Apolipoprotein (apo) E2 is
often associated with low levels of low density lipoprotein (LDL)
cholesterol and high levels of plasma triglycerides in humans. Mice
expressing apoE2 also have low LDL levels. To evaluate the possible
role of the LDL receptor in the cholesterol-lowering effect of apoE2,
we bred transgenic mice expressing low levels of apoE2 with LDL
receptor-null mice (hE2+/0,LDLR
/
).
Even in the absence of the LDL receptor, plasma total and LDL cholesterol levels decreased progressively with increasing levels of
plasma apoE2. At plasma apoE2 levels >20 mg/dl, LDL cholesterol was
~45% lower than in LDLR
/
mice. Thus, the LDL
cholesterol-lowering effect of apoE2 is independent of the LDL
receptor. In contrast, plasma triglyceride levels increased (mostly in
very low density lipoproteins (VLDL) and intermediate density
lipoproteins (IDL)) progressively as apoE2 levels increased. At plasma
apoE2 levels >20 mg/dl, triglycerides were ~150% higher than in
LDLR
/
mice. Furthermore, in apoE-null mice
(hE2+/0, mE
/
), apoE2 levels also
correlated positively with plasma triglyceride levels, suggesting
impaired lipolysis in both
hE2+/0,LDLR
/
and
hE2+/0,mE
/
mice. Incubating VLDL or IDL
from the hE2+/0,LDLR
/
or the
hE2+/0,mE
/
mice with mouse
postheparin plasma inhibited lipoprotein lipase-mediated lipolysis of
apoE2-containing VLDL and IDL by ~80 and ~70%, respectively, versus normal VLDL and IDL. This observation was confirmed
by studies with triglyceride-rich emulsion particles, apoE2, and purified lipoprotein lipase. Furthermore, apoE2-containing VLDL had
much less apoC-II than normal VLDL. Adding apoC-II to the incubation
partially corrected the apoE2-impaired lipolysis in apoE2-containing
VLDL or IDL and corrected it completely in apoE2-containing emulsion
particles. Thus, apoE2 lowers LDL cholesterol by impairing lipoprotein
lipase-mediated lipolysis of triglyceride-rich lipoproteins (mostly by
displacing or masking apoC-II). Furthermore, the effects of apoE2 on
both plasma cholesterol and triglyceride levels are dose dependent and
act via different mechanisms. The increase in plasma cholesterol caused
by apoE2 is due mostly to impaired clearance, whereas the increase in
plasma triglycerides is caused mainly by apoE2-impaired lipolysis
of triglyceride-rich lipoproteins.
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INTRODUCTION |
Apolipoprotein (apo)1 E
polymorphism is one of the common genetic factors responsible for
interindividual differences in plasma lipid and lipoprotein levels in
humans. The majority of apoE2 homozygotes, who do not display overt
type III hyperlipoproteinemia, have lower plasma cholesterol, low
density lipoprotein (LDL) cholesterol, and apoB levels but higher
plasma triglyceride and apoE levels than apoE3 homozygotes. In
contrast, apoE4 is associated with higher plasma cholesterol, LDL
cholesterol, and apoB levels but lower apoE levels (1-4). It also has
been suggested that apoE4 is associated with a higher incidence of
coronary heart disease (1, 4-9), probably due either to higher plasma
cholesterol and LDL cholesterol levels or to potentially different
roles of apoE isoforms within the lesions (5, 6, 10, 11). In contrast,
apoE2 seems to reduce the risk for coronary heart disease, probably due
to lower plasma and LDL cholesterol levels, but only in subjects
without type III hyperlipoproteinemia (1, 4, 7, 9). Those apoE2
homozygotes who develop type III hyperlipoproteinemia because of
secondary precipitating factors are at increased risk for both coronary
and peripheral artery atherosclerosis (1).
Although the cholesterol-lowering effect of apoE2 has been confirmed in
several populations, the mechanism is not completely understood. One
hypothesis is that defective LDL receptor binding of apoE2 lowers the
transport of cholesterol-rich remnant particles into the liver,
up-regulating hepatic LDL receptors and thereby accelerating the
clearance of plasma LDL (1, 4). However, some experimental evidence
shows that the number of hepatic LDL receptors is not increased in
either human apoE2 (hE2) transgenic rabbits or apoE-null
(mE
/
) mice (12, 13). Another hypothesis is that
apoE2-containing very low density lipoproteins (VLDL) compete poorly
with apoB-containing LDL for binding to the hepatic LDL receptor,
speeding LDL clearance (13) and thereby decreasing plasma LDL. A third
hypothesis is that apoE2 impairs lipolytic conversion of VLDL to LDL
(14-17), perhaps by directly inhibiting lipoprotein lipase (LPL)
activity (16).
Recently, we generated apoE2 transgenic mice in which moderate
expression of apoE2 (10-30 mg/dl) on the wild-type mouse background produced a hypolipidemic phenotype, somewhat mimicking the
hypocholesterolemia in humans with apoE2; lower expression of apoE2
(<10 mg/dl) did not alter plasma lipid levels significantly (18).
Crossing hypolipidemic apoE2 mice (apoE2 ~20 mg/dl) with LDL
receptor-null (LDLR
/
) mice resulted in a typical type
III hyperlipoproteinemic phenotype, characterized by increased levels
of total cholesterol and triglycerides and by the accumulation of both
apoE2 (50-60 mg/dl) and
-VLDL in plasma (19). These findings
indicate that low LDL receptor number is one genetic factor
responsible for converting apoE2-induced hypolipidemia into an overt
type III hyperlipoproteinemic phenotype. Furthermore, the LDL
cholesterol levels were lower in the LDLR
/
mice
expressing apoE2 than in the LDLR
/
mice, suggesting
that the LDL cholesterol-lowering effect of apoE2 might occur even in
the absence of the LDL receptor (19). However, this latter conclusion
was tenuous because of the marked increase in remnants and intermediate
density lipoproteins (IDL) in these mice.
In the present study, we evaluated in detail the potential role of the
LDL receptor in apoE2-induced low LDL cholesterol by crossing
transgenic mice expressing low levels of apoE2 (2-10 mg/dl) with
LDLR
/
mice. Our data demonstrate that apoE2
consistently lowered the LDL cholesterol level in the absence of the
LDL receptor. In vitro lipolytic studies indicated that low
LDL cholesterol is caused by impairment of LPL-mediated lipolysis of
triglyceride-rich lipoproteins. Furthermore, data obtained from
hE2+/0,mE
/
mice demonstrated that the
effects of apoE2 on plasma cholesterol and triglyceride levels are dose
dependent and act via different mechanisms.
 |
EXPERIMENTAL PROCEDURES |
Materials--
A Superose 6 column, purchased from Pharmacia
(Uppsala, Sweden), was used on a Pharmacia fast protein liquid
chromatography system. Centricon concentration filters were from Amicon
(Lexington, MA). Cholesterol and triglyceride standards were from
Abbott (North Chicago, IL) and Boehringer Mannheim (Indianapolis, IN),
respectively. An automated system (Kinetic Microplate Reader, Molecular
Devices, Menlo Park, CA) was used for lipid analysis. Bovine milk LPL
was from Sigma. Human apoC-II was provided by Dr. Karl H. Weisgraber (Gladstone Institute of Cardiovascular Disease, San Francisco, CA). The
LFS Lipogel assay kit was from Zaxis (Hudson, OH). All reagents for
lipoprotein-agarose gels were from Ciba Corning (Palo Alto, CA). The
ECL chemiluminescence detection kit for Western blots was from Amersham
Life Science (Little Chalfont, Buckinghamshire, United Kingdom).
Preparation of Transgenic Mice--
Hemizygous human apoE2
transgenic mice with endogenous mouse apoE (hE2+/0, ICR
strain) were produced previously in our laboratory (18). LDLR
/
and mE
/
mice (both C57BL/6
strain) were purchased from Jackson Laboratories (Bar Harbor, ME).
Female apoE2 mice from three low-expressing lines (apoE2 = 2, 5, and 10 mg/dl) were crossed with male LDLR
/
mice. The
resulting obligate heterozygotes (LDLR+/
)
expressing apoE2 (hE2+/0, LDLR/
) were
then crossed with LDLR
/
mice to yield apoE2 transgenic
mice lacking LDL receptors (hE2+/0,LDLR
/
).
Thus, the genetic background of these mice was 75% C57BL/6 and 25%
ICR. To create apoE2 transgenic mice without endogenous mouse
apoE (hE2+/0,mE
/
), female apoE2 mice
from three different lines (apoE2 = 2, 10, and 20 mg/dl) were
crossed with male mE
/
mice. The resulting obligate
heterozygotes (mE+/
) expressing apoE2
(hE2+/0,mE+/
) were then crossed with
mE
/
mice to yield apoE2 transgenic mice without mouse
apoE (hE2+/0,mE
/
). Thus, the genetic
background of the mice was also 75% C57BL/6 and 25% ICR.
The human apoE2 transgene was identified by immunoblotting of 1 µl of
plasma with human-specific anti-apoE antiserum (18, 20). In the Western
blot assay, human apoE2 was semiquantitated by comparing the
densitometry readings of the sample bands with those of different
concentrations of purified human apoE2. LDL receptor deficiency was
assessed by polymerase chain reaction with specific primers designed
according to the knockout gene construct. Mouse apoE deficiency was
assessed by Western blotting with mouse-specific anti-apoE antiserum
(19). All experiments were performed under protocols approved by the
Committee on Animal Research, University of California, San
Francisco.
Lipoprotein Separation and Analysis--
Blood was collected
from the tails of 8-12-week-old mice that had been fasted for 5 h. EDTA was used as anticoagulant (final concentration, 10 mM). Plasma was obtained by centrifugation at 14,000 rpm
(microcentrifuge) for 10 min at 4 °C, and samples were stored for no
more than 3 days at 4 °C in the presence of 1 mM phenylmethylsulfonyl fluoride as a protease inhibitor. Cholesterol and
triglycerides were measured on total plasma and on chromatographic fractions by an enzymatic colorimetric method adapted for use with a
microplate reader (21, 22).
Lipoproteins in 100 µl of plasma were separated by chromatography on
a Superose 6 column, as described previously (18, 20, 22). The major
lipoprotein classes eluted from the column were pooled and concentrated
with Centricon filters (fractions 16-18, VLDL; fractions 19-22, IDL;
fractions 23-27, LDL and a subclass of high density lipoproteins
(HDL1); and fractions 28-33, HDL). For agarose gel
electrophoresis, 2-µl aliquots of concentrated lipoproteins were run
on precast agarose gels (1%) for 45 min at 90 V. The gels were dried
and stained with Fat Red 7B. In some cases, to analyze the distribution
of apoE2 in various lipoproteins, the pooled samples representing
different lipoprotein classes were separated on a 12%
polyacrylamide-sodium dodecyl sulfate gel followed by immunoblotting
with anti-human apoE antiserum. For analysis of chemical compositions
and lipolysis of VLDL, the VLDL (d < 1.006 g/ml) were
isolated from plasma by ultracentrifugation at 98,000 rpm for 2 h
at 4 °C in a Beckman TL100 ultracentrifuge (23). Apolipoproteins
were separated on 3-20% polyacrylamide-sodium dodecyl sulfate
gradient gels. The amounts of human apoE2, mouse apoE, or mouse apoC-II
were determined by Western blotting with polyclonal antibodies against
human apoE, mouse apoE, or mouse apoC-II, respectively. Purified human
apoE2, mouse apoE, and mouse apoC-II were used as standards,
respectively.
Alternatively, lipoproteins in 8 µl of plasma were separated by LFS
Lipogel electrophoresis (Zaxis, Hudson, OH), fractionated according to
their particle size in a 0.5-30% polyacrylamide gradient gel, and
stained for cholesterol. The distribution of cholesterol in various
lipoproteins was determined by densitometry at 600 nm. The cholesterol
levels in various lipoproteins were calculated from the plasma total
cholesterol determined by an enzymatic colorimetric method. For five
normal human plasma standards, the intra-assay coefficients of
variation were 9.8, 5.3, and 6.4% for VLDL/IDL, LDL, and HDL
cholesterol, respectively; the inter-assay coefficients of variation
were 14.2, 7.1, and 8.3%, respectively.
Lipolysis of VLDL and IDL in Vitro--
To determine the ability
of normal and apoE2 VLDL to serve as substrates for lipase-mediated
lipolysis, 30 µg of VLDL or IDL triglyceride was incubated for 30 min
at 37 °C with 10 µl of VLDL-depleted postheparin mouse plasma,
which was collected from normal mice 10 min after intravenous injection
of heparin (50 units/kg). The incubation was performed in the presence
of 1.2 M NaCl (to measure hepatic lipase) or in its absence
(to measure total lipolytic activity). In some cases, specific amounts
of purified human apoC-II were included in the incubation. After
incubation, the levels of released free fatty acids were determined by
an enzymatic colorimetric method (24) (Wako Chemicals, Richmond, VA).
The LPL-mediated lipolysis was calculated as the difference between
total lipolysis and hepatic lipase-mediated lipolysis (25).
Lipolysis of Triglyceride-rich Emulsion Particles in
Vitro--
The triglyceride-rich emulsion particles were prepared
as described previously (26-28). Briefly, 100 mg of triolein (Sigma) and 25 mg of egg yolk phosphatidylcholine were mixed together and dried
under nitrogen. The pellets were resuspended in 5 ml of 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM
EDTA and 100 mM KCl and then sonicated. The resulting
emulsion particles were similar in size to native human VLDL (27, 29).
To determine the effect of apoE2 on LPL-mediated lipolysis, the
triglyceride-rich emulsion particles (50 µl) were incubated at
37 °C first with 4 µg of human apoC-II for 30 min and then with
different amounts of apoE2 and 0.5 µg of bovine milk LPL (Sigma) for
30 min. To determine the effect of apoC-II on LPL-mediated lipolysis,
the triglyceride-rich emulsion particles (50 µl) were incubated at 37 °C first with 4 µg of human apoE2 for 30 min and then with different amounts of apoC-II and 0.5 µg of bovine milk LPL (Sigma) for 30 min. After incubation, the lipolytic activity was determined as
described above.
Statistical Analysis--
Mean lipid levels are reported as the
mean ± S.D. Differences in lipid levels were evaluated by
t test. Correlations of plasma apoE2 and lipid levels were
assessed by regression analysis.
 |
RESULTS |
Effects of ApoE2 Expression on Plasma Lipids and Lipoproteins in
the Absence of LDL Receptors--
To generate LDLR
/
mice expressing different levels of apoE2, we mated
LDLR
/
mice with three mice expressing low levels of
apoE2. Table I summarizes the plasma
levels of lipids and lipoproteins in
hE2+/0,LDLR
/
and
hE20/0,LDLR
/
mice. As reported previously
(30), both male and female LDLR
/
mice had significant
hypercholesterolemia (total cholesterol levels about double those of
nontransgenic controls) and slightly increased triglyceride levels
(25% higher than in nontransgenic controls) (data not shown for
nontransgenic mice). Gel filtration chromatography on a Superose 6 column (Fig. 1A) and
nondenaturing polyacrylamide gradient gel electrophoresis (Fig.
2) demonstrated that the
hypercholesterolemia in the LDLR
/
mice was due mainly
to an accumulation of LDL cholesterol.
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Table I
Plasma levels (mg/dl) of lipids and lipoproteins in LDLR /
mice expressing different levels of apoE2
The abbreviations are the following: hE2, human apoE2; TC, total
cholesterol; TG, triglyceride; HDL-C, HDL cholesterol; LDL-C, LDL
cholesterol; VLDL/IDL-C, VLDL/IDL cholesterol. Differences were
evaluated by t test.
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Fig. 1.
Superose 6 chromatography of 100 µl of
mouse plasma. The cholesterol and triglyceride distributions in
the plasma of individual male mice were analyzed as described under
"Experimental Procedures." Each panel is one representative profile
of several analyzed in each group of mice. A,
hE20/0,LDLR / mouse plasma.
B-D, plasma from LDLR / mice
expressing different levels of apoE2. TC, total cholesterol;
TG, triglyceride. The units for apoE2, TC, and TG are
mg/dl.
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Fig. 2.
Polyacrylamide gradient gel electrophoresis
of plasmas from LDLR / male mice expressing different
levels of apoE2. Lipoproteins in 8 µl of plasma were separated
on a 0.5-30% polyacrylamide gradient gel as described under
"Experimental Procedures." Lane 1, LDLR /
mouse; lanes 2-4, LDLR / mice with apoE2
<10 mg/dl; lanes 5 and 6, LDLR /
mice with 10-20 mg/dl apoE2; lane 7, LDLR /
mouse with apoE2 >20 mg/dl. The migration positions of HDL, LDL, and
VLDL/IDL are indicated.
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|
The hE2+/0,LDLR
/
mice expressing apoE2 at
<10 mg/dl had lower total cholesterol (9 and 7% for males and
females, respectively) than hE20/0,LDLR
/
mice (Table I). The decrease was due to reductions of about 15% in
both LDL and HDL cholesterol, which were partially offset by an
increase in VLDL/IDL cholesterol (males, 39%; females, 30%) (Table I,
Fig. 1B, and Fig. 2). Plasma triglyceride levels were higher
in both males (32%) and females (42%) than in
hE20/0,LDLR
/
mice (Table I). Mice
expressing apoE2 at 10-20 mg/dl had a greater decrease in total
cholesterol (14 and 13% for males and females, respectively) than
hE20/0,LDLR
/
mice (Table I). Again, the
decrease was due to reductions of both LDL (males, 32%; females, 29%)
and HDL (males, 24%; females, 19%) cholesterol; however, the
hE2+/0,LDLR
/
mice had higher VLDL/IDL
cholesterol (males, 64%; females, 40%) (Table I, Fig. 1C,
and Fig. 2). Plasma triglyceride levels were increased significantly in
both male (62%) and female (85%) mice (Table I), and
triglyceride-enriched LDL appeared (Fig. 1C). Mice
expressing apoE2 at more than 20 mg/dl had an even greater decrease in
total cholesterol (males, 20%; females, 17%) but more severe
hypertriglyceridemia (males, 131%; females, 167%) than hE20/0, LDLR
/
mice (Table I). Their LDL and
HDL cholesterol levels were about half of those in
hE20/0,LDLR
/
mice (Table I); most
significantly, the distribution of plasma cholesterol was shifted
predominantly to VLDL/IDL fractions (Fig. 1D and Fig. 2).
Thus, in general, there was a strong negative correlation between
plasma apoE2 concentrations and plasma total cholesterol levels (males,
r =
0.81, p < 0.001; females,
r =
0.87, p < 0.001) (Fig.
3A). Likewise, there was a
negative correlation between apoE2 and LDL cholesterol (males,
r =
0.89, p < 0.001; females,
r =
0.92, p < 0.001) or HDL
cholesterol (males, r =
0.72, p < 0.001; females, r =
0.81, p < 0.001)
(data not shown). On the other hand, there was a strong positive
correlation between plasma apoE2 concentrations and VLDL/IDL
cholesterol (males, r = 0.68, p < 0.001; females, r = 0.78, p < 0.001)
and plasma triglyceride levels (males, r = 0.74, p < 0.001; females, r = 0.84, p < 0.001) (Fig. 3B). We have previously
shown that hE2+/0,LDLR
/
mice expressing
50-60 mg/dl apoE2 have a hypercholesterolemia characterized by a
marked increase in
-VLDL (19).

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Fig. 3.
Correlation of plasma apoE2 and lipids.
A, apoE2 versus cholesterol in
hE2+/0,LDLR / mice. B, apoE2
versus triglycerides in
hE2+/0,LDLR / mice. C, apoE2
versus cholesterol in hE2+/0,mE /
mice. D, apoE2 versus triglycerides in
hE2+/0,mE / mice.
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Column fractions representing various lipoproteins were pooled,
concentrated, and subjected to agarose gel electrophoresis (Fig.
4). As expected,
hE20/0,LDLR
/
mice accumulated a large
amount of
-migrating LDL (Fig. 4A). The
hE2+/0, LDLR
/
mice expressing <20 mg/dl
apoE2 had a significant decrease in LDL and a slight increase in VLDL
(Fig. 4B). In mice expressing >20 mg/dl apoE2, the decrease
in LDL was more pronounced, and the VLDL increased dramatically and
migrated more slowly toward the
-position (Fig. 4C).
Taken together, all the above data demonstrate that apoE2 has an LDL
cholesterol-lowering effect independent of the LDL receptor in
transgenic mice.

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Fig. 4.
Agarose gel electrophoresis of lipoprotein
fractions from male LDLR / mice expressing different
levels of apoE2. The VLDL are Superose 6 fractions 16-18, and the
IDL are fractions 19-22. The origin and migration positions of
-migrating (HDL), pre- -migrating (VLDL), and -migrating (LDL)
lipoproteins are indicated.
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Distribution of ApoE2 among Lipoproteins--
The distribution of
apoE2 among various lipoprotein classes is shown in Table
II. In the LDLR
/
mice
expressing apoE2 at <10 mg/dl, most of the apoE2 was in the HDL
fraction, with lesser amounts in the LDL/HDL1 fractions and
very little in the VLDL fraction. In contrast, LDLR
/
mice expressing apoE2 at >20 mg/dl had less apoE2 in the HDL fraction
but almost three times more apoE2 in the VLDL fraction than the mice
expressing apoE2 at <10 mg/dl. Mice expressing apoE2 at 10-20 mg/dl
had an apoE2 distribution pattern in between those of mice expressing
lower or higher levels of apoE2.
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Table II
Distribution of apoE2 (percent ± S.D.) in various lipoproteins of
LDLR / mice expressing different levels of apoE2
Aliquots of pooled Superose 6 fractions representing the major
lipoprotein classes were separated on 12% polyacrylamide-sodium
dodecyl sulfate gels and detected by anti-human apoE immunoblotting.
The distribution of apoE2 in the different lipoproteins was determined
by densitometric scanning.
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Effect of ApoE2 Expression on Plasma Lipids in the Absence of
Endogenous Mouse ApoE--
The positive correlation between plasma
apoE2 and triglyceride levels in the
hE2+/0,LDLR
/
mice mimics a condition
observed in humans (31, 32). However, because
hE2+/0,LDLR
/
mice lack LDL receptors, it is
difficult to determine whether increased plasma triglyceride levels are
caused solely by an increase in apoE2 or by an increase in apoE2 in
combination with the absence of LDL receptors. To address this
question, we generated apoE2 transgenic mice on the mE
/
background but with a normal level of LDL receptors. Previously, we
showed that high levels of apoE2 on the mE
/
background
lead to a type III hyperlipoproteinemic phenotype (19). In the present
study, we generated hE2+/0,mE
/
mice
expressing low and intermediate levels of apoE2, which has enabled us
to investigate the dose-dependent effect of apoE2 on plasma
cholesterol and triglycerides in the absence of endogenous mouse apoE.
Compared with plasma cholesterol levels in apoE
/
mice
(males, 402 ± 4 mg/dl; females, 398 ± 39 mg/dl),
cholesterol levels in the hE2+/0,mE
/
mice
decreased gradually as apoE2 increased from ~4 to 25 mg/dl (Fig.
3C). In fact, at apoE2 concentrations of 20-25 mg/dl,
plasma cholesterol levels in the
hE2+/0,LDLR
/
mice were only slightly higher
than in nontransgenic mice (males, 93 ± 15 mg/dl; females,
83 ± 10 mg/dl), indicating that, as far as cholesterol catabolism
is concerned, having defective apoE2 is better than having no apoE at
all and that increasing the amount of apoE2 up to a critical level may
compensate for the lower LDL receptor binding activity of apoE2. In
contrast, plasma triglyceride levels increased linearly with increasing
levels of apoE2 (4-25 mg/dl) (Fig. 3D), indicating that the
increase in triglycerides was independent of the effect of apoE2 on
cholesterol and likely reflected a direct effect of apoE2 on lipolytic
processing. At apoE2 levels >30 mg/dl, positive correlations were
observed for both plasma cholesterol and triglycerides (Fig. 3,
C and D). These data indicate that the effects of apoE2
on plasma cholesterol and triglyceride levels are dose dependent and
may act via different mechanisms.
Effect of ApoE2 on Lipolysis of VLDL and IDL--
The simultaneous
increase in plasma triglycerides and VLDL/IDL cholesterol and the
decrease in LDL cholesterol in the presence of an increasing level of
apoE2 in hE2+/0,LDLR
/
mice raise the
possibility that apoE2 impairs the lipolytic conversion of VLDL to LDL,
as previously hypothesized (16). To address this question, we
determined the ability of normal and apoE2-containing VLDL and IDL to
serve as substrates for lipase-mediated lipolysis in
vitro (Fig. 5). The presence of
apoE2 in the VLDL and IDL from the
hE2+/0,LDLR
/
mice inhibited LPL-mediated
lipolysis by 84 and 73%, respectively, compared with VLDL and IDL from
nontransgenic mice, while hepatic lipase-mediated lipolysis appeared to
be affected to a lesser degree. These results indicate that
apoE2-containing VLDL and IDL are not good substrates for LPL-mediated
lipolysis compared with normal VLDL. The VLDL and IDL from
LDLR
/
mice were, however, good substrates for LPL.

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Fig. 5.
Effect of apoE2 and apoC-II on the lipolysis
of VLDL and IDL. Various VLDL (A) or IDL (B)
(30 µg of triglycerides) were incubated with 10 µl of VLDL-depleted
postheparin mouse plasma for 30 min at 37 °C with or without 1.2 M NaCl. In some cases, various VLDL or IDL (30 µg of
triglycerides) were first incubated with 8 µg of apoC-II for 30 min
at 37 °C and then with 10 µl of VLDL-depleted postheparin mouse
plasma for 30 min at 37 °C with or without 1.2 M NaCl.
Lipase activities were calculated as described under "Experimental
Procedures." For the apoE2 content of the various VLDL, see Table
III. Results are the mean ± S.D. of determinations in four mice.
FFA, free fatty acid.
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The apoE2-containing VLDL from hE2+/0,LDLR
/
mice contained much more cholesterol than normal VLDL, which was
reflected by a much lower ratio of triglyceride to cholesterol (Table
III). To determine if the higher
cholesterol content of apoE2-containing VLDL, rather than apoE2 itself,
is responsible for the impaired lipolysis, we isolated
hE20/0,mE
/
VLDL, which contained much more
cholesterol than either normal VLDL or apoE2-containing VLDL from
hE2+/0, LDLR
/
mice. We compared the
LPL-mediated lipolysis of hE20/0,mE
/
VLDL
with hE2+/0,mE
/
VLDL, which had a
cholesterol content similar to that of
hE2+/0,LDLR
/
VLDL (Table III). Although the
VLDL from mE
/
mice was a poorer substrate for
LPL-mediated lipolysis than normal VLDL (~60% of normal VLDL), it
was more susceptible to LPL-mediated lipolysis than VLDL from
hE2+/0,mE
/
mice (~20% of normal VLDL).
These data suggest that at least a significant portion of the impaired
lipolysis for the apoE2-containing VLDL is due to apoE2, although a
possible effect on lipolysis due to a higher cholesterol content of the
particles cannot be ruled out.
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Table III
Characteristics of VLDL (d < 1.006 g/ml) from various
transgenic mice
Apolipoproteins of VLDL pooled from 3 to 5 mice were separated on
3-20% polyacrylamide-sodium dodecyl sulfate gradient gels. The
amounts of human apoE2 (hApoE2), mouse apoE (mApoE), or mouse apoC-II
(mApoC-II) were determined by Western blotting with polyclonal
antibodies against human apoE, mouse apoE, or mouse apoC-II,
respectively. TC, total cholesterol; TG, triglyceride.
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Effect of the ApoE2:ApoC-II Ratio on the Lipolysis of VLDL and
Triglyceride-rich Emulsion Particles--
Another difference in the
VLDL composition was that apoE2-containing VLDL had much more apoE, but
much less apoC-II, than normal VLDL (Table III). The apoC-II content of
the d < 1.006 g/ml lipoproteins was 5-6-fold higher
in nontransgenic mice than in apoE2-expressing mice. Since previous
studies had shown that increasing apoE in VLDL displaces apoCs from the
particles (33), and vice versa (33-36), the impairment of VLDL
lipolysis by apoE2 could occur via a displacement or masking of
apoC-II, which is an absolute cofactor for LPL activity (37, 38). To
evaluate this possibility, we added purified human apoC-II to various
VLDL and determined its effect on lipolysis (Fig. 5A).
Adding apoC-II to apoE2-containing VLDL stimulated LPL-mediated
lipolysis 3-fold, but did not significantly affect the lipolysis of
normal VLDL. As expected, apoC-II did not affect hepatic
lipase-mediated lipolysis of either normal or apoE2-containing VLDL
(Fig. 5A). Similar results were also obtained from various
IDL (Fig. 5B). These data indicate that apoE2-impaired
lipolysis of VLDL can be partially corrected by increasing the amount
of apoC-II on the particles. The addition of apoC-II to nontransgenic
or LDLR
/
VLDL or IDL had only a very small stimulatory
effect.
Since adding apoE2 to normal VLDL inhibited lipolysis in a
dose-dependent fashion (data not shown), it is probable
that the apoE2:apoC-II ratio is a major factor that modulates the
lipolysis. To evaluate the importance of this ratio as a determinant of
lipolysis, VLDL-like triglyceride-rich emulsion particles were prepared
with increasing amounts of apoE2 and a constant amount of apoC-II. Increasing the apoE2:apoC-II ratio progressively inhibited lipolysis, reaching 90% at a ratio of 3.0 (Fig.
6A). In an additional set of
experiments, we reisolated the particles after the addition of the
apoproteins. We found that at lower levels of added apoE2, the
apoE2:apoC-II ratio was only slightly lower than predicted, and at
higher levels of added apoE2, the apoE2:apoC-II ratio was higher than
predicted. At the highest level of added apoE2, where lipolysis was
inhibited >90% (Fig. 6A), the apoE2:apoC-II ratio was
10.4. This is quite similar to the apoE2:apoC-II ratio in the
d < 1.006 g/ml particles from
hE2+/0,LDLR
/
mice, where the ratio was 12.4 considering both mouse and human apoE2 or 10.2 considering only the
human apoE2; in hE2+/0,mE
/
mice, the ratio
was 9.2 (Table III). When increasing amounts of apoC-II were added to
the emulsion particles in the presence of a constant amount of apoE2,
lipolysis was stimulated to 3-fold at ratios above 2.0 (Fig.
6B).

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Fig. 6.
Effect of the interaction of apoE2 with
apoC-II on LPL-mediated lipolysis of triglyceride-rich emulsion
particles. A, 50 µl of triglyceride-rich emulsion
particles was incubated first with 4 µg of apoC-II for 30 min at
37 °C and then with 10 µl of bovine milk LPL (0.5 µg protein)
and different amounts of apoE2 for 30 min at 37 °C. B, 50 µl of triglyceride-rich emulsion particles was incubated first with 4 µg of apoE2 for 30 min at 37 °C and then with 10 µl of bovine
milk LPL (0.5 µg of protein) and different amounts of apoC-II for 30 min at 37 °C. After incubations in both experiments (A
and B), LPL activity was calculated as described under
"Experimental Procedures." Results are the mean ± S.D. of
determinations in triplicate experiments. FFA, free fatty
acid.
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DISCUSSION |
The LDL cholesterol-lowering effect of apoE2 has been offered as
the explanation for reduced risk for coronary heart disease in subjects
with apoE2 who do not have type III hyperlipoproteinemia (1-4, 7, 9).
Thus, defining the mechanism of this phenomenon will clarify the role
of different apoE isoforms in plasma lipoprotein metabolism and in the
development of atherosclerosis. Since up-regulation of hepatic LDL
receptors due to a delayed liver uptake of apoE2-containing remnant
lipoproteins has long been suggested to explain the LDL cholesterol-lowering effect of apoE2 (4), we crossed transgenic mice
expressing low levels of apoE2 (2-10 mg/dl) with LDLR
/
mice to evaluate the involvement of the LDL receptor in this phenomenon. Our data demonstrating that apoE2 lowers LDL cholesterol in
apoE2 transgenic mice lacking LDL receptors suggests that there is a
mechanism independent of the LDL receptor to explain this phenomenon.
Evidence from other studies supports this conclusion: neither apoE2
transgenic rabbits nor mE
/
mice have more hepatic LDL
receptors than nontransgenics (12, 13). Previously, apoE2 has been
shown to impair the lipolytic conversion of VLDL to LDL (14-17). Our
present studies further indicate that apoE2 lowers LDL cholesterol by
impairing the lipolysis of triglyceride-rich lipoproteins both in
vivo and in vitro.
High density lipoprotein levels in humans homozygous for apoE2 are
usually low, especially in type III hyperlipoproteinemic subjects
(4, 39). Impairment of VLDL lipolysis by apoE2 provides a logical
explanation for the HDL-lowering effect in apoE2 transgenic mice on the
wild-type, mE
/
, or LDLR
/
background
(18, 19). Since the formation of at least a portion of HDL particles
requires the surface components of triglyceride-rich lipoproteins
released during lipolytic processing (40), apoE2-associated impairment
of lipolysis would lead to a lesser release of surface components from
the triglyceride-rich lipoproteins, which in turn would decrease the
production of HDL particles. This possibility is supported by the
observation that patients with homozygous LPL deficiency have extremely
high levels of chylomicrons and VLDL and reduced levels of LDL and HDL
(41). Hypertriglyceridemia with reduced LDL and HDL can also be induced
by infusing antibodies that inhibit LPL into monkeys (42). In addition,
plasma LPL activity correlates positively with HDL cholesterol levels
(43).
Population studies have demonstrated that apoE2 is associated with
increased plasma triglyceride levels (31, 32), which is confirmed in
this study. This phenomenon has been ascribed to defective LDL receptor
binding of apoE2, which impairs the clearance of triglyceride-rich
lipoproteins from the circulation (1, 32). However, the normal or only
mildly elevated triglyceride levels in mE
/
mice with
severe hypercholesterolemia argues against this hypothesis (44, 45).
The present studies of the hE2+/0,LDLR
/
mice provide evidence supporting the hypothesis that apoE2-induced hypertriglyceridemia is caused not only by impaired remnant lipoprotein clearance but also to a major extent by impaired lipolysis of triglyceride-rich lipoproteins (1, 32). Likewise, our previous studies
(19) and the present expanded studies demonstrate that moderate and
high expression of apoE2 on the mE
/
background
(hE2+/0,mE
/
) increased plasma triglyceride
levels 2-5-fold above those in mE
/
mice (Fig.
3D), indicating that as far as triglyceride catabolism is
concerned, having defective apoE2 is worse than having no apoE at all.
On the other hand, since plasma total cholesterol levels were lower in
the hE2+/0,mE
/
than in the
mE
/
mice (Fig. 3C and Ref. 19), it seems
that the hypercholesterolemia and hypertriglyceridemia associated with
apoE2 occur via two separate mechanisms: hypercholesterolemia caused by
defective binding of apoE2 to the LDL receptor and hypertriglyceridemia
caused mostly by impaired lipolysis of triglyceride-rich lipoproteins
by apoE2.
The present study also shows that the apoE2:apoC-II ratio in VLDL
particles affects lipolysis. Increasing this ratio in VLDL or
triglyceride-rich emulsion particles impaired LPL-mediated lipolysis.
Conversely, decreasing this ratio in these apoE2-containing particles
corrected or enhanced LPL-mediated lipolysis. Thus, it seems that
apoE2-containing VLDL are poor substrates for lipolysis partly because
of displacement of apoC-II, although a direct effect of apoE2 on
lipolysis cannot be excluded (46, 47). Taken together with data showing
that the apoE:apoC ratio is also an important determinant for
apoE-mediated clearance of remnant lipoproteins via the hepatic
receptors (33-36), our present data indicate that the apoE2:apoC-II
ratio is also an important determinant for LPL-mediated lipolysis of
triglyceride-rich lipoproteins. Thus, as remnant lipoproteins
accumulate in the plasma, the level of apoE2 in the remnant fraction
increases, resulting in impaired lipolysis and a blockade in the
VLDL
LDL lipolytic cascade. We should point out that displacement
of apoC-II and impairment of lipolysis by apoE2 is not specific for
this isoform. In vitro, all three apoE isoforms inhibit
lipolysis to a similar
extent.2 Thus, under the
right genetic and physiological conditions, high plasma levels of any
of the apoE isoforms might have severe consequences. However, these
consequences most likely manifest themselves in the presence of the
apoE2 isoform, since this is the isoform that accumulates significantly
in the plasma because it binds defectively to lipoprotein
receptors.
Based on these studies and our previous studies (19) with increasing
concentrations of apoE2 expression on the mE
/
background, we have defined how apoE2 modulates both cholesterol and
triglyceride levels in mice. We have shown that the effects of apoE2 on
both cholesterol and triglyceride levels are dose dependent and act via
different mechanisms. Low expression of apoE2 (4-8 mg/dl) in the
hE2+/0,mE
/
mice does not provide enough
apoE2 molecules to the remnant fractions to mediate clearance of the
particles (because of the defective receptor binding of apoE2), leading
to only a slight reduction of the hypercholesterolemia of the
mE
/
mice. Furthermore, low levels of apoE2 in VLDL or
remnant lipoproteins are not sufficient to displace or mask the
apoC-II; thus, the lipolysis is effective and plasma triglyceride
levels are normal. However, intermediate levels of apoE2 (15-25 mg/dl)
at least partially compensate for the absence of normal apoE by
overloading the particles with apoE2, which even though partially
defective, can mediate receptor binding and remnant clearance. Thus,
cholesterol levels decrease toward normal. However, triglyceride levels
begin to rise sharply as the increased apoE2 displaces or masks the
apoC-II and impairs LPL-mediated lipolysis of VLDL and IDL. At high
levels of apoE2 expression (>25 mg/dl), the remnants become saturated with apoE2, markedly impairing LPL-mediated lipolysis and remnant clearance and leading to severe hypertriglyceridemia and
hypercholesterolemia. Both the incomplete lipolysis of the remnants and
the defective receptor-mediated clearance combine to result in the
severe hyperlipidemia characterizing type III hyperlipoproteinemia.
Similarly, human apoE2 homozygotes who are hypolipidemic have low
levels of LDL cholesterol and only a slight accumulation of remnant
lipoprotein cholesterol and triglycerides (Fig.
7). Presumably the low plasma levels of
apoE2 mimic what is seen in the intermediate-expressing
hE2+/0,mE
/
mice, i.e. the apoE2
level is sufficient to mediate at least some remnant clearance either
by the LDL receptor (even though apoE2 has defective binding) or by the
heparan sulfate proteoglycan/LDL receptor-related protein pathway
(apoE2 binding is fairly normal) (48-50). However, the LDL cholesterol
is lower than normal in these hypolipidemic apoE2 homozygotes,
indicating that the apoE2 levels are sufficient to displace or mask
apoC-II and impair lipolysis of the triglyceride-rich lipoproteins. On
the other hand, overt type III hyperlipoproteinemia with marked
hypercholesterolemia and hypertriglyceridemia can be precipitated when
other factors are present, such as a high rate of VLDL production, a
low number of LDL receptors as demonstrated in this and a previous
study (19), or low estrogen status, which decreases both LDL receptor numbers and lipolytic activities (12). These factors further stress the
already compromised remnant catabolism in the apoE2 homozygotes,
resulting in a further impairment of clearance and lipolytic processing
and an accumulation of apoE2. As with the high-expressing
hE2+/0,mE
/
mice, the enrichment of the
remnant lipoproteins with apoE2 would severely displace or mask the
apoC-II, further impairing lipolysis and further disrupting remnant
lipoprotein clearance (Fig. 7).

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Fig. 7.
Receptor-mediated clearance and lipolytic
processing: VLDL LDL. In normolipidemic subjects, apoE3 plays
a key role in mediating the clearance of remnant lipoproteins from
plasma by the liver. In hypolipidemic apoE2 homozygotes, the plasma
levels of apoE2 are increased due to its defective receptor binding.
These increased levels may compensate for apoE2's defective receptor
binding and allow it to mediate efficient remnant clearance from the
plasma by the LDL receptor (even though apoE2 has defective binding) or
by the heparan sulfate proteoglycan/LDL receptor-related protein
pathway (apoE2 binding is fairly normal), leading to only a small
increase in remnant lipoproteins. However, these increased levels of
apoE2 are sufficient to displace or mask apoC-II and impair lipolytic
processing of VLDL, leading to decreased LDL formation and decreased
LDL levels. In the presence of secondary factors, the already
compromised remnant catabolism in the apoE2/2 homozygotes is further
stressed, resulting in a further impairment of clearance and an
accumulation of apoE2. The enrichment of remnant lipoproteins with
apoE2 would severely displace or mask the apoC-II, further impairing
lipolysis and disrupting remnant clearance. CII, apoC-II;
E2, apoE2; E3, apoE3. Dashed lines
indicate blockage of either LDL formation or remnant
clearance.
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We thank Dr. Karl H. Weisgraber for providing
apoC-II, human apoE, and mouse apoE antisera, Sylvia Richmond for
manuscript preparation, Gary Howard and Stephen Ordway for editorial
assistance, and John C. W. Carroll and Amy Corder for
graphics.