Overexpressed lipoprotein lipase protects against
atherosclerosis in apolipoprotein E knockout mice
Hiroaki Yagyu,* Shun Ishibashi,1,* Zhong Chen,* Jun-ichi Osuga,* Mitsuyo Okazaki,†
Stéphane Perrey,* Tetsuya Kitamine,* Masako Shimada,* Ken Ohashi,* Kenji Harada,*
Futoshi Shionoiri,* Naoya Yahagi,* Takanari Gotoda,* Yoshio Yazaki,* and Nobuhiro Yamada*
Department of Internal Medicine,* Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113, Japan, and Laboratory of Chemistry,† Department of General Education, Tokyo Medical
and Dental University, Chiba, Japan

Abstract Lipoprotein lipase (LPL) is known to play a crucialrole in lipoprotein metabolism by hydrolyzing triglycerides;
however its role in atherogenesis has yet to be determined.
We have previously shown that low density lipoprotein receptor knockout mice overexpressing LPL are resistant todiet-induced atherosclerosis due to the suppression of remnant lipoproteins. Plasma lipoproteins and atherosclerosis
of apolipoprotein (apo) E knockout mice which overexpress
the human LPL transgene (LPL/APOEKO) were comparedwith those of control apoE knockout mice (APOEKO). On
a normal chow diet, LPL/APOEKO mice showed marked
suppression of the plasma triglyceride levels compared withAPOEKO mice (54 vs. 182 mg/dl), but no significant changes
in plasma cholesterol and apoB levels. Non-high density
lipoproteins (HDL) from LPL/APOEKO mice had lower
triglyceride content, a smaller size, and a more positive
charge compared with those from APOEKO mice. Cholesterol, apoA-I, and apoA-IV were increased in HDL. Although both groups developed hypercholesterolemia to acomparable degree in response to an atherogenic diet, the
LPL/APOEKO mice developed 2-fold smaller fatty streak lesions in the aortic sinus compared to the APOEKO mice.
In conclusion, overproduction of LPL is protective against
atherosclerosis even in the absence of apoE.—Yagyu, H., S.
Ishibashi, Z. Chen, J. Osuga, M. Okazaki, S. Perrey, T. Kitamine, M. Shimada, K. Ohashi, K. Harada, F. Shionoiri, N.
Yahagi, T. Gotoda, Y. Yazaki, and N. Yamada. Overexpressed
lipoprotein lipase protects against atherosclerosis in apolipoprotein E knockout mice. J. Lipid Res. 1999. 40: 1677–
1685.
Supplementary key words lipoprotein lipase • apolipoprotein E •knockout mice • atherosclerosis • triglycerides • cholesterol • foamcells • remnant lipoproteins • apolipoprotein A-I • apolipoprotein A-IV

Lipoprotein lipase (LPL) hydrolyzes triglycerides inplasma lipoproteins such as chylomicrons and very low
density lipoproteins (VLDL) and causes a wide variety ofalterations in lipoprotein metabolism. These effects include: i) the conversion of triglyceride-rich lipoproteins todenser lipoproteins such as chylomicron remnants, intermediate density lipoproteins (IDL), and low density lipo-

proteins (LDL) ii) stimulation of the hepatic removal of
the lipolyzed lipoproteins in concert with apolipoprotein(apo) E, and iii) transfer of surface components of triglyceride-rich lipoproteins to high density lipoproteins (HDL)(See refs: 1–3 for review).
The effects of LPL on atherosclerosis have been controversial. As atheromatous plaques contain substantial
amounts of LPL in situ, Zilversmit (4) proposed that local
LPL is atherogenic. Furthermore, LPL mediates the lipolytic conversion of triglyceride-rich lipoproteins to atherogenic cholesterol-rich lipoproteins such as LDL and chylomicron remnants (1–3). Supporting this, LPL deficiency inhumans, a common genetic cause of chylomicronemia syndrome, results in very low plasma levels of LDL-cholesterol,
and is believed to cause resistance to premature atherosclerosis (1). However, Benlian et al. (5) have recently reported that several LPL-deficient patients have developed
relatively advanced atherosclerosis. It has also been reported that even individuals who are heterozygous for
LPL mutations are predisposed to premature atherosclerosis (6–9). Furthermore, several clinical studies haveshown that fibric acid derivatives induce LPL activity, lower
plasma triglycerides, and suppress atherosclerosis (10 –12). This leads to the question of what the exact role of
LPL in atherosclerosis is as a whole.
In order to address this issue, we have previously established a line of transgenic mice that overexpress LPL
ubiquitously (13), and cross-bred these animals to the
LDL receptor knockout mice to generate mice that lack

Abbreviations: LPL, lipoprotein lipase; apo, apolipoprotein;
APOEKO, apoE knockout mice; LPL/APOEKO, apoE knockout miceoverexpressing human lipoprotein lipase, VLDL, very low density lipoproteins; LDL, low density lipoproteins; HDL, high density lipoproteins; LPDS, lipoprotein-deficient serum; LRP, LDL low density lipoprotein receptor-related protein; PCR, polymerase chain reaction;
PMSF, phenylmethylsulfonyl fluoride; HPLC, high performance liquid
chromatography.
1 To whom correspondence should be addressed.

Journal of Lipid Research Volume 40, 19991677

the LDL receptor and overproduce LPL (LPL/LDLRKO)(14). LPL/LDLRKO mice had reduced plasma levels not
only of triglycerides but also of total cholesterol, and werevery resistant to diet-induced atherosclerosis (14). In thisanimal model, a subfraction of VLDL was selectively reduced and HDL-cholesterol levels were not changed.
Therefore we ascribed the anti-atherogenic effects of theoverexpressed LPL to the reduction of remnant lipoproteins. There were not significant alterations in either
plasma apoB concentrations or plasma clearance of apoBcontaining lipoproteins in LPL/LDLRKO mice. Therefore
it is unlikely that the overproduced LPL stimulates the interaction of apoB-containing lipoproteins with hepatic receptors distinct from the LDL receptor, an apparent contradiction to the reported function of LPL in vitro (15–19).
In order to further characterize the effects of the overexpressed LPL on lipoprotein metabolism and atherosclerosis in the setting of apoE deficiency, we have generated
APOEKO mice that overexpress LPL (LPL/APOEKO) by
cross-breeding to apoE knockout (APOEKO) mice (20,21), and analyzed their lipoprotein profiles and susceptibility to atherosclerosis as compared to APOEKO mice
without the overexpression of LPL.

EXPERIMENTAL PROCEDURES
Mice. The LPL transgenic mice (13) and APOEKO mice (20)were created as described in the indicated references. The LPL
transgenic mice express human LPL cDNA under control of achicken b-actin enhancer/cytomegalovirus (CMV) promoter ina wide variety of tissues such as the heart, skeletal muscle, adipose
tissue and, to a lesser extent, in other tissues including the aorta(13). To obtain LPL/APOEKO mice, the LPL transgenic mice
were bred to the homozygous APOEKO mice. The resultant offspring, which overexpressed LPL and were obligatorily heterozygous for the mutant apoE locus, were further bred to the homozygous APOEKO mice to obtain homozygous APOEKO micewith or without the LPL transgene. Male offspring of brother–sister matings of each line were used for experiments. PCR wasused to assign the genotype. The parental APOEKO mice wereF2 hybrids between 129/Sv and C57BL/6, and parental LPL
transgenic mice were F2 hybrids between C57BL/6 and DBA/2,
which had been back-crossed to C57BL/6 three times. Therefore, 72%, 25%, and 3% of the genetic background of the mice
was derived from C57BL/6, 129/Sv, and DBA/2 strains, respectively. Two diets were used: i) a normal chow diet (MF diet from
Oriental Yeast Co., Tokyo) that contained 5.6% (w/w) fat with
0.09% (w/w) cholesterol was provided, and ii) an atherogenic
diet: MF diet containing 0.15% (w/w) cholesterol and 15% (w/w)
butter. Before killing, mice were anesthetized with pentobarbital.
The current experiments were performed in accord with institutional guidelines for animal experiments at University of Tokyo.

Northern blot analysis
Total RNA was isolated from the cells by Trizol (Gibco-BRL)
according to the manufacturer’s instructions. Ten mg of total
RNA was subjected to electrophoresis in 1% agarose gel containing formamide, and transferred to a nylon filter (Hybond N,
Amersham Pharmacia). cDNA probes were radiolabeled with[a-32P]deoxy-CTP. After prehybridization for 2 h, blots were hybridized in a RapidhybR buffer (Amersham Pharmacia) for 1 h at
658C with the probes.

1678

Journal of Lipid Research Volume 40, 1999

Human LPL mass in plasma
After a 16-h fast, heparin (100 units/kg) was injected intravenously as a bolus. After 3 min, post-heparin plasma was taken.
Human LPL mass was determined as previously described (22).

Plasma lipids and lipoproteins
Blood was collected from the retro-orbital venous plexus into
tubes containing EDTA. VLDL (d , 1.006 g/ml), IDL (d1.006– 1.019 g/ml), LDL (d 1.019– 1.063 g/ml), HDL (d 1.063–
1.21 g/ml), HDL2 (d 1.063– 1.125 g/ml), and HDL3 (d 1.125–
1.21 g/ml) were isolated from the pooled plasma by sequential
ultracentrifugation (23). Total cholesterol and triglyceride concentrations in plasma or lipoprotein fractions were determined
enzymatically by kits (Determiner TC555 and Determiner TG555,
Kyowa Medex, Tokyo), and their concentrations in originalplasma were calculated as mg/dl. Plasma apoB concentrations
were measured by single radial immunodiffusion method using a
kit designed for measurement of human apoB levels (Apo B
plate ‘Daiichi’; Daiichi Pure Chemicals, Tokyo, Japan), according
to the manufacturer’s instruction (24, 25).
Agarose gel electrophoresis of plasma was performed using
Universal Gel/8 electrophoresis system (Ciba Corning Diagnostic Corp) according to the manufacturer’s instruction.
Immunoblot of apoA-I was performed using an ECL kit and ananti-rat apoA-I antibody as described previously (25).
For apolipoprotein analyses, each lipoprotein fraction was dialyzed against a solution containing 0.15 m NaCl, 1 mm EDTA, and
1 mm PMSF at pH 7.4 and delipidated with ethanol/ether (26).
Apolipoproteins in each lipoprotein fraction equivalent to 10 mlof plasma were loaded onto each well of 5–15% gradient SDS/
polyacrylamide gel and proteins were visualized by staining with
Coomassie Brilliant Blue.

High performance liquid chromatography(HPLC) analyses
HPLC analyses of plasma lipoproteins were performed as described (27, 28). In brief, 100-ml samples of the diluted plasma
were applied onto four columns of TSK gel Lipopropak XL(TOSOH, Tokyo) connected in tandem. This system provides
high resolution especially in the range of large lipoproteins. The
elution was performed at a flow rate of 0.6 ml/min using an TSK
eluent LP-2 (TOSOH, Tokyo) and 0.3 ml/min for an enzyme solution (Determiner L TC, Kyowa Medex Co., Tokyo). The detection of cholesterol in a post-column effluent was carried out by
A550 following the enzymatic reaction in an on-line system.

Pathology
Atheromatous plaques in the aortas were visualized by staining
with Sudan IV as described (29). The luminal side of the stained
aortas was photographed. Image capture and analysis was performed using Adobe Photoshop™ 3 image analysis software, as
essentially described. The extent of atherosclerosis was determined using selection threshold ranges in the three basic colors
and was expressed as the percent of surface area of the entire
aorta covered by lesions; this was designated as the en face surface lesion area (30). The cross-sectional lesion area was evaluated according to a modified method of Paigen et al. (31). In
brief, the hearts were perfused with saline containing 4% (w/v)
formalin and fixed for more than 48 h in the same solution. The
basal half of the hearts was embedded in Tissue-Tek OCT compound (Miles, Inc.), and the serial sections were captured using
Cryostat (6 mm thick) as described (13). Four sections, each separated by 60 mm, were used to evaluate the lesions: two at the endof the aortic sinus and two at the junctional site of sinus and ascending. The sections were stained with Oil Red O and counterstained with hematoxylin.

Cells
One ml of thioglycolate broth was injected into the peritoneal
cavities of mice aged 3 months. After 4 days, the peritoneal cavities were lavaged with 10 ml/mouse of ice-cold saline. The cells
were washed three times with PBS and resuspended in DMEM
and 2 3 106 cells were plated in one well of 12-well plates (Corning). After incubation at 378C for 2 h, the non-adherent cells
were removed by washing three times with pre-warmed PBS. The
adherent cells were incubated with DMEM containing 5 mg/ml
of LPDS for 24 h at 378C in the atmosphere of 5% CO2 and 95%
air unless otherwise stated.

Cholesteryl ester formation assay
VLDL and d , 1.063 g/ml lipoproteins were isolated from theplasma by ultracentrifugation at densities of 1.006 g/ml and 1.063
g/ml, respectively. b-VLDL, apoE- and cholesterol-enriched lipoproteins were isolated from LDLRKO mice fed an atherogenic
diet. The lipoproteins were re-floated at the same density and dialyzed against a solution containing 0.15 m NaCl, 1 mm EDTA,
and 1 mm PMSF at pH 7.4. After dialysis, the lipoproteins weresterilized by filtration through Milex 0.45 mm and their protein
concentrations were measured by method of Lowry et al. (32).
Cholesteryl ester formation from [1-14C] oleate was essentiallydetermined as described (33).
Briefly, after incubation with a medium containing 5 mg/ml of
LPDS for 24 h, the cells were incubated with a medium containing 12.5 mg/ml of lipoproteins, the [1-14C]oleate-albumin complex, and 5 mg/ml of LPDS at 378C for 24 h. Plates were washedtwice with PBS containing 2 mg/ml BSA and once with PBS. Cholesteryl ester was extracted with hexane–isopropanol 3:2 towhich [3H]cholesteryl-oleate and unlabeled oleic acid wereadded as the internal standard and carrier, respectively. The
organic phase was evaporated to dryness under flowing N2. Cholesterol [1-14C]-oleate was separated on silica-coated TLC plates(#5583, Merck, Germany) using a solvent system composed ofheptane–ethyl ether–acetic acid 90:30:1 (v/v/v), their position
was identified using a BAS2000 phosphoimager (Fuji Film, Tokyo).
The spots corresponding to cholesteryl esters were scraped andtheir radioactivities were quantified by liquid scintillation. Cellular proteins were dissolved in 0.1 N NaOH and determined by
the Lowry method (32).

Statistics
Student’s t -test was used to compare mean values between two
sets of mice.

RESULTS
Expression of the LPL transgene
In order to compare the expression levels of the human
LPL transgene in various organs, Northern blot analysis
was performed (Fig. 1). The human LPL transgene wasexpressed in skeletal muscle, heart, adipose tissue and, toa lesser extent, in the aorta as previously reported (13).
Plasma lipoprotein profile of mice fed normal chow
Plasma lipid concentrations and LPL mass were compared between APOEKO and LPL/APOEKO mice fed anormal chow diet (Table 1). LPL/APOEKO mice hadhigh concentrations of human LPL, while APOEKO mice
did not. As expected, LPL/APOEKO mice had 69% lower
plasma triglyceride levels than APOEKO mice. Although a

Fig. 1. Expression of human LPL transgene in various tissues of
LPL/APOEKO mice. Total RNA was isolated from the indicatedorgans and subjected to Northern blot analysis.

relatively large variation exists in the plasma total cholesterol levels in both APOEKO and LPL/APOEKO mice, nosignificant difference was observed between the two groups.
LPL/APOEKO mice had higher plasma HDL cholesterollevels than APOEKO mice, though they were not statistically significant.
In order to determine the size distribution of lipoproteins which accumulated in LPL/APOEKO mice, we performed HPLC analyses on the total plasma. The non-HDL
cholesterol peak of LPL/APOEKO mice was smaller thanthat of APOEKO mice (retention time: 46.9 vs. 45.6 min)TABLE 1. Human LPL mass and lipoproteins in the plasmaof LPL/APOEKO and APOEKO mice

Normal chown
Post-heparin LPL (ng/ml)
Pre-heparin LPL (ng/ml)
Total cholesterol (mg/dl)
Triglycerides (mg/dl)
HDL-cholesterol (mg/dl)
ApoB (mg/dl)
Atherogenic dietnTotal cholesterol (mg/dl)
Triglycerides (mg/dl)
HDL-cholesterol (mg/dl)
ApoB (mg/dl)

APOEKO

LPL/APOEKO

17
,20
,20
636 6 253
182 6 90
22 6 12131 6 49101288 6 328
174 6 40654 6 170
54 6 16a
29 6 26
142 6 28171164 6 409
206 6 125
15 6 8
198 6 63101129 6 330
51 6 9a
13 6 7
224 6 63

Blood was collected from the mice before and 8 weeks after consumption of an atherogenic diet. Plasma cholesterol and triglyceride
levels were determined enzymatically. HDL cholesterol levels were determined from the HPLC profiles and total cholesterol levels. The
mice were 8–12 weeks of age at the start of the feeding. Values aremean 6 SD.
a P , 0.01, APOEKO vs. LPL/APOEKO.

Yagyu et al. LPL overexpression in apoE knockout mice

1679

Fig. 2. HPLC lipoprotein profiles of APOEKO (solid line) and LPL/APOEKO (dashed line) mice fed anormal chow (A, C) and atherogenic diet (B, D). Cholesterol (A, B) and triglycerides (C, D) contents were
measured enzymatically. The original volume of the plasma applied to the columns was as follows: A, 5 ml; B,
1 ml; C, 20 ml; D, 4 ml.

(Fig. 2A), while the size distribution of HDL cholesterol
was not different between the two groups. For non-HDL
triglycerides, there were two peaks in both types of mice:
one peak in the VLDL range and another peak in the
IDL/LDL range (Fig. 2C). A significant size reduction wasobserved in both peaks in LPL/APOEKO mice comparedto APOEKO mice. In contrast to the cholesterol profile,the differences in the retention time for each peak wereless remarkable between the two groups (47.7 vs. 48.2 mm
for the peaks with smaller molecular size and 42.0 vs. 42.7
min for the peaks with larger molecular size).
Agarose gel electrophoresis of the plasma fromAPOEKO mice showed a broad band whose migratory position was between b and pre-b (Fig. 3, lane 2), while
LPL/APOEKO mouse plasma had a sharper band with bmobility (Fig. 3, lane 3).
In support of the results of HPLC analyses, lipoprotein
analyses by sequential ultracentrifugation showed that
more than 90% of total cholesterol was distributed in nonHDL fractions in both types of mice (Table 2). VLDL cholesterol was decreased and LDL cholesterol was increasedin LPL/APOEKO mice.
Immunoblot analysis revealed that there was an approximately 2-fold increase in the plasma apoA-I levels in LPL/
1680

Journal of Lipid Research Volume 40, 1999

APOEKO mice compared with those in APOEKO mice(data not shown). SDS/PAGE analyses were performed toestimate the distribution of apolipoproteins in lipoprotein
fractions (Fig. 4A and B). ApoB-48 was present in VLDL

Fig. 3. Agarose gel electrophoresis of lipoproteins from wild-type(lane 1), APOEKO (lane 2) and LPL/APOEKO (lane 3) mice fed anormal chow, and APOEKO (lane 4) and LPL/APOEKO (lane 5)
mice fed an atherogenic diet for 8 weeks. Two ml of plasma was applied to Universal Gel/8. Migratory positions for a, pre-b, and b
mobility and origin are indicated.

TABLE 2. Distribution of cholesterol between lipoprotein
fractions from APOEKO and LPL/APOEKO mice

Normal chow
APOEKO
LPL/APOEKO
Atherogenic dietAPOEKO
LPL/APOEKO

VLDL

IDL

LDL

HDL

523
479

134
193

122
155

10
32

614
520

298
335

226
249

25
25

Lipoproteins were isolated from pooled plasma by sequential ultracentrifugation. Cholesterol content in each lipoprotein fraction wasdetermined enzymatically and expressed as mg/dl of the originalplasma. Overall recovery of cholesterol during ultracentrifugation was
calculated to be 82–89%.

through LDL, but not in HDL. ApoA-IV and apoA-I were
present in all lipoprotein classes. ApoB-48 appears to be
decreased in VLDL and increased in LDL fractions in
LPL/APOEKO mice compared to the APOEKO mice(compare Fig. 4A with Fig. 4B). LPL/APOEKO mice exhibited a 3-fold increase in apoA-I content in HDL com-pared to APOEKO mice. Similarly, LPL/APOEKO mice
had increased apoA-IV contents in HDL compared to
APOEKO mice. The same result was obtained in a separate experiment using different animals (data not shown).
Effects of atherogenic diet on the lipoprotein profile
Both APOEKO and LPL/APOEKO mice were fed an
atherogenic diet for 8 weeks, and the changes in lipoprotein profiles and the extent of atherosclerosis were compared between the two types of mice. After 8 weeks offeeding, plasma cholesterol levels increased 1.74-fold in
APOEKO and 1.8-fold in LPL/APOEKO mice, with no significant difference between the two groups (Table 1). On
the other hand, LPL/APOEKO mice had lower plasma
triglyceride levels than APOEKO mice (51 vs. 206 mg/dl,
P , 0.01). Upon HPLC lipoprotein analyses, the nonHDL cholesterol peaks from the mice fed an atherogenic
diet were larger than those from mice fed a normal chowdiet (Fig. 2B). However, no differences were observed ineither the retention time or the amount of non-HDL cholesterol between the two groups. Notably, non-HDL tri-

Fig. 4. Apolipoproteins in lipoprotein fractions isolated from APOEKO (A) and LPL/APOEKO (B) micefed either a normal chow (A and B) or an atherogenic diet for 2 (C) and 8 weeks (D). The indicated lipoproteins were isolated from pooled plasma by sequential ultracentrifugation. In panels C and D, APOEKO andLPL/APOEKO are indicated by LPL (2) and LPL (1), respectively. Each fraction was dialyzed, delipidated,and subjected to SDS/5–15% polyacrylamide gel electrophoresis. Proteins were visualized by staining with
Coomassie Brilliant Blue. The molecular weights of apoB-100, apoB-48, apoA-IV, apoA-I, and apoA-II1C’s areindicated.

Yagyu et al. LPL overexpression in apoE knockout mice

1681

glycerides of both groups consisted of two peaks with retention times that were identical to those of the mice fed anormal chow diet (Fig. 2D). A significant reduction wasobserved in both peaks in LPL/APOEKO mice comparedto APOEKO mice.
Agarose gel electrophoresis of the plasma showed patterns which were essentially similar to those observed in
the mice fed a normal chow (Fig. 3). APOEKO mice had abroad band which migrated between b and pre-b positions(lane 4), and LPL/APOEKO mice had a sharper band withb mobility (lane 5).
Two weeks after feeding mice an atherogenic diet,apoA-I and apoA-IV were preferentially distributed in the
HDL2 fraction (Fig. 4C). LPL/APOEKO mice had higher
concentrations of apoA-I than APOEKO mice particularlyin the HDL2 fraction. At this point, HDL2 cholesterol
content was increased by 2-fold in LPL/APOEKO mice ascompared to APOEKO mice (25 vs. 12 mg/dl). After 8
weeks, the preferential distribution of apoA-I in the HDL2
fraction became less prominent (Fig. 4D). There was nodifference in the HDL2 cholesterol content at this point
as shown in Tables 1 and 2. LPL/APOEKO mice had ahigher concentration of apoA-I than APOEKO mice in
VLDL and IDL, but not in HDL2. A similar increase of
apoA-I in non-HDL fractions from LPL/APOEKO mice
was observed in a separate experiment using different animals (data not shown).
Diet-induced atherosclerosis
Atherosclerosis was evaluated by two different methods:
the en face surface lesion area of the aorta and crosssectional analysis of aortic roots (Fig. 5). The en face surface lesion areas of LPL/APOEKO mice were significantly
smaller than those of APOEKO mice by 30% (14.6 6 5.1
vs. 20.6 6 6.9%, P , 0.05) (Fig. 5A). Cross-sectional lesion
areas of LPL/APOEKO mice were significantly smaller
than those of APOEKO mice by 51% (290,000 6 150,000
vs. 590,000 6 160,000 mm2, P , 0.001) (Fig. 5B). It isnoteworthy that the two different methods showed comparable differences between the two groups.
Cholesteryl ester formation in mouse
peritoneal macrophages
We isolated both VLDL and d , 1.063 g/ml lipoproteins from APOEKO and LPL/APOEKO mice, and compared their abilities to stimulate cholesteryl ester formation in peritoneal macrophages (Fig. 6). We used two
types of mice as sources of macrophages: C57BL/6 wildtype and APOEKO mice. Both VLDL and d , 1.063 g/ml
lipoproteins stimulated cholesteryl ester formation in
macrophages prepared from wild-type C57BL/6 mice.
The cholesteryl ester formation stimulated by the lipoproteins isolated from LPL/APOEKO mice was significantly
smaller, by 30%, than that from APOEKO mice (P , 0.05).
In contrast, neither VLDL nor d , 1.063 g/ml lipoproteins stimulated cholesteryl ester formation in macrophages prepared from APOEKO mice (,0.01 nmol/h per
mg cell protein), irrespective of the lipoproteins used.
In contrast, b-VLDL obtained from LDLRKO mice fed
1682

Journal of Lipid Research Volume 40, 1999

Fig. 5. En face (A) and cross-sectional (B) lesion area in the aortic sinus from APOEKO (open circle) and LPL/APOE KO mice(closed circle) fed an atherogenic diet. Male mice 8 – 12 weeks ofage were fed an atherogenic diet for 8 weeks and the fatty streak lesion areas were determined as previously described (14, 29, 30).
Mean 6 SD is indicated by bold lines and error bars; * P , 0.001,** P , 0.05.

an atherogenic diet markedly stimulated cholesteryl ester
formation in wild-type macrophages (1.4 6 0.1 nmol/h
per mg cell protein).

DISCUSSION
In the present study, we have demonstrated that overexpression of LPL protects against diet-induced atherosclerosis in the absence of apoE. We postulate that twochanges in the lipoprotein profiles underlie the suppression of atherosclerosis in LPL/APOEKO mice: changes in
HDL and non-HDL.
HDL-cholesterol levels were slightly elevated in LPL/APOEKO mice fed a normal chow diet (Table 1). Plasma
apoA-I levels were also increased in LPL/APOEKO mice.
With regard to distribution of apolipoproteins, apoA-I
and apoA-IV were present in both HDL and non-HDL
lipoproteins probably due to the need for VLDL surface
components (34, 35). They appeared to be increased in
HDL of LPL/APOEKO mice (compare Fig. 4B with Fig.
4A). Two weeks after feeding an atherogenic diet, apoA-I

Fig. 6. Cholesteryl ester formation stimulated by either VLDL ord , 1.063 g/ml lipoproteins. Plasma was obtained from either
APOEKO (open bar) or LPL/APOE KO (closed bar) mice (n 5 5)which had been fed an atherogenic diet for 8 weeks. VLDL or d ,1.063 g/ml lipoproteins were isolated from pooled plasma by ultracentrifugation. Thioglycollate-elicited peritoneal macrophages
were prepared from wild-type C57BL/6 mice. After treatment withLPDS for 24 h, the cells were incubated with 12.5 mg/ml of the indicated lipoproteins for 24 h, and cholesteryl ester formation wasdetermined as described in Experimedntal Procedures. Errors bars
denote SD; * P , 0.05 (n 5 3).

and apoA-IV were preferentially distributed in HDL2 inboth LPL/APOEKO and APOEKO mice (Fig. 4C). This
may result from the increased transfer of these apolipoproteins from chylomicrons whose production is stimulated upon high fat feeding. The stimulated lipolysis of chylomicrons may account for the increased amounts ofcholesterol as well as apoA-I in HDL2 in LPL/APOEKO
mice. After 8 weeks, their preferential distribution in HDL
disappeared probably because of reaching a new steadystate (Fig. 4D). In the setting of apoE deficiency, even aslight change in apoA-I levels is detectable because baseline HDL cholesterol concentrations are significantly
lower than those observed in the setting of LDL receptor
deficiency where a subtle change may be easily masked. It
is plausible that the increases in the levels of either apoAI, apoA-IV or both in HDL mediate the suppression of atherosclerosis in LPL/APOEKO mice. In support of this,anti-atherogenic effects of the overexpressed apoA-I has
been shown in the presence (36) or absence of apoE (37,38) and overexpression of apoA-IV has been demonstrated to protect against diet-induced atherosclerosis (39,40). With regard to the mechanisms underlying the increase in apoA-I levels, it is reasonable to consider that
LPL-mediated lipolysis of triglyceride-rich lipoproteinscauses transfer of surface apolipoprotein components tonascent HDL, thereby increasing apoA-I on HDL. Indeed,
there is an inverse correlation between plasma triglycerideand HDL-cholesterol levels (41), and production of HDL

is partly determined by LPL activity (42). However, HDL
cholesterol levels vary greatly from no change (43, 44) toincreases (13) or decreases (45, 46) among the different LPL transgenic mice containing different promoters.
Probably, the tissues where the LPL transgenes are expressed are important determinants of HDL cholesterollevels.
In LPL/APOEKO mice, non-HDL lipoproteins weretriglyceride-poor, small, and positively charged as compared to those in APOEKO mice (Figs. 2 and 3). In orderto determine whether these physical changes were responsible for the suppression of atherosclerosis in LPL/APOEKO mice, we compared the ability of non-HDL lipoproteins to elicit cholesteryl ester formation in macrophages. As expected, non-HDL lipoproteins did not elicit
any detectable cholesteryl ester formation in macrophages obtained from APOEKO mice. This is reasonablebecause the non-HDL lipoproteins completely lacked
apoE and contained only a trace of apoB-100. Both apoB100 and apoE are ligands for the LDL receptor (47), andapoE is one of the important ligands for LRP (48). Despite the failure of atherogenic lipoproteins to stimulatecholesteryl ester accumulation in apoE-deficient macrophages, APOEKO mice develop rampant atherosclerotic lesions that are composed of cholesteryl ester-filled
macrophages. These observations indicate that modificationof non-HDL lipoproteins is necessary for the development of
atherosclerosis in this model. Interestingly, non-HDL lipoproteins from either APOEKO or LPL/APOEKO mice induced a significant degree of cholesteryl ester formationin macrophages obtained from wild-type mice (Fig. 6). It
is possible that apoE secreted by wild-type macrophages isattached to the lipoprotein surface and is recognized by
the LDL receptor expressed in these cells, as proposed previously (49). Cholesteryl ester formation elicited by nonHDL obtained from LPL/APOEKO mice was significantly
reduced from that of APOEKO mice by 30% (Fig. 6). We
would speculate that the non-HDL of LPL/APOEKO mice
have limited capacity to accommodate apoE secreted frommacrophages. It is noteworthy that an overabundance of
LPL did not stimulate cholesteryl ester formation, in lightof the belief that LPL bridges lipoproteins and cells in
vitro and thus promotes their clearance (50, 51).
Despite these alterations in the physical properties ofthe lipoproteins mentioned above, no difference was observed in the plasma concentrations of apoB between
LPL/APOEKO and APOEKO mice. These results indicatethat the excessive lipolysis did not stimulate the hepatic
removal of the lipolyzed lipoproteins containing apoB-48.
As our LPL/APOEKO mice expressed the LPL transgene in the aorta, we cannot completely rule out the possibility that the LPL expressed in the vascular wall contributed to the anti-atherogenic effects of the transgene.
However, there was an approximately 10-fold difference inthe degree of suppression of atherosclerotic lesions when
LPL was overexpressed on a background of LDLR deficiency versus apoE deficiency. Therefore, it is plausiblethat lipoprotein profiles play a more important role in determining athereosclerosis than local expression of LPL.
Yagyu et al. LPL overexpression in apoE knockout mice1683

In summary, the LPL overexpression is protective
against diet-induced atherosclerosis even in the setting of
apoE deficiency. The reduced triglyceride content of the
non-HDL, the increase in the plasma apoA-I levels, orboth, may account for the anti-atherogenic effects of LPL
overexpression.
We thank K. Sasamoto, K. Saito, E. Herai, and M. Kusubae forexpert technical assistance, and A. M. Hasty for critical readingof the manuscript. We are indebted to Dr. N. Maeda (Universityof North Carolina) for APOEKO mice. This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, the Promotion of Fundamental Studies in Health Science of The Organization for
Pharmaceutical Safety and Research (OPSR) and Health Sciences Research Grants (Research on Human Genome andGene Therapy) from the Ministry of Health and Welfare.
Manuscript received 1 December 1998 and in revised form 4 May 1999.

REFERENCES
1. Brunzell, J. D. 1995. Familial lipoprotein lipase deficiency andother causes of the chylomicronemia syndrome. In The Metabolicand Molecular Basis of Inherited Disease. R. S. Scriver, A. L. Beaudet, W. S. Sly, D. Valli, editors. McGraw-Hill Book Co., New York.
1913–1932.
2. Eckel, R. H. 1989. Lipoprotein lipase. A multifunctional enzyme
relevant to common metabolic diseases. N. Engl. J. Med. 320: 1060–
1068.
3. Goldberg, I. J. 1996. Lipoprotein lipase and lipolysis: central roles inlipoprotein metabolism and atherogenesis. J. Lipid Res. 37: 693–707.
4. Zilversmit, D. B. 1973. A proposal linking atherosclerosis to the interaction of endothelial lipoprotein lipase with triglyceride-richlipoproteins. Circ. Res. 33: 633–638.
5. Benlian, P., J. Luc De Gennes, L. Foubert, H. Zhang, S. E. Gagne,and M. Hayden. 1996. Premature atherosclerosis in patients withfamilial chylomicronemia caused by mutations in the lipoprotein
lipase gene. N. Engl. J. Med. 335: 848–854.
6. Reymer, P. W. A., E. Gagne, B. E. Groenemeyer, H. Zhang, I. Forsyth, H. Jansen, J. C. Seidell, D. Kromhout, K. E. Lie, J. Kastelein,and M. R. Hayden. 1995. A lipoprotein lipase mutation(Asn291Ser) is associated with reduced HDL cholesterol levels inpremature atherosclerosis. Nat. Genet. 10: 28–34.
7. Jukema, J. W., A. J. van Boven, B. Groenemeijer, A. H. Zwinderman, J. H. Reiber, A. V. Bruschke, J. A. Henneman, G. P. Molhoek,
T. Bruin, H. Jansen, E. Gagne, M. R. Hayden, and J. J. Kastelein.
1996. The Asp9 Asn mutation in the lipoprotein lipase gene is associated with increased progression of coronary atherosclerosis.
REGRESS Study Group, Interuniversity Cardiology Institute, Utrecht,
The Netherlands. Regression Growth Evaluation Statin Study. Circulation. 94: 1913–1918.
8. Wittrup, H. H., A. Tybjörg-Hansen, S. Abildgaard, R. Steffensen, P.
Schnohr, and B. G. Nordestgaard. 1997. A common substitution(Asn291Ser) in lipoprotein lipase is associated with increased riskof ischemic heart disease. J. Clin. Invest. 99: 1606–1613.
9. Nordestgaard, B. G., S. Abildgaard, H. H. Wittrup, R. Steffensen,
G. Jensen, and A. Tybjaerg-Hansen. 1997. Heterozygous lipoprotein lipase deficiency: frequency in the general population, effecton plasma lipid levels, and risk of ischemic heart disease. Circulation. 96: 1737–1744.
10. Frick, M. H., O. Elo, K. Haapa, O. P. Heinonen, P. Heinsalmi, P.
Helo, J. K. Huttunen, P. Kaitaniemi, P. Koskinen, V. Manninen, H.
Mäenpää, M. Mälkönen, M. Mänttäri, S. Norola, A. Pasternack, J.
Pikkarainen, M. Romo, T. Sjöblom, and E. A. Nikkilä. 1987. Helsinki heart study: primary-prevention trial with gemfibrozil in
middle-aged men with dyslipidemia. Safety of treatment, changes
in risk factors, and incidence of coronary heart disease. N. Engl. J.
Med. 317: 1237–1245.
11. Manninen, V., L. Tenkanen, P. Koskinen, J. K. Huttunen, M. Mänt-

1684

Journal of Lipid Research Volume 40, 1999

täri, O. P. Heinonen, and M. H. Frick. 1992. Joint effects of serum
triglyceride and LDL cholesterol and HDL cholesterol concentrations on coronary heart disease risk in the Helsinki heart study.
Implication for treatment. Circulation. 85: 37–45.
12. Ericsson, C-G., A. Hamsten, J. Nilsson, L. Grip, B. Svane, and U. deFaire. 1996. Angiographic assessment of effects of bezafibrate on
progression of coronary artery disease in young male postinfarction patients. Lancet. 347: 849–853.
13. Shimada, M., H. Shimano, T. Gotoda, K. Yamamoto, M. Kawamura, T. Inaba, Y. Yazaki, and N. Yamada. 1993. Overexpression ofhuman lipoprotein lipase in transgenic mice. Resistance to dietinduced hypertriglyceridemia and hypercholesterolemia. J. Biol.
Chem. 268: 17924–17929.
14. Shimada, M., S. Ishibashi, T. Inaba, H. Yagyu, K. Harada, J-i. Osuga,
K. Ohashi, Y. Yazaki, and N. Yamada. 1996. Suppression of dietinduced atherosclerosis in low density lipoprotein receptor knockout mice overexpressing lipoprotein lipase. Proc. Natl. Acad. Sci.
USA. 93: 7242–7246.
15. Beisiegel, U., W. Weber, and G. Bengtsson-Olivecrona. 1991. Lipoprotein lipase enhances the binding of chylomicrons to low density lipoprotein receptor-related protein. Proc. Natl. Acad. Sci. USA.
88: 8342–8346.
16. Willnow, T., J. L. Goldstein, K. Orth, M. S. Brown, and J. Herz.
1992. Low density lipoprotein receptor-related protein (LRP) and
gp330 bind similar ligands, including plasminogen activator-inhibitor complexes and lactoferrin, an inhibitor of chylomicron remnant clearance. J. Biol. Chem. 267: 26172–26180.
17. Chappell, D. A., G. L. Fry, M. A. Waknitz, P-H. Iverius, S. E. Williams, and D.K. Strickland. 1992. The low density lipoprotein
receptor-related protein/a2-macroglobulin receptor binds andmediates catabolism of bovine milk lipoprotein lipase. J. Biol.
Chem. 267: 25764–25767.
18. Nykjaer, A., G. Bengstsson-Olivecrona, A. Lookene, S. K. Moestrup,
C. M. Petersen, W. Weber, U. Beisiegel, and J. Gliemann. 1993.
The a2-macroglobulin receptor/low density lipoprotein receptorrelated protein binds lipoprotein lipase and b-migrating very low
density lipoprotein associated with the lipase. J. Biol. Chem. 268:
15048–15055.
19. Chappell, D. A., G. L. Fry, M. A. Waknitz, L. E. Muhonen, M. W.
Pladet, P-H. Iverius, and D. K. Strickland. 1993. Lipoprotein lipaseinduces catabolism of normal triglyceride-rich lipoproteins via thelow density lipoprotein receptor-related protein/a2-macroglobulin receptor in vitro. A process facilitated by cell-surface proteoglycans. J. Biol. Chem. 268: 14168–14175.
20. Zhang, S. H., R. L. Reddick, J. A. Piedrahita, and N. Maeda. 1992.
Spontaneous hypercholesterolemia and arterial lesions in micelacking apolipoprotein E. Science. 258: 468–471.
21. Plump, A. S., J. D. Smith, T. Hayek, K. Aalto-Setala, A. Walsh, J. G.
Verstuyft, E. M. Rubin, and J. L. Breslow. 1992. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient micecreated by homologous recombination in ES cells. Cell. 71: 343–
353.
22. Kawamura, M., H. Shimano, T. Gotoda, K. Harada, M. Shimada, J.
Ohsuga, T. Inaba, Y. Watanabe, K. Yamamoto, K. Kozaki, Y. Yazaki,
and N. Yamada. 1994. Overexpression of human lipoprotein lipaseenhances uptake of lipoproteins containing apolipoprotein B-100
in transfected cells. Arterioscler. Thromb. 14: 235–242.
23. Havel, R. J., H. A. Eder, and J. H. Bragdon. 1955. The distribution
and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest. 34: 1345–1353.
24. Noma, A., T. Yokosuka, and K. Kitamura. 1983. Plasma lipids andapolipoproteins as determinatiors for presence and severity ofangiographycally defined coronary artery disease. Atherosclerosis. 49:
1–7.
25. Osuga, J-J., M. Yonemoto, N. Yamada, H. Shimano, H. Yagyu, K.
Ohashi, K. Harada, T. Kamei, Y. Yazaki, and S. Ishibashi. 1998.
Cholesterol lowering in low density lipoprotein receptor knockout
mice overexpressing apolipoprotein E. J. Clin. Invest. 102: 386–
394.
26. Bergeron, N., L. Kotite, and R. J. Havel. 1996. Simultaneous quantification of apolipoprotein B-100, B-48, and E separated by SDSPAGE. Methods Enzymol. 262: 82–94.
27. Okazaki, M., K. Sasamoto, T. Muramatsu, and S. Hosaki. 1997.
Analysis of plasma lipoproteins by gel permeation chromatography. In Handbook of Lipoprotein Testing. N. Rifai, G. R. Warnick,and M. H. Dominiczk, editors. AACC Press, Washington, DC. 531–
548.

28. Okazaki, M., N. Komoriya, H. Tomoike, N. Inoue, S. Usui, S. Itoh,and S. Hosaki. 1998. Quantitative detection method of triglycerides in serum lipoproteins and serum-free glycerol high-performance liquid chromatography. J. Chromatogr. 709: 179–187.
29. Ishibashi, S., J. L. Goldstein, M. S. Brown, J. Herz, and D. K. Burns.
1994. Massive xanthomatosis and atherosclerosis in cholesterol-fed
low density lipoprotein receptor-negative mice. J. Clin. Invest. 93:
1885–1893.
30. Tangirala, R. K., E. M. Rubin, and W. Palinski. 1995. Quantitationof atherosclerosis in murine models: correlation between lesions
in the aortic origin and in the entire aorta, and differences in theextent of the lesions between sexes in LDL receptor-deficient andapoE-deficient mice. J. Lipid Res. 36: 2320–2328.
31. Paigen, B., A. Morrow, P. A. Holmes, D. Mitchell, and R. A. Williams. 1987. Quantitative assessment of atherosclerotic lesions in
mice. Atherosclerosis. 68: 231–240.
32. Lowry, O. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall.
1951. Protein measurement with Folin phenol reagent. J. Biol.
Chem. 193: 265–275.
33. Goldstein, J. L., S. K. Basu, and M. S. Brown. 1983. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol. 98: 241–260.
34. Zhang, S. H., R. L. Reddick, B. Burkey, and N. Maeda. 1994. Dietinduced atherosclerosis in mice heterozygous and homozygous forapolipoprotein E gene disruption. J. Clin. Invest. 94: 937–945.
35. Spangenberg, J., and L. K. Curtiss. 1997. Influence of macrophage-derived apoprotein E on plasma lipoprotein distribution of
apolipoprotein A-I in apoprotein E-deficient mice. Biochim. Biophys. Acta. 1349: 109–121.
36. Rubin, E. M., R. M. Krauss, E. A. Spangler, J. G. Verstuyft, and S. M.
Clift. 1991. Inhibition of early atherogenesis in transgenic mice byhuman apolipoprotein AI. Nature. 353: 265–267.
37. Paszty, C., N. Maeda, J. Verstuyft, and E. M. Rubin. 1994. Apolipoprotein AI transgene corrects apolipoprotein E deficiencyinduced atherosclerosis in mice. J. Clin. Invest. 94: 899–903.
38. Plump, A. S., C. J. Scott, and J. L. Breslow. 1994. Human apolipoprotein A-I gene expression increases high density lipoprotein andsuppresses atherosclerosis in the apolipoprotein E-deficientmouse. Proc. Natl. Acad. Sci. USA. 91: 9607–9611.
39. Duverger, N., G. Tremp, J-M. Caillaud, F. Emmanuel, G. Castro, J-C.
Fruchart, A. Steinmetz, and P. Denefle. 1996. Protection against
atherosclerosis in mice mediated by human apolipoprotein A-IV.
Science. 273: 966–968.
40. Cohen, R. D., L. W. Castellani, H. Qiao, B. J. Van Lenrten, A. J. Lusis,

and K. Reue. 1997. Reduced aortic lesions and elevated high density lipoprotein levels in transgenic mice overexpressing mouseapolipoprotein A-IV. J. Clin. Invest. 99: 1906–1916.
41. Austin, M. A. 1991. Plasma triglyceride and coronary heart disease.
Arterioscler. Thromb. 11: 2–14.
42. Eisenberg, S. 1984. High density lipoprotein metabolism. J. Lipid
Res. 25: 1017–1058.
43. Liu, M-S., F. R. Jirik, R. C. LeBoeuf, H. Henderson, L. W. Castellani, A. J. Lusis, Y. Ma, I. J. Forsythe, H. Zhang, E. Kirk, J. D. Brunzell, and M. R. Hayden. 1994. Alteration of lipid profiles in plasmaof transgenic mice expressing human lipoprotein lipase. J. Biol.
Chem. 269: 11417–11424.
44. Zsigmond, E., E. Scheffler, T. M. Forte, R. Potenz, W. Wu, and L.
Chan. 1994. Transgenic mice expressing human lipoprotein lipase
driven by the mouse metallothionein promoter. A phenotype associated with increased perinatal mortality and reduced plasma very low
density lipoprotein of normal size. J. Biol. Chem. 269: 18757–18766.
45. Levak-Frank, S., H. Radner, A. Walsh, R. Stollberger, G. Knipping,
G. Hoefler, W. Sattler, P. H. Weinstock, J. L. Breslow, and R. Zechner.
1995. Muscle-specific overexpression of lipoprotein lipase causes a
severe myopathy characterized by proliferation of mitochondria
and peroxisomes in transgenic mice. J. Clin. Invest. 96: 976–986.
46. Levak-Frank, S., P. H. Weinstock, T. Hayek, R. Verdery, W. Hofmann,
R. Ramakrishnan, W. Sattler, J. L. Breslow, and R. Zechner. 1997. Induced mutant mice expressing lipoprotein lipase exclusively in muscle have subnormal triglycerides yet reduced high density lipoprotein cholesterol levels in plasma. J. Biol. Chem. 272: 17182–17190.
47. Brown, M., and J. L. Goldstein. 1986. A receptor-mediated pathway
for cholesterol homeostasis. Science. 232: 34–47.
48. Brown, M., J. Herz, R. C. Kowal, and J. L. Goldstein. 1991. The low
density lipoprotein receptor-related protein: double agent or decoy? Curr. Opin. Lipidol. 2: 65–72.
49. Ishibashi, S., N. Yamada, H. Shimano, N. Mori, H. Mokuno, T.
Gotoda, M. Kawakami, T. Murase, and F. Takaku. 1990. Apolipoprotein E and lipoprotein lipase secreted from human monocytederived macrophages modulate very low density lipoprotein uptake. J. Biol. Chem. 265: 3040–3047.
50. Saxena, U., M. G. Klein, T. M. Vanni, and I. J. Goldberg. 1992.
Lipoprotein lipase increases low density lipoprotein retention by
subendothelial cell matrix. J. Clin. Invest. 89: 373–380.
51. Eisenberg, S., E. Sehayek, T. Olivecrona, and I. Vlodavsky. 1992.
Lipoprotein lipase enhances binding of lipoproteins to heparansulfate on cell surfaces and extracellular matrix. J. Clin. Invest. 90:
2013–2021.

Yagyu et al. LPL overexpression in apoE knockout mice

1685