Cholesterol Lowering in Low Density Lipoprotein Receptor Knockout Mice
Overexpressing Apolipoprotein E
Jun-ichi Osuga,* Mari Yonemoto,‡ Nobuhiro Yamada,* Hitoshi Shimano,* Hiroaki Yagyu,* Ken Ohashi,* Kenji Harada,*
Toshio Kamei,‡ Yoshio Yazaki,* and Shun Ishibashi**The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655,
Japan; and ‡Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., 3 Okubo, Tsukuba, Ibaragi 300-33, Japan

Abstract
Apo E is a key molecule in the lipoprotein metabolism; thus,
genetic manipulation of apo E may prove useful in thetreatment of hypercholesterolemia. To test the feasibility ofthis idea, we have generated low density lipoprotein receptor (LDLR) knockout mice that overexpress the rat apo E
transgene (ETg1/1:LDLRKO), and compared their plasma
lipoprotein profiles with those of nonexpressing LDLR
knockout mice (ETg2/2:LDLRKO). On a normal chow diet,the mean plasma cholesterol level of ETg1/1:LDLRKO mice
was significantly lower than that of ETg2/2:LDLRKO mice
(189 versus 240 mg/dl, P , 0.01). The LDL fraction was selectively reduced in the ETg1/1:LDLRKO mice. Despite thechallenge with an atherogenic diet, cholesterol lowering was
persistently observed and fatty streak lesions in the aortic
sinus were significantly suppressed in the mice overexpressing apo E. These results imply that stimulation of hepatic
production of apo E may be used as a promising adjunctive
therapy for homozygous familial hypercholesterolemia. (J.
Clin. Invest. 1998. 102:386–394.) Key words: apo E • low
density lipoprotein receptor • hypercholesterolemia • knockout mouse • atherosclerosis

Introduction
Apo E plays a crucial role in the metabolism of plasma lipoproteins (1). Apo E is produced primarily by the liver and distributed in all classes of lipoproteins. In particular, the plasma
metabolism of triglyceride (TG)1-rich lipoproteins such as chy-

J. Osuga and M. Yonemoto contributed equally to this work.
Address correspondence to Shun Ishibashi, The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Phone: 81-3-38155411 (Ext. 3129); FAX: 81-3-5802-2955; E-mail: ishibash-tky@umin.
u-tokyo. ac.jp
Received for publication 7 July 1997 and accepted in revised form
21 May 1998.
1. Abbreviations used in this paper: CR, chylomicron remnants; FCR,
fractional catabolic rate; HPLC, high performance liquid chromatography; IDL, intermediate density lipoprotein; LDLR, low density lipoprotein receptor; SRID, single radial immunodiffusion; TC, totalcholesterol; TG, triglyceride.
J. Clin. Invest.
© The American Society for Clinical Investigation, Inc.
0021-9738/98/07/0386/09 $2.00
Volume 102, Number 2, July 1998, 386–394
http://www.jci.org
386

Osuga et al.

lomicron, VLDL, and intermediate density lipoprotein (IDL)
is profoundly influenced by apo E. Several distinct mechanisms have been proposed to explain the role of apo E in themetabolism of TG-rich lipoproteins. First, apo E binds to thelow density lipoprotein receptor (LDLR) with a high affinity,
thereby facilitating the LDLR-mediated hepatic lipoprotein
uptake (2, 3). Second, apo E binds to cell surface proteoglycans, thereby sequestering the apo E–containing lipoproteinsto the surface of cells such as hepatocytes and vascular endothelial cells in the space of Disse of the liver (4). Third, evidence suggests that other endocytic lipoprotein receptors are
involved in the hepatic uptake of chylomicron remnants (CR),lipoproteins extremely rich in apo E (5, 6). Herz and his colleagues have shown that LDLR-related protein, a multifunctional behemoth receptor belonging to the LDLR gene family,
is responsible for the hepatic uptake of CR in the absence of
the LDLR (7–9).
Therapeutic application of this property of apo E has been
vigorously sought. We and Mahley et al. demonstrated a transient acute reduction in plasma cholesterol levels by z 20–40%
after intravenous administration of purified apo E (6–70 mg)
into Watanabe heritable hyperlipidemic rabbits, an animallacking normal LDLR (10, 11). Repeated injection of apo E(10 mg) three times a week, however, did not affect the plasmacholesterol levels, even though the progression of atherosclerosis was suppressed by 50% (12).
To know whether sustained supply of exogenous apo E has
a cholesterol-lowering effect, we developed a line of transgenic
mice that overexpress rat apo E under control of metallothionein promoter (ETg) (13). The plasma levels of total cholesterol(TC) and TG were reduced in these mice particularly after
treatment with zinc, which induces the expression of the transgene, even after feeding with a high-fat, high-cholesterol diet.
The cholesterol lowering may result from augmented hepatic
uptake of the lipoproteins, because 125I-labeled VLDL or LDL
were cleared from the plasma faster in these ETg mice (14). In
addition, the plasma excursions of retinyl ester, an indicator of
chylomicron and CR, were markedly suppressed in the ETg
mice, after intragastric bolus administration of retinol (14).
Based on these results, we proposed that the overexpressed
apo E enhances the plasma clearance of lipoproteins of both
hepatic and intestinal origin. However, whether or not thepathway by which this clearance is mediated involves the
LDLR remains unknown.
The present study was undertaken to test the feasibility of
the idea that genetic manipulation of apo E is useful for thetreatment of hypercholesterolemia arising from the LDLR deficiency. We have cross-bred the ETg mice to LDLR knockout(LDLRKO) mice, generated LDLRKO mice possessing the
apo E transgene in a heterozygous (ETg1/2:LDLRKO) or homozygous form (ETg1/1:LDLRKO), and compared theirplasma lipoprotein metabolism and atherosclerosis with thoseof nonexpressing LDLRKO (ETg2/2:LDLRKO) mice.

Methods
Mice. Generation of ETg (13) and LDLRKO mice (15) were described in the indicated references. PCR was performed to determine
the wild-type and mutated LDLR allele using the following sets ofprimers: primer A (59-GATGCCAGGATGGCAA-39) and primer B(59-CGCAGTGCTCCTCATCTGAC-39), yielding a 354-bp fragment for the wild-type allele and a 1.7-kb fragment for the disrupted allele; and primer C (59-GATTGGGAAGACAATAGCAGGCATGC-39) and primer D (59-GGCAAGATGGCTCAGCAAGCAAGCAAAGGC-39), yielding a 1.49-kb fragment for the disrupted allele. Southern blot analyses for the apo E transgene wasdescribed previously (13). The radioactivities were measured by
BAS2000 (Fuji Film, Tokyo, Japan) to discriminate between the heterozygotes and homozygotes. Male LDLRKO mice were mated tofemale ETg mice. The resulting offspring, which were heterozygous
for the apo E transgene and were obligatorily heterozygous for the
disrupted LDLR locus (ETg1/2:LDLR1/2), were further bred to LDLRKO mice to produce LDLRKO mice heterozygous for the apo E
transgene (ETg1/2:LDLR2/2 5 ETg1/2:LDLRKO). Brother–sistermating of the ETg1/2:LDLRKO mice was performed to generate
ETg2/2:LDLRKO, ETg1/2:LDLRKO, and ETg1/1:LDLRKO micewith comparable genetic background: 50% from C57BL/6J, 37.5%
from 129Sv, and 12.5% from DBA/2.
Mice were maintained on a 12-h dark/light cycle and were allowed access to food and water ad libitum. Two diets were used: (a) anormal chow diet (Lab Diet 5002, PMI Feeds, Inc., St. Louis, MO)
that contained 4.5% animal fat and 0.025% cholesterol, 1.16 mM Zn,
1.36 mM Mn, 0.27 mM Cu, 0.01 mM Fe, and 0.04 mM Cr; (b) a 1.25%
cholesterol/atherogenic diet (three parts of the normal diet mixed
with one part of a cholesterol, cocoa butter, casein, and sodium cholate diet [Thomas Hartroft diet; PMI Foods Inc.]). The final diet contained 1.25% cholesterol, 7.5% cocoa butter, 7.5% casein, and 0.5%
sodium cholate (16). For induction of the metallothionein promoter,water supplemented with 20 mM ZnSO4 was administered for . 2 wk
(13). Experiments were performed in accordance with institutionalguidelines. Unless otherwise stated, mice aged between 12–16 wk
were used for the experiments.
Plasma lipid analysis. Blood was collected from the retroorbital
plexus into a tube containing EDTA after a 12-h fast, and plasma wasseparated by centrifugation and mixed with 1/500 vol of a solution
containing 60 mg/ml of benzamidine. TC and TG concentrations were
determined enzymatically using kits (Determiner TC555 and Determiner TG555; Kyowa Medex, Tokyo, Japan). For lipoprotein analyses, the plasma was immediately applied to high performance liquid
chromatography (HPLC) or sequential ultracentrifugation. HPLC
analyses were performed according to a modified method of Hara
and Okazaki (16). 5 ml of plasma was applied to a combined column
system composed of two TSK gel Lipopropacks (TOSOH; Tokyo, Japan) in tandem, and eluted with the supplied buffer (TSK eluent LP1;TOSOH) at a rate of 0.5 ml/min. TC concentrations in the effluentswere monitored using a kit (Determiner TC555). Areas under theelution curves for HDL and non-HDL peaks were determined. These
values and plasma TC levels were used to calculate non–HDL- andHDL-cholesterol concentrations.
Quantitation of apo B. Plasma apo B concentrations were determined by isopropanol precipitation method as described (17). In
brief, 100 ml of plasma was obtained from an individual mouse and ultracentrifuged at a density of 1.063 g/ml. An aliquot of the top d ,1.063 g/ml lipoprotein fraction was mixed with an equal volume ofisopropanol in a microfuge tube, incubated at room temperature for
1 h, and centrifuged at 10,000 g for 15 min. The resulting protein pellets were solubilized in 0.1 N NaOH, and protein concentrations were
determined by a kit (BCA protein assay reagent; Pierce, Rockford,
IL). In parallel, apo B concentrations were determined by a single radial immunodiffusion (SRID) technique using a kit designed for measurement of human apo B levels (Apo B plate ‘Daiichi’; Daiichi Pure
Chemicals, Tokyo, Japan), according to a manufacturer’s instruction(18). When the same amounts of LDL apo B were applied to theplates, mouse LDL apo B produced a 8.7-fold larger precipitation
ring in diameter than human LDL apo B. The results of the isopropanol precipitation method were correlated with those of the SRID
method with r2 . 0.99; n 5 9.
Quantitation of apo E. Immunoblot analyses were performed todetermine plasma apo E levels. 1 ml of plasma was subjected to both
SDS/3–15% PAGE and isoelectric focusing (IEF) (Phenotyping apo
E IEF system; Joko, Tokyo, Japan) (19). After proteins were electrophoretically transferred to nitrocellulose filters (Hybond ECL®; Amersham Corp., Arlington Heights, IL) in a buffer containing 20 mM
Tris, 150 mM glycine, 0.02% SDS, and 20% methanol at 200 mA for 6 h,immunoblotting was performed using a rabbit anti–rat apo E polyclonal antibody as the first antibody and 125I-protein A (2,000 cpm/ng,
0.5 mg/ml; Amersham Corp.) as described (13). The radioactive images of the filters were analyzed by BAS2000. Apo E was purified
from either rat or mouse plasma, as previously described (10), andused for calibration.
The following four variables were measured: X, relative radioactivity of the sample apo E compared to the standard rat apo E on
SDS-PAGE; Y, proportion of the radioactivities in rat apo E (threebands together) in all the apo E radioactivities (rat plus mouse) on
IEF; A, radioactivity of the standard rat apo E; and B, the antibody’s
immunoreactivity for rat apo E compared to that for mouse apo E.
Rat and mouse apo E concentrations were calculated from the following formulas: Rat apo E concentration 5 X 3 Y/A; Mouse apo E
concentration 5 B 3 X 3 (1 2 Y)/A.
Analyses of plasma lipoprotein and apolipoproteins. Preparative
ultracentrifugation was performed to obtain CM1VLDL (d , 1.006g/ml), IDL (d 1.006–1.019 g/ml), LDL (d 1.019–1.063 g/ml) and HDL(d 1.063–1.21 g/ml) from the pooled plasma as described (20). TC
contents were measured and represented as mg/dl of original plasma.
After dialysis against saline containing 0.2 mM EDTA and 1 mM
PMSF, each lipoprotein fraction was delipidated overnight at 2208C
with 20 vol of ice-cold ethanol-diethyl ether (3:1, vol/vol), and theapolipoproteins were pelleted by centrifugation for 20 min at 2108C,
1,000 g (21). The pellets were extracted twice with cold anhydrous diethyl ether, and subjected to SDS/3–15% PAGE. Proteins were visualized by staining with Commassie brilliant blue. The intensities ofthe bands were determined by densitometric scanning and quantitated by National Institutes of Health Image software (Bethesda, MD).
Plasma lipoprotein kinetics. After a 12-h fast, blood was obtained
from ETg2/2:LDLRKO or ETg1/1:LDLRKO mice (n 5 10) that had
been maintained on water supplemented with zinc and a normalchow, and pooled for lipoprotein isolation. VLDL (d , 1.006 g/ml)
and LDL (d 1.019–1.063 g/ml) were isolated by ultracentrifugation
and refloated at the same density to increase the purity. VLDL obtained from ETg2/2:LDLRKO or ETg1/1:LDLRKO mice are denoted as E2VLDL or E1VLDL, respectively. These lipoproteins andthe infranatant d . 1.063 g/ml fraction were dialyzed against saline
containing 1 mM sodium phosphate, pH 7.4, 0.2 mM EDTA, and 1 mM
PMSF. The lipoproteins were labeled with 125I by iodine monochloride method (22), and dialyzed as described above. After removing
EDTA and PMSF by dialysis against PBS, the labeled lipoproteins
were mixed with the d . 1.063 g/ml fraction, and diluted with PBS
containing 10 mg/ml BSA to 40 mg/ml. 100 ml (equivalent to 4 mg protein of the lipoprotein) was injected as a bolus into the jugular vein ofmice that had been maintained on water containing zinc for 2 wk, under anesthesia with pentobarbital. Subsequently, blood was collectedfrom the retroorbital plexus at the indicated times. Apo B–associatedradioactivities were determined by the isopropanol precipitation
method as described (14). The value at 1 min after injection was used
as 100%. Fractional catabolic rate (FCR) and production rate were
calculated according to Matthews (23).
Vitamin A fat tolerance test. After a 4-h fast, 100 ml of Chocola
A® (Eisai Co., Ltd.; Tokyo, Japan) containing 2 mg of retinyl palmitate and 10.6 mg castor oil was administered as a bolus through a gastric cannula to mice that had been maintained on water suppleOverexpression of Apo E in LDL-receptor Knockout Mice387

mented with zinc, without anesthesia. Blood was collected from theretroorbital plexus at the indicated times. Plasma was immediately
separated and stored wrapped in foil at 2208C. After extraction withhexane from pooled plasma, the lipids were subjected to HPLC for
the measurement of retinyl palmitate and retinol, as previously described (14).
Pathology. The hearts and attached aortas were removed, fixedwith saline containing 4% formalin, embedded in 25% gelatin, andsectioned as described (24). Briefly, 10 mm cross-sections were taken
sequentially, starting just above the aortic valve and moving along theascending aorta. Five sections, each separated by 120 mm, were used
to evaluate the lesions. Fatty streak lesions were stained using Oil
Red O. The lesion area of each section was estimated by a digitizing
tablet (NEC PC-8875 personal TABLET).
Statistics. Student’s t test was used to compare the values between two groups.

Results
Expression of rat apo E. Immunoblot analyses were performed to measure the plasma concentration of apo E (Fig. 1).
Fig. 1 A compares the intensities of bands corresponding to
apo E purified from mice and rats, when the same amounts (75ng) were loaded to the gels; the antibody had twofold higher
immunoreactivity for rat apo E than for mouse apo E. Fig. 1 Bshows immunoblots of SDS-PAGE (top) and IEF (bottom) forthe plasma apo E of the three types of mice before and after
zinc treatment. SDS-PAGE revealed that plasma apo E levels
were higher in the mice having the rat apo E transgene thanthose without it in the following order before zinc treatment:
ETg1/1:LDLRKO . ETg1/2:LDLRKO . ETg2/2:LDLRKO.
After zinc treatment, plasma apo E levels were further increased in the ETg1/2:LDLRKO mice. Upon IEF, mouse apo
E was focused as a single band, and rat apo E was focused as
three bands that were more acidic than mouse apo E. Presumably, the most basic band of rat apo E is nonsialidated apo E
and the second and third bands are with one and two molecules of sialic acid, respectively. Furthermore, the IEF results
confirm that rat apo E was present only in the ETg1/2:LDLRKO and ETg1/1:LDLRKO mice, but not in the ETg2/2:
LDLRKO.
Table I summarizes the calculated values for the plasma
levels of rat and mouse apo E. Before zinc treatment, theETg1/2:LDLRKO and ETg1/1:LDLRKO mice had rat apo E
in the plasma in amounts comparable to 50% of mouse endogenous apo E. The ETg1/1:LDLRKO mice had 40% higher
concentrations of rat apo E than the ETg1/2:LDLRKO mice.
After zinc treatment, the rat apo E levels were increased by
38% in the ETg1/2:LDLRKO mice and by 23% in the ETg1/1:

Figure 1. Immunoblot analyses of plasma apo E in ETg2/2:LDLRKO (2/2), ETg1/2:LDLRKO (1/2), and ETg1/1:LDLRKO mice (1/1)
maintained on a normal chow. (A) Difference of the immunoreactivity of the antibody. The same amount of rat and mouse apo E (75 ng) was
subjected to SDS-PAGE. (B) Immunoblots of apo E separated by SDS-PAGE and IEF. Blood was collected from four mice with the indicatedgenotypes after a 12-h fast, before and after treatment with zinc. 1 ml of pooled plasma was subjected to SDS-PAGE and IEF analyses. (C) Immunoblots of apo E in VLDL, IDL, LDL, and HDL. Lipoproteins were separated from pooled plasma (n 5 10) after treatment with zinc by sequential ultracentrifugation. Each lipoprotein fraction equivalent to 2 ml of original plasma was subjected to SDS-PAGE.
388

Osuga et al.

Table I. Plasma Apo E Levels
Tg

n

Zn

Total apoE

Table II. Plasma Lipid Levels
Mouse apoE

Rat apoE

Tg

mg/dl

2/2

4

1/2

4

1/1

4

BeforeAfter
BeforeAfter
BeforeAfter19.763.7
18.461.8
22.862.7
26.561.2‡i
27.463.5*
26.262.3‡

19.763.7
18.461.8
15.560.7
16.661.4§
17.161.9
13.661.1‡i

n

Zn

TC

m/f

0
0
7.262.9
9.962.5i
10.362.0
12.763.2

2/2

15
Before 260668.6(8/7) After 240660.8*
1/2
29
Before 254654.9(12/17) After 203650.0§
1/1
16Before 251646.5(7/9) After 189631.2§i

Non–HDL-C

HDL-C

TG

mg/dl

137652.6
122644.1
132638.1
105632.5§
128633.887617.4§i121628.1 127655.5
117628.3 104639.0
122628.6 124636.0
108627.9‡ 128640.2
122634.7 133642.8
101631.1 132643.8

ETg2/2:LDLRKO (2/2), ETg1/2:LDLRKO (1/2), and ETg1/1:LDLRKO mice (1/1) were maintained on a normal chow diet. Blood wascollected before and after treatment with zinc for 2 wk. Plasma apo E
levels were determined by immunoblot analyses as described in Methods. The values are expressed as means6SD. *P , 0.05 versus before of
2/2; ‡ P , 0.001 versus after of 2/2; § P , 0.01 versus after of 1/2 and
2/2; i P , 0.05 after versus before.

ETg2/2:LDLRKO (2/2), ETg1/2:LDLRKO (1/2), and ETg1/1:LDLRKO mice (1/1) were maintained on a normal chow diet. Blood wascollected before and after treatment with zinc for 2 wk. Plasma TC andTG levels were determined. HPLC analyses were performed for eachsample. Non–HDL-cholesterol (non–HDL-C) and HDL-C were calculated from the TC levels and the HPLC profiles. The values are expressed as means6SD. *P , 0.05, ‡P , 0.01, § P , 0.0001 after versus
before. i P , 0.01 versus after of 2/2.

LDLRKO mice. But the mouse apo E levels were decreasedin the ETg1/1:LDLRKO mice; therefore, the combined apo E
concentrations were not changed.
Fig. 1 C illustrates the combined apo E levels in each lipoprotein fraction from the three types of mice treated with zinc.
In any lipoprotein classes, the LDLRKO mice with rat apo E
transgene had more apo E than the LDLRKO mice without
the transgene. The apo E contents were highest in LDL fraction irrespective of the presence of the transgene. Mild genedosage effects were seen in VLDL and IDL fraction.
Plasma lipoprotein profiles. To determine whether constitutive expression of the apo E transgene alters plasma lipid
levels, the plasma TC and TG levels were compared between
the three types of mice, before and after zinc treatment (Table
II). Before zinc treatment, neither plasma TC nor TG differedsignificantly between these mice. After zinc treatment for 2 wk,
the plasma TC levels decreased by 20% in the ETg1/2:LDL-RKO and by 25% in the ETg1/1:LDLRKO mice, but not in
the ETg2/2:LDLRKO mice. Such cholesterol lowering waslargely attributable to the reduction in the non–HDL-C as
shown in Fig. 2; the non–HDL-C was reduced by 20% in the
ETg1/2:LDLRKO mice, and by 32% in the ETg1/1:LDLRKO
mice (Table II).
To further determine which lipoprotein fraction was affected by the expression of rat apo E, we measured the TC and
apo B contents in the lipoproteins isolated from the three
types of mice treated with zinc (Table III). The most outstanding changes were observed in the TC and apo B-100 contents
in the LDL fraction. TC contents in the LDL were reduced by
25% in the ETg1/2:LDLRKO mice and by 39% in the ETg1/1:
LDLRKO mice; apo B-100 contents in the LDL were reduced
by 23% in the ETg1/2:LDLRKO mice, and by 43% in the
ETg1/1:LDLRKO mice. Thus, the changes in the LDL–TC appeared comparable to those in the LDL–apo B-100, indicating

Figure 2. HPLC profiles of plasma lipoproteins from mice maintained on a normal chow. Plasma was obtained from five male ETg2/2:LDLRKO(A, 2/2), ETg1/2:LDLRKO (B, 1/2), and ETg1/1:LDLRKO mice (C, 1/1) maintained on a normal chow and water supplemented with zinc.
Pooled plasma from each group was subjected to HPLC lipoprotein analysis, and cholesterol contents in effluents were monitored.
Overexpression of Apo E in LDL-receptor Knockout Mice389

Table III. Cholesterol, Apo B-100, and Apo B-48 Compositionin the Plasma Lipoproteins
Tg

VLDL

IDL

LDL

HDL

115
86
70
89.5/7.0
69.2/5.3
50.6/3.8

75
82
80
N.D.
N.D.
N.D.

mg/dl

Cholesterol

Apo B-100/Apo B-48

2/2
1/2
1/1
2/2
1/2
1/1

26
35
25
0.8/1.0
0.8/1.2
1.3/1.420
12
11
0.2/0.5
0.2/0.7
0.3/0.7

ETg2/2:LDLRKO (2/2), ETg1/2:LDLRKO (1/2), and ETg1/1:LDLRKO mice (1/1) were maintained on a normal chow diet. Blood wascollected after treatment with zinc for 2 wk. VLDL, IDL, LDL, and
HDL were isolated by sequential ultracentrifugation and cholesterol
contents were determined enzymatically. Isopropanol precipitation
method was used to determine the total apo B concentrations. Lipoproteins were subjected to SDS-PAGE, stained with Coomassie brilliant
blue, and scanned by a densitometer. The concentrations of apo B-100
and apo B-48 were calculated from the values for total apo B and relative ratio between apo B-100 and apo B-48. N.D. 5 not determined.

that the overexpression of rat apo E reduced the particle number of the apo B-100–containing lipoproteins primarily in the
LDL fraction.
SDS/PAGE of apolipoproteins shows that apo B-100, a
predominant apo B species, in the LDL fraction was reducedin the ETg1/2:LDLRKO and ETg1/1:LDLRKO mice, compared to the ETg2/2:LDLRKO mice (Fig. 3). VLDL and IDL
contained less apo B-100 than LDL, and there was no signifi-

cant difference in its contents between the three types of mice.
Consistent with the immunoblot results (Fig. 1), the ETg1/2:
LDLRKO and ETg1/1:LDLRKO mice contained more apo E
than the ETg2/2:LDLRKO mice, especially in the VLDL fraction. No significant difference was observed in the compositionof the other apolipoproteins such as apo A-I and apo B-48 between the three types of mice.
Lipoprotein kinetics studies. To clarify how the overexpression of rat apo E reduced plasma cholesterol levels even in
the absence of the LDLR, lipoprotein kinetics studies were
performed (Fig. 4). When injected into wild-type mice with thefunctional LDLR, 125I-labeled E1VLDL was cleared fasterthan 125I-labeled E2VLDL; their t1/2 were z 15 and 25 min, respectively (Fig. 4 A). The mean FCR of 125I-labeled E1VLDL
were 1.6 times larger than that of 125I-labeled E2VLDL (2.17
versus 1.33 pools/h; P , 0.05).
Next, 125I-labeled E1VLDL or 125I-labeled E2VLDL were
injected into the ETg1/1:LDLRKO or ETg2/2:LDLRKO
mice, respectively (Fig. 4 B). Within the initial 3 h, no significant
difference in plasma clearance of the autologous lipoproteins
was observed; t1/2 5 90 min. After 6 h, however, 125I-labeled
E1VLDL cleared faster than 125I-labeled E2VLDL. Overall,
the mean FCR of 125I-labeled E1VLDL was 1.45 times larger
than that of 125I-labeled E2VLDL (0.33 versus 0.23 pools/h,
P , 0.05). The mean production rate of apo B was calculated
to be 21.2 and 18.4 nmol/h/g body weight for the ETg2/2:LDLRKO and ETg1/1:LDLRKO mice, respectively (P , 0.05).
A similar kinetic study was performed using autologous
LDL. There was no difference in the plasma clearance of LDL
between the ETg2/2:LDLRKO and ETg1/1:LDLRKO mice
up to 24 h (data not shown).
A vitamin A fat tolerance test did not reveal any differencein the plasma excursion of retinyl ester between the three

Figure 3. Apolipoprotein composition of lipoproteinsin ETg2/2:LDLRKO (2/2),
ETg1/2:LDLRKO (1/2), andETg1/1:LDLRKO mice (1/1)
maintained on a normal chow
and water supplemented withzinc. Pooled plasma isolated
from 10 mice with indicated genotypes was subjected to sequential ultracentrifugation toisolate VLDL, IDL, LDL, andHDL. Delipidated apoproteins
equivalent to 20 ml (VLDL andIDL) or 10 ml (LDL and HDL)of original plasma were subjected to SDS/3–15% PAGE.
Two separate gels were used;
one for VLDL, IDL, and HDL,
and the other for LDL. Protein
staining with Coomassie brilliant
blue was performed separately
for each gel. Therefore, comparison of the band intensities between VLDL, IDL, and HDL
was valid, but not between theselipoproteins and LDL. Gels
were calibrated with Rainbow protein molecular weight markers. The migration positions of apo B-100, apo B-48, apo A-IV, apo E, apo A-I,
and apo A-II 1 C’s are noted on the right.
390

Osuga et al.

Figure 4. Plasma kinetics of 125I-labeled VLDL. VLDL were isolated
from either ETg2/2:LDLRKO (s) or ETg1/1:LDLRKO mice (d)that had been maintained on a normal chow and water supplemented
with zinc, by ultracentrifugation at a density of 1.006 g/ml, and refloated at same density. The same lipoprotein preparations as shownin Fig. 1 C were used for radioiodination by iodine monochloride
method. Specific activities of labeled lipoproteins were 50–200 cpm/
ng protein. 4 mg protein were injected into the jugular vein of threemice, together with the autologous fraction (d . 1.063 g/ml) equivalent to the same amount of plasma from which VLDL originated. At
the indicated times, blood was collected. Apo B–associated radioactivity was determined using the isopropanol precipitation method as
previously described (9). The value at 1 min after injection was used
as 100%. The recipients were wild-type mice (A), and autologous
ETg2/2:LDLRKO and ETg1/1:LDLRKO mice that had been maintained on a normal chow and water supplemented with zinc (B).

types of mice (Fig. 5 A). The plasma excursion curves for freeretinol were also similar to each other, indicating that no difference in the absorption of vitamin A existed (Fig. 5 B).
Resistance against diet-induced hypercholesterolemia. The
response of plasma TC and TG levels to the feeding with a

Figure 5. Vitamin A tolerance test. Aqueous retinyl palmitate (2 mg
per animal) was administered as a bolus through a gastric cannula to
the ETg2/2:LDLRKO (s), ETg1/2:LDLRKO (d), and ETg1/1:
LDLRKO mice (n) that had been maintained on a normal chow andwater supplemented with zinc, after a 4-h fast (n 5 5). Blood was collected at the indicated times. Plasma levels of retinyl palmitate (A)and free retinol (B) were determined in the pooled plasma.

1.25% cholesterol/atherogenic diet was shown in Table IV.
During the feeding, the mice had been maintained on watersupplemented with zinc. The ETg2/2:LDLRKO mice developed severe hypercholesterolemia with a mean plasma TC
level of 3,116 mg/dl, which is similar to the previously reported
values (16). The ETg1/2:LDLRKO and ETg1/1:LDLRKO
mice exhibited milder hypercholesterolemia compared with
the ETg2/2:LDLRKO mice, indicating that the overexpressionof rat apo E suppressed diet-induced hypercholesterolemia by
23% in the ETg1/2:LDLRKO mice and by 38% in the ETg1/1:
LDLRKO mice, even in the absence of the functional LDLR.
Lipoprotein analyses showed that the TC contents were reduced in CM1VLDL and in IDL, but not in LDL (Table V).
Overexpression of Apo E in LDL-receptor Knockout Mice391

Table IV. Effects of Cholesterol Feeding on the Plasma
Lipid Levels
Tg

n

Zn

m/f

2/2
1/2
1/1

15
(8/7)
27
(11/16)
15
(6/9)

TC

Table V. Cholesterol Contents in the Lipoprotein Fraction after
Cholesterol Feeding
TG

Tg

CM/VLDL

mg/dl

BeforeAfter
BeforeAfter
BeforeAfter297673311661025
28464823946893*
294647
19186632‡

IDL

LDL

HDL

336
362
331

8
29
48

mg/dl

12163310165911162873655
13164066640

ETg2/2:LDLRKO (2/2), ETg1/2:LDLRKO (1/2), and ETg1/1:LDLRKO mice (1/1) were initially maintained on a normal chow, and thenon a 1.25% cholesterol/atherogenic diet and water supplemented withzinc for 14 wk. Blood was collected before and after the feeding. Plasma
TC and TG levels were determined enzymatically. HPLC analyses were
performed for each sample. Non–HDL-cholesterol (non–HDL-C) and
HDL-C were calculated from the TC levels and the HPLC profiles. The
values are expressed as means6SD. *P , 0.05, ‡ P , 0.01.

The concentrations of plasma apo B measured by SRID wereas follows: 3,05561,103 mg/dl (n 5 6) in the ETg2/2:LDLRKO, 1,50561,093 mg/d (n 5 8) in the ETg1/2:LDLRKO, and
8876715 mg/dl (n 5 11) in the ETg1/1:LDLRKO mice. ETg1/1:
LDLRKO and ETg1/2:LDLRKO mice had significantly lower
plasma apo B levels than ETg2/2:LDLRKO mice (P , 0.05
and , 0.001, respectively).
Suppression of diet-induced atherosclerosis. We compared
the cross-sectional lesion areas in the aortic sinus from theETg2/2:LDLRKO and ETg1/1:LDLRKO mice after feedingwith a 1.25% cholesterol/atherogenic diet (Fig. 6). The lesion
areas of the ETg1/1:LDLRKO mice were significantly
smaller than those of the ETg2/2:LDLRKO mice by 45%(124,603641,464 [n 5 14] versus 68051630525 mm2 [n 5 16],
P , 0.001).

2/2
1/2
1/1

968
475
284

1010
990
694

ETg2/2:LDLRKO (2/2), ETg1/2:LDLRKO (1/2), and ETg1/1:LDLRKO mice (1/1) were initially maintained on a normal chow, and thenon a 1.25% cholesterol/atherogenic diet and water supplemented withzinc for 14 wk. Blood was collected after the feeding. VLDL, IDL, LDL,
and HDL were isolated by sequential ultracentrifugation, and their cholesterol contents were determined.

Plasma apo B-100 concentrations were reduced in micethat overexpressed apo E (Fig. 3 and Table III), corroborating
the idea that the reduction of non–HDL-C levels is due to adecrease in the particle number of apo B–containing lipoproteins, but not to a decrease in the cholesterol contents in eachlipoprotein particle. E1VLDL was enriched with rat apo E
compared to E2VLDL (Fig. 1), and cleared faster than
E2VLDL when injected into wild-type mice with the functional LDLR (Fig. 4 A), indicating that E1VLDL has higher
affinity for the LDLR than E2VLDL. When E2VLDL or
E1VLDL were injected back into the LDLRKO mice fromwhich the respective VLDL were isolated (Fig. 4 B), theirplasma clearance rates were four- and sixfold slower than thevalues for the experiments where wild-type mice were used asrecipients. These results indicate that the LDLR has overwhelming contribution to the clearance of these lipoproteins.
The difference in the plasma clearance between E2VLDL
and E1VLDL was not discernible in the initial 3 h, but
E1VLDL was cleared faster than E2VLDL after 6 h. As awhole, E1VLDL had higher FCR and lower production rate

Discussion
The present study has demonstrated that overexpression of
apo E reduces plasma cholesterol levels even in the absence ofthe functional LDLR. The cholesterol lowering appears to result from the reduction of the apo B-100–containing lipoproteins in LDL. From the results of the lipoprotein kinetics studies, we speculate that both the decreased production and theincreased clearance of VLDL, but not the changes in chylomicron metabolism, account for the cholesterol-lowering in the
LDLRKO mice overexpressing rat apo E. Furthermore, theoverexpression of rat apo E suppressed diet-induced hypercholesterolemia as well as atherosclerosis.
We have previously shown that mice overexpressing apo E
have decreased levels of plasma TC and TG, and are resistant
to diet-induced hypercholesterolemia (13, 14). Lipoproteinskinetics studies showed the increased plasma clearance of lipoproteins such as VLDL and LDL. However, it remains unknown whether the increased clearance of the lipoproteins wasmediated through the LDLR. The present study has clearly
demonstrated that the overexpression of rat apo E has cholesterol-lowering effects even in the absence of the functional
LDLR, indicating that the non-LDLR pathway is also involved in the cholesterol lowering by the apo E transgene.
392

Osuga et al.

Figure 6. Lipid staining lesion area in the mouse aorta. After feeding
a 1.25% cholesterol/atherogenic diet and water supplemented withzinc for 14 wk, the hearts with aortic roots were taken for the evaluation of fatty streak lesion areas. The mean lesion area per section isplotted in each of the ETg2/2:LDLRKO (s) and ETg1/1:LDLRKO
mice (d). *denotes the difference at P , 0.001.

than E2VLDL, even when injected into mice lacking theLDLR.
Apo E secreted from the liver has been thought to be an
important determinant of the clearance of lipoproteins. Thissecretion–recapture model was initially proposed to explain
the local autocrine and paracrine-like function of apo E (1, 25),
and was subsequently extended to explain the rapid clearanceof CR (26). In agreement with this model, the enhanced clearance of E1VLDL via the non-LDLR pathway may, at least inpart, reduce the apo B-100 levels in the LDLRKO mice expressing rat apo E. In addition, VLDL production was reducedin these animals. We speculate that nascent VLDL acquires
excess apo E in the space of Disse, and then is sequesteredonto the surface of the hepatocytes followed by endocytosis
through a pathway distinct from the LDLR, presumably LRPor heparan sulfate proteoglycans.
In contrast to our previous report showing that the plasma
TG levels were markedly reduced in mice expressing apo E(13), the plasma TG levels in the ETg1/2:LDLRKO and ETg1/1:
LDLRKO were similar to those in the ETg2/2:LDLRKO
mice. This discrepancy can be explained by assuming that the
TG-lowering effect of the apo E transgene requires the LDLR,
indicating that the apo E transgene preferentially stimulates
the catabolism of TG-rich lipoproteins through the LDLRmediated pathway.
With respect to chylomicron metabolism, the excursion of
the plasma retinyl ester after vitamin A load was delayed ninefold in the LDLRKO mice, compared to wild-type mice (13).
These observations are consistent with our previous findingsthat the LDLR plays a pivotal role in the metabolism of chylomicron (27). In the present study, however, no significant difference was observed in the plasma excursion of retinyl ester
between the LDLRKO mice with and without the apo E transgene (Fig. 5), which is apparently contradictory to the changes
in the VLDL metabolism. This can be explained by the facts
that apo E is not overproduced in the intestine (13), and thatthe intestine lacks such space for enrichment with apo E as thespace of Disse.
Diet-induced hypercholesterolemia was suppressed in the
LDLRKO mice overexpressing rat apo E in both a homozygous or heterozygous state (Table IV). In parallel, diet-induced
fatty streak lesion formation was suppressed in the ETg1/1:
LDLRKO mice (Fig. 6). Because the lesion formation and hypercholesterolemia were suppressed to a similar degree, thecholesterol-lowering effects of the apo E transgene may largely
account for the antiatherogenic effects.
Recently, we and other investigators have reported that the
introduction of genes such as lipoprotein lipase (11), VLDL
receptor (28, 29), APOBEC-1 (30), or cholesterol 7a-hydroxylase (31), which are apparently irrelevant to the LDLR, ameliorated hypercholesterolemia in the LDLRKO mice. Thiskind of strategy may help avoid potential problems such as autoantibody formation against genetically introduced proteins.
Pharmaceutical or genetic manipulation of the hepatic apo E
production should be a promising adjunctive therapy for homozygous familial hypercholesterolemia.

Acknowledgments
We would like to thank Kimiko Saito and Mihoko Kusubae for technical assistance.

References
1. Mahley, R.W. 1988. Apolipoprotein E: cholesterol transport protein withexpanding role in cell biology. Science. 240:622–630.
2. Brown, M.S., and J.L. Goldstein. 1986. A receptor pathway for cholesterol homeostasis. Science. 232:34–37.
3. Yamada, N., D.M. Shames, J.B. Stoudmire, and R.J. Havel. 1987. Metabolism of lipoproteins containing apolipoprotein B-100 in blood plasma of rabbits: heterogeneity related to the presence of apolipoprotein E. Proc. Natl.
Acad. Sci. USA. 83:3479–3483.
4. Ji, Z.-S., S. Fazio, Y.-L. Lee, and R.W. Mahley. 1994. Secretion-capturerole for apolipoprotein E in remnant lipoprotein metabolism involving cell surface heparan sulfate proteoglycan. J. Biol. Chem. 269:2764–2772.
5. Kita, T., M.S. Brown, D.W. Bilheimer, and J.L. Goldstein. 1982. Delayed
clearance of very low density and intermediate density lipoproteins with enhanced conversion to low density lipoprotein in WHHL rabbits. Proc. Natl.
Acad. Sci. USA. 79:5693–5697.
6. Ishibashi, S., J. Herz, N. Maeda, J.L. Goldstein, and M.S. Brown. 1994.
The two-receptor model of lipoprotein clearance: tests of the hypothesis in“knockout” mice lacking the low density lipoprotein receptor, apolipoprotein
E, or both proteins. Proc. Natl. Acad. Sci. USA. 91:4431–4435.
7. Herz, J., U. Hamann, S. Rogne, O. Myklebost, H. Gausepohl, and K.K.
Stanley. 1988. Surface location and high affinity for calcium of a 500 kDa liver
membrane protein closely related to the LDL-receptor suggest a physiologicalrole as lipoprotein receptor. EMBO (Eur. Mol. Biol. Organ.) J. 7:4119–4127.
8. Willnow, T.E., Z. Sheng, S. Ishibashi, and J. Herz. 1994. Inhibition of hepatic chylomicron remnant uptake by gene transfer of a receptor antagonist.
Science. 264:1471–1474.
9. Rohlmann, A., M. Gotthardt, R.E. Hammer, and J. Herz. 1998. Inducible
inactivation of hepatic LRP by Cre-mediated recombination confirms role of
LRP in clearance of chylomicron remnants. J. Clin. Invest. 101:689–695.
10. Yamada, N., H. Shimano, H. Mokuno, S. Ishibashi, T. Gotohda, K.
Kawakami, Y. Watanabe, Y. Akanuma, T. Murase, and F. Takaku. 1989. Increased clearance of plasma cholesterol after injection of apolipoprotein E into
Watanabe heritable hyperlipidemic rabbits. Proc. Natl. Acad. Sci. USA. 86:665–669.
11. Mahley, R.W., K.H. Weisgraber, M.M. Hussain, B. Greenman, M.
Fisher, T. Vogel, and M. Gorecki. 1989. Intravenous infusion of apolipoprotein
E accelerates clearance of plasma lipoproteins in rabbits. J. Clin. Invest. 83:
2125–2130.
12. Yamada, N., I. Inoue, M. Kawamura, K. Harada, Y. Watanabe, H. Shimano, T. Gotoda, M. Shimada, K. Kohzaki, T. Tsukada. M. Shiomi, Y. Watanabe, and Y. Yazaki. 1992. Apolipoprotein E prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits. J. Clin. Invest. 89:
706–711.
13. Shimano, H., N. Yamada, M. Katsuki, M. Shimada,T. Gotoda, K.
Harada, T. Murase, F. Fukazawa, F. Takaku, and Y. Yazaki. 1992. Overexpression of apolipoprotein E in transgenic mice: a marked reduction in plasma lipoproteins except high density lipoprotein, and resistance against diet-induced hypercholesterolemia. Proc. Natl. Acad. Sci. USA. 89:1750–1754.
14. Shimano, H., N. Yamada, M. Katsuki, K. Yamamoto, T. Gotoda, K.
Harada, M. Shimada, and Y. Yazaki. 1992. Plasma lipoprotein metabolism intransgenic mice overexpressing apolipoprotein E. Accelerated clearance of lipoproteins containing apolipoprotein B. J. Clin. Invest. 90:2084–2091.
15. 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 adenovirus-mediated gene delivery. J. Clin.
Invest. 92:883–893.
16. Shimada, M., S. Ishibashi, T. Inaba, H. Yagyu, K. Harada, J. Osuga, K.
Ohashi, Y. Yazaki, and N. Yamada. 1996. Suppression of diet-induced atherosclerosis in low density lipoprtein receptor knockout mice overexpressing lipoprotein lipase. Proc. Natl. Acad. Sci. USA. 93:7242–7246.
17. Kotite, L., N. Bergeron, and R.J. Havel. 1995. Quantitation of apolipoprotein B-100, B-48, and E in human triglyceride-rich lipoproteins. J. Lipid Res.
36:890–900.
18. Noma, A., T. Yokosuka, and K. Kitamura. 1983. Plasma lipids and apolipoproteins as determinators for presence and severity of angioraphycally defined coronary artery disease. Atherosclerosis. 49:1–7.
19. Kataoka, S., M. Paidi, and B.V. Howard. 1994. Simplified isoelectric focusing/immunoblotting determination of apolipoprotein E phenotype. Clin.
Chem. 40:11–13.
20. Havel, R.J., H.A. Eder, and J.H. Bragdon. 1955. The distribution andchemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest. 34:1345–1353.
21. Bergeron, N., L. Kotite, and R.J. Havel. 1996. Simultaneous quantitation of apolipoproteins B-100, B-48, and E separated by SDS-PAGE. Methods
Enzymol. 262:82–94.
22. Goldstein, J.L., S.K. Basu, and M.S. Brown. 1983. Receptor-mediatedendocytosis of LDL in cultured cells. Methods Enzymol. 98:241–260.
23. Matthews, C.M.E. 1957. The theory of tracer experiments with 131I-labeledplasma proteins. Phys. Med. Biol. 2:36–53.
24. Paigen, B., A. Morrow, P.A. Holmes, D. Mitchell, and R.A. Williams.
1987. Quantitative assessment of atherosclerotic lesions in mice. Atherosclero-

Overexpression of Apo E in LDL-receptor Knockout Mice393

sis. 68:231–240.
25. Ishibashi, S., N. Yamada, H. Shimano, N. Mori, K. Mokuno, T. Gptoda,
M. Mawamaki, T. Murase, and F. Takaku. 1990. Apolipoprotein E and lipoprotein lipase secreted from human monocyte-derived macrophages modulate very
low density lipoprotein uptake. J. Biol. Chem. 265:3040–3047.
26. Shimano, H., Y. Namba, J. Ohsuga, M. Kawamura, K. Yamamoto, M.
Shimada, T. Gotoda, K. Harada, Y. Yazaki, and N. Yamada. 1994. Secretionrecapture process of apolipoprotein E in hepatic uptake of chylomicron remnants in transgenic mice. J. Clin. Invest. 93:2215–2223.
27. Ishibashi, S., S. Perrey, Z. Chen, J. Osuga, M. Shimada, K. Ohashi, K.
Harada, Y. Yazaki, and N. Yamada. 1996. Role of the low density lipoprotein
receptor pathway in the metabolism of chylomicron remnants: a quantitative
study in knockout mice lacking the LDL receptor, apolipoprotein E or both. J.
Biol. Chem. 271:22422–22427.
28. Kobayashi, K., K. Oka, T. Forte, B. Ishida, B. Teng, K. Ishimura-Oka,
M. Nakamuta, and L. Chan. 1996. Reversal of hypercholesterolemia in low den-

394

Osuga et al.

sity lipoprotein receptor knockout mice by adenovirus-mediated gene transferof the very low density lipoprotein receptor. J. Biol. Chem. 271:6852–6856.
29. Kozarsky, K.F., K. Jooss, M. Donahee, J.F. Strauss, III, and J.M. Wilson. 1996. Effective treatment of familial hypercholesterolemia in the mousemodel using adenovirus-mediated transfer of the VLDL receptor gene. Nat.
Genet. 13:54–62.
30. Teng, B., B. Ishida, T.M. Forte, S. Blumenthal, L.-Z. Song, A.M. Gotto,Jr., and L. Chan. 1997. Effective lowering of plasma, LDL, and esterified cholesterol in LDL receptor-knockout mice by adenovirous-mediated gene delivery of apo B mRNA editing enzyme (Apobec1). Arterioscler. Thromb. Vasc.
Biol. 17:889–897.
31. Spady, D.K., J.A. Cuthbert, M.N. Willard, and R.S. Meidell. 1998. Overexpression of cholesterol 7a-hydroxylase (CYP7A) in mice lacking the low density lipoprotein (LDL) receptor gene: LDL transport and plasma LDL concentrations are reduced. J. Biol. Chem. 273:126–132.