–30848.
19. Ohashi, K., J. Osuga, R. Tozawa, T. Kitamine, H. Yagyu, M. Sekiya,
S. Tomita, H. Okazaki, Y. Tamura, N. Yahagi, et al. 2003. Early embryonic lethality caused by targeted disruption of the 3-hydroxy-3methylglutaryl-CoA reductase gene. J. Biol. Chem. 278: 42936–42941.
20. Tsukamoto, K., P. Smith, J. M. Glick, and D. J. Rader. 1997. Liverdirected gene transfer and prolonged expression of three major
human ApoE isoforms in ApoE-deficient mice. J. Clin. Invest. 100:
107–114.
21. Okazaki, H., J. Osuga, K. Tsukamoto, N. Isoo, T. Kitamine, Y.
Tamura, S. Tomita, M. Sekiya, N. Yahagi, Y. Iizuka, et al. 2002.
Elimination of cholesterol ester from macrophage foam cells by
adenovirus-mediated gene transfer of hormone-sensitive lipase. J.
Biol. Chem. 277: 31893–31899.
22. Ishibashi, S., M. S. Brown, J. L. Goldstein, R. D. Gerard, R. E.
Hammer, and J. Herz. 1993. Hypercholesterolemia in low density
lipoprotein receptor knockout mice and its reversal by adenovirusmediated gene delivery. J. Clin. Invest. 92: 883–893.
23. Yang, J., J. L. Goldstein, R. E. Hammer, Y. A. Moon, M. S. Brown,
and J. D. Horton. 2001. Decreased lipid synthesis in livers of micewith disrupted Site-1 protease gene. Proc. Natl. Acad. Sci. USA. 98:
13607–13612.
24. Cohen, L. H., A. M. Griffioen, R. J. Wanders, C. W. Van Roermund,C. M. Huysmans, and H. M. Princen. 1986. Regulation of squalene
synthetase activity in rat liver: elevation by cholestyramine, but nodiurnal variation. Biochem. Biophys. Res. Commun. 138: 335–341.
25. Yagyu, H., T. Kitamine, J. Osuga, R. Tozawa, Z. Chen, Y. Kaji, T. Oka,
S. Perrey, Y. Tamura, K. Ohashi, et al. 2000. Absence of ACAT-1
attenuates atherosclerosis but causes dry eye and cutaneous xanthomatosis in mice with congenital hyperlipidemia. J. Biol. Chem. 275:
21324–21330.
26. Folch, J., M. Lees, and G. H. Sloane Stanley. 1957. A simple method
for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226: 497–509.
27. Heider, J. G., and R. L. Boyett. 1978. The picomole determination offree and total cholesterol in cells in culture. J. Lipid Res. 19: 514–518.
28. Eisele, B., R. Budzinski, P. Muller, R. Maier, and M. Mark. 1997.
Effects of a novel 2,3-oxidosqualene cyclase inhibitor on cholesterol
biosynthesis and lipid metabolism in vivo. J. Lipid Res. 38: 564–575.
29. Horton, J. D., H. Shimano, R. L. Hamilton, M. S. Brown, and J. L.
Goldstein. 1999. Disruption of LDL receptor gene in transgenic