From the Whitaker Cardiovascular Institute,
Department of Medicine, Boston University School of Medicine,
Boston, Massachusetts 02118, the ¶ Department of Human and
Clinical Genetics, Leiden University Medical Center, 2333AL Leiden, The
Netherlands,
TNO-PG Prevention and Health, Gaubius Laboratory,
Leiden 2333CK, The Netherlands, and ** Department of Biochemistry and
Institute of Molecular Biology and Biotechnology, University of Crete,
Heraklion, Crete 71110, Greece
Received for publication, January 17, 2001
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ABSTRACT |
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Apolipoprotein (apo) E has been
implicated in cholesterol and triglyceride homeostasis in humans. At
physiological concentration apoE promotes efficient clearance of
apoE-containing lipoprotein remnants. However, high apoE plasma levels
correlate with high plasma triglyceride levels. We have used
adenovirus-mediated gene transfer in apoE-deficient mice
(E Apolipoprotein (apo)1 E
is the ligand for several cell receptors and promotes the catabolism of
apoE-containing lipoprotein remnants by the liver (1-5). Mutations in
apoE that prevent binding of apoE-containing lipoproteins to the LDL
receptor and possibly other receptors and heparan sulfate proteoglycans
are associated with type III hyperlipoproteinemia and premature
atherosclerosis (6-14).
Studies in human patients with type III hyperlipoproteinemia and in
animal models with apoE deficiency or defective apoE forms (15-24)
showed that apoE is required for the clearance of cholesterol ester-rich lipoprotein remnants found in the VLDL and IDL region (15-27). The accumulation of such remnants in plasma is associated with premature atherosclerosis (15, 17-19). ApoE may also be involved
in cholesterol efflux processes (28-31). These functions of apoE
contribute to cell and tissue cholesterol homeostasis (28-31) and may
explain why, when expressed locally in macrophages or endothelial
cells, apoE protects from atherosclerosis (32-34).
In humans, plasma apoE levels correlate with plasma triglyceride
levels (26). Animal studies have shown that increased plasma apoE
levels inhibit lipolysis of triglyceride-rich lipoproteins in
vivo and result in hypertriglyceridemia (35-39). Lipolysis of VLDL in vitro could be partially restored by the addition of
apoCII (35, 36). Other studies have shown that overexpression of apoE
also stimulates hepatic VLDL-triglyceride production in vivo (39) and in cell cultures (35), possibly by promoting the assembly
and/or secretion of apoB-containing lipoproteins. In contrast, lack of
apoE is associated with decreased VLDL-triglyceride secretion (40).
Such studies provide compelling evidence that apoE contributes both to
cholesterol as well as triglyceride homeostasis in vivo.
To identify the domains of apoE that contribute to cholesterol and
triglyceride homeostasis in vivo, we used
adenovirus-mediated gene transfer in apoE-deficient mice. We have found
that overexpression of full-length apoE4 is associated with high
cholesterol and triglyceride levels, whereas overexpression of
apoE4-202 normalizes the cholesterol levels of apoE-deficient mice and
does not trigger hypertriglyceridemia. ApoE secretion by cells, hepatic
apoE mRNA expression, and association of apoE with VLDL are not
affected by the apoE truncation. In contrast, apoE4, but not
apoE4-202, greatly increases hepatic VLDL-triglyceride secretion. Our
findings suggest that truncated forms of apoE may find useful gene
therapy applications in the future in correcting remnant removal disorders.
Construction of Recombinant Adenoviruses Expressing ApoE4 and
ApoE4-202--
The construction of pUC-apoE4 cDNA has been
described previously (41). pUC-apoE4-202 was generated by
overlap-extension PCR that resulted in mutagenesis of codon 203 (GTA)
to a stop codon (TAA) using pUC-apoE4 as a template and the four sets
of oligonucleotides indicated in Table I
as primers. The set of external primers OUTPR1-S (sense) and OUTPR2-A
(antisense) correspond to nucleotides encoding amino acids 103-111 and
208 -215 of apoE, respectively, and contain the restriction sites
NgoMI and BstEII, respectively. The set of
mutagenic oligonucleotides extending 10 residues 5' and 10 residues 3'
of codon 203 has been altered in its sequence to a stop TAA codon
(Table I). The PCR-based mutagenesis of codon 203 involved two separate
amplification reactions. The first reactions used the 5' external
primer and the antisense mutagenic primer (Internal-A, Table I)
covering codon 203. The second reaction used the 3' external primer and
the sense mutagenic primer (Internal-S, Table I) covering codon 203. An
aliquot of 4% of the volume of each PCR was mixed, and the sample was
amplified by the 5' and the 3' external primers. The amplified fragment was then digested with NgoMI and BstEII and was
used to replace the WT sequence of the pUC-E4 plasmid. To incorporate
the PCR-generated mutations to exon IV of the apoE gene, a two-step
procedure was followed. The EcoRI fragment of the human
apoE4 gene, which includes the entire exon IV sequence, was cloned into
the EcoRI site of the pBS vector to generate the vector
pBlue-exIV. The pUC-apoE4-202 plasmid was digested with
StyI/BbsI, and the mutated sequence was exchanged
for the WT sequence of the pBlue-E4-exIV plasmid to generate plasmid
pBlue-E4-202-exIV plasmid.
The recombinant viruses were constructed using the Ad-Easy-1 system
where recombinant adenovirus construct is generated in bacteria BJ-5183
cells (42). The 1507-bp MscI-EcoRI fragment of
apoE genomic DNA (nucleotides 1853-3360), which contains exons 2 and
3, was cloned into the SmaI-EcoRI sites of pGEM7
vector, resulting in the pGEM7-apoE-Ex II, III vector. The 1911-bp
EcoRI fragment of apoE4 or apoE4-202 gene (that contains
the stop mutation at codon 203) was then excised from pBlue-E4-Ex IV or
the pBlue-E4-202-Ex IV vector, respectively, and cloned into the
EcoRI site of the pGEM7-apoE-Ex II, III vector. This
generated the pGEM7-apoE4g or pGEM7-apoE4g-202 vector, respectively,
that contains exons II-IV of the apoE gene. The correct orientation of
the 1911-bp EcoRI insert was checked by restriction digest
with NotI and XbaI. The entire
HindIII-XbaI fragment from pGEM7-apoE4g or
pGEM7-apoE4-202-g vector was cloned into the corresponding sites of
the pAd Track-cytomegalovirus adenovirus shuttle plasmid. The
recombinant vector was used to electroporate BJ 5183 Escherichia
coli cells along with the pAd Easy-1 helper vector. pAdEasy-1
contains the viral genome and the long terminal repeats of the
adenovirus and allows for the formation by homologous recombination of
the recombinant virus containing the gene of interest. The vector also
contains the green fluorescence protein gene, which enables detection
of the infection of cells and tissues by their green fluorescence.
Recombinant bacterial clones resistant to kanamycin were selected and
screened for the presence of the gene of interest by restriction
endonuclease analysis and DNA sequencing. The viruses expressing WT
apoE4 and apoE4-202 forms are designated as AdGFP-E4 and
AdGFP-E4-202, respectively. Correct clones were propagated in RecA
DH5 Cell Culture Studies--
Human HTB13 cells (SW1783, human
astrocytoma) grown to confluence in medium containing 10% fetal calf
serum were infected with AdGFP-E4 or AdGFP-E4-202, at a multiplicity
of infection of 20. Twenty four hours post-infection, cells were washed
twice with phosphate-buffered saline (PBS) and preincubated in
serum-free medium for 2 h. Following an additional wash with PBS,
fresh serum-free medium was added. After 24 h of incubation,
medium was collected and analyzed by enzyme linked immunoabsorbent
assay (ELISA) and SDS-PAGE for apoE expression. In some experiments,
60-mm diameter cultures were labeled metabolically with 0.5 mCi of
[35S]methionine for 2 h, in which case medium was
collected 2 h after addition of the radiolabeled amino acid for
further analysis.
Association of ApoE Secreted into the Culture Medium of Cells
with Plasma Lipoproteins following Density Gradient
Ultracentrifugation--
For this analysis, following the labeling of
cells with [35S]methionine, 1 ml of medium from one
100-mm diameter dish was mixed with 1 ml of lipoprotein fractions of
densities (d) in the range of 1.006-1.21 g/ml, which were
previously separated from plasma by density gradient
ultracentrifugation, the mixture was adjusted to density 1.24 with KBr
and overlaid with 4 ml each of KBr solutions of d = 1.20 and 1.06, and 2 ml of KBr solution of d = 1.019 g/ml. The mixture was centrifuged for 24 h in a SW-41 rotor at
32,000 rpm. Following ultracentrifugation, 12 1-ml fractions were
collected and analyzed by SDS-PAGE and autoradiography. This analysis
showed that both apoE4 and apoE4-202 can associate with exogenous
lipoproteins that float in the IDL to HDL region.
Isolation of VLDL by Density Gradient
Ultracentrifugation--
One ml of serum sample obtained from apoE-
and LDLR-deficient mice were overlaid on a KBr density composed of 1 ml
of 1.21 g/ml KBr, 1 ml of 1.063 g/ml KBr, 1 ml of 1.019 g/ml KBr, and 1 ml of saline. Samples were subjected to ultracentrifugation at 30,000 rpm in an SW-55 rotor for 16 h, and then the top 1 ml of the
gradient containing the VLDL fraction was isolated.
Binding of ApoE to VLDL Particles--
Two hundred fifty
microliters of VLDL, isolated from the plasma of apoE-deficient mice,
was mixed with 750 µl of culture medium containing 15 µg of apoE4
or apoE4-202 secreted by HTB cells that were infected with AdGFP-E4 or
AdGFP-E4-202, respectively, and the mixtures were brought to a final
volume of 1 ml with saline. Mixtures were incubated on a shaker at
37 °C for 30 min and then subjected to density gradient
centrifugation to separate free apoE from the VLDL-bound apoE, as
described in the VLDL purification step above. Then the apoE-enriched
VLDL and free apoE fractions were isolated and analyzed for apoE
concentration by SDS-PAGE and immunoblotting.
Animal Studies--
Female apoE-deficient mice 20-25 weeks old
were used in these studies (44). Groups of mice were formed based on
their plasma cholesterol and triglyceride levels before initiation of
the experiments to ensure similar mean cholesterol and triglyceride
levels in each group. The mice were injected intravenously through the
tail vein with doses ranging from 5 × 108 to 1 × 1010 pfu of AdGFP (control adenovirus), AdGFP-E4, or
AdGFP-E4-202 virus, as indicated. Each group contained 8-10 mice.
Blood was obtained from the tail vein or retro-orbital plexus after a
4-h fast preceding adenoviral injection. On the indicated days after injection (0, 4 5, 8, and 12), blood was collected into a CB300 or
CB1000 blood collection tube (Sarstedt). Aliquots of plasma were stored
at 4 and FPLC Analysis--
For FPLC analysis of serum samples, 12 µl
of serum were diluted 1:5 with PBS. Then sample was loaded onto a
Sepharose 6 column in a SMART micro FPLC system (Amersham Pharmacia
Biotech) and eluted with PBS. A total of 25 fractions of 50-µl volume
each were collected for further analysis.
Triglyceride and Cholesterol Analysis--
Ten µl of serum
sample were diluted with 40 µl of phosphate-buffered saline (PBS),
and 7.5 µl of the dilute sample were analyzed for triglycerides and
cholesterol using the GPO-Trinder Kit (Sigma) and CHOL-MPR3 kit (Roche
Molecular Biochemicals), according to the manufacturer's instructions.
Triglyceride and cholesterol concentrations were determined
spectrophotometrically at 540 and 492 nm, respectively. Triglyceride
analysis of the FPLC fractions was performed using the TG Buffer
(Sigma), and concentrations were determined spectrophotometrically at
492 nm according to the manufacturer's instructions. Cholesterol
analysis of the FPLC fractions was performed as described above for the
serum samples.
Quantification of Human ApoE--
Serum human apoE4
concentrations were measured by using sandwich ELISA (45).
Affinity-purified polyclonal goat anti-human apoE antibodies were used
for coating microtiter plates, and polyclonal goat anti-human apoE
coupled to horseradish peroxidase was used as the secondary antibody.
The immunoperoxidase procedure was employed for the colorimetric
detection of apoE at 450 nm, using tetramethylbenzidine as substrate.
Pooled plasma from healthy human subjects with known apoE level was
used as a standard.
RNA Isolation and Hybridization Analysis--
Total RNA was
isolated from livers of mice five days post infection using the RNA
Easy solution (RNA Insta-Pure, Eurogentec Belgium) according to the
manufacturer's instructions. For Northern blot analysis, RNA samples
(15 µg) were denatured and separated by electrophoresis on 1.0%
formaldehyde-agarose gels. RNA was stained with ethidium bromide to
verify integrity and equal loading and then transferred to GeneScreen
Plus (PerkinElmer Life Sciences). RNA was cross-linked to the membrane
by UV irradiation (Stratalinker, Stratagene) at 0.12 J/cm2
for 30 s. Probes, prepared by random priming, were used as
described previously, and 2.0 × 106 cpm/ml
32P-labeled DNA was employed. Quantitation by scanning
densitometry was performed using a Molecular Dynamics PhosphorImager
(model 400B). ApoE mRNA expression was normalized for GAPDH
mRNA levels and reported in the form of a bar graph. Experiments
were performed in triplicate, and data are reported as mean value ± S.D.
Rate of VLDL-Triglyceride Production in Mice Infected with
Different ApoE Forms--
To determine the effects of the apoE
truncations on hepatic VLDL-triglyceride secretion, four apoE-deficient
mice for each group were infected with a dose of 2 × 109 pfu of AdGFP-E4 and 1 × 1010 pfu of
the AdGFP-E4-202 or the control AdGFP viruses, respectively. Four days
post-infection, mice were fasted for 4 h and then injected with
Triton WR1339 at a dose of 500 mg/kg of body weight, using a 15%
solution (w/v) in 0.9% NaCl (Triton WR1339 has been shown to inhibit
completely VLDL catabolism (46)). Serum samples were isolated 5, 10, 20, 30, 40, 50, and 60 min after injection with Triton WR1339. As
control, serum samples were isolated 1 min immediately after the
injection with the detergent. Serum triglyceride levels were determined
as described under the "Experimental Procedures," and a linear
graph of serum triglyceride versus time was generated. The
rate of VLDL-triglyceride secretion expressed in mg/dl/min was
calculated from the slope of the linear graph for each individual mouse. Then slopes were grouped together and reported as mean ± S.D. in the form of a bar graph.
Expression and Flotation Properties of ApoE Secreted by HTB-13 Cell
Cultures Infected with Control and Recombinant Adenovirus Containing
ApoE4 or ApoE4-202--
HTB-13 cells that do not synthesize
endogenous apoE were infected with recombinant adenoviruses expressing
apoE4 or apoE4-202, designated AdGFP-E4 and AdGFP-E4-202,
respectively, at a multiplicity of infection of 20. Analysis of the
culture medium by SDS-PAGE and sandwich ELISA showed that both apoE4
and apoE4-202 are secreted efficiently at comparable levels in the
range of 60-80 µg of apoE per ml 24 h after infection with
AdGFP-E4 or AdGFP-E4-202. To study the flotation properties of apoE4
and apoE4-202, 100-mm diameter dishes of HTB-13 cultures that were
infected with AdGFP-E4 or AdGFP-E4-202, respectively, were
metabolically labeled with 0.5 mCi of [35S]methionine for
2 h, and then 1 ml of medium from each dish was mixed with ~1 mg
of lipoprotein fractions of densities (d) in the range of
1.006-1.21 g/ml, which were previously separated from plasma by
density gradient ultracentrifugation. The mixture was subjected again
to density gradient ultracentrifugation, and 12 fractions were
collected and fractionated on SDS-PAGE, followed by autoradiography.
This analysis showed that the majority of apoE4 and a considerable
fraction of apoE4-202 floats in the d > 1.21 g/ml
lipoprotein fraction, although a large quantity of apoE-202 was also
found in the lipid-poor or lipid-free fraction (Fig.
1A). To establish further the
ability of apoE4 and apoE4-202 to associate with VLDL, 15 µg of
apoE4 or apoE4-202 were mixed with VLDL fractions isolated from the
plasma of apoE- and LDLR-deficient mice by ultracentrifugation, and the
mixtures were incubated at 37 °C for 30 min. The mixture was then
subjected to density gradient ultracentrifugation. The amounts of the
free and lipoprotein-associated apoE were assessed by fractionation on
SDS-PAGE followed by Western blot analysis for apoE. As shown in Fig.
1B, both the full-length apoE4 and the truncated apoE4-202
associate with particles with densities in the VLDL to LDL region.
ApoE4 in this analysis is found in the VLDL to IDL region, and
apoE4-202 appears to associate with particles in the VLDL to
HDL3 region. The data indicate that both the wild-type apoE4 as
well as the truncated apoE form, apoE4-202, have the ability to
associate with pre-existing lipoprotein particles, a process that is
required for receptor-mediated lipoprotein clearance.
The Carboxyl-terminal 203-299 Segment of ApoE Contributes to
Hypertriglyceridemia in ApoE-deficient Mice--
To assess the effects
of apoE4 and apoE4-202 on hyperlipidemia in vivo,
apoE-deficient mice (E The Hepatic ApoE4 and ApoE4-202 mRNA Levels Are Similar in
Infected Mice, under Conditions of ApoE4-induced
Hypertriglyceridemia--
To assess the expression of apoE4 and
apoE4-202 in the mice infected with AdGFP-E4 and AdGFP-E4-202,
respectively, at least three infected mice from each group were
sacrificed on day 5 post-infection, and their livers were collected.
Total RNA was isolated from these livers and analyzed for apoE mRNA
levels by Northern blot analysis. In agreement with cell culture data,
where we see similar levels of apoE4 and apoE4-202 protein expression
following adenovirus infection, the apoE mRNA levels in mice
infected with a dose of 2 × 109 pfu AdGFP-E4 are
similar to those in mice infected with either 2 × 109
pfu or 1 × 1010 pfu AdGFP-E4-202 (Fig.
3, A and B).
However, apoE4-202 clears efficiently the cholesterol from the plasma
of E Cholesterol, Triglyceride, and ApoE FPLC Profiles of Plasma
Isolated from Mice Infected with AdGFP-E4, AdGFP-E4-202, or the
Control Virus AdGFP--
FPLC analysis of plasma from
adenovirus-infected mice showed that in mice expressing apoE4 5 days
post-infection, cholesterol levels were high. Approximately 70% of
cholesterol was distributed in VLDL and ~20% in HDL. On day 8 post-infection, the ratio of VLDL/HDL cholesterol was ~1:1 to 2:1
(Fig. 4A, upper
panel). In mice infected with AdGFP-E4-202, cholesterol was
distributed in the VLDL and HDL, and the ratio VLDL cholesterol to HDL
cholesterol was ~1:1 on either day, when the dose of adenovirus used
was either 2 × 109 or 1010 pfu (Fig.
4A, lower panels). In mice infected with
AdGFP-E4, as expected, triglyceride levels were very high in the VLDL
fractions and barely detectable in the rest of the lipoprotein
fractions, whereas in mice infected with AdGFP-E4-202 triglyceride
levels were very low in all the lipoprotein fractions either 5 or 8 days post-infection (Fig. 4B). As an additional control,
infection with 2 × 109 pfu of the control virus
AdGFP, did not result in any change in the cholesterol and triglyceride
profiles of the apoE-deficient mice (data not shown).
Analysis of FPLC fractions by sandwich ELISA showed that in mice
infected with 2 × 109 pfu AdGFP-E4, 5 days
post-infection ~50% of the total apoE was distributed in HDL and
25% in VLDL, and the remaining apoE was distributed across the other
FPLC fractions. In contrast, in mice infected with 1010 pfu
AdGFP-E4-202, apoE was uniformly distributed in all lipoprotein fractions (Fig. 5). The levels of apoE4
and apoE4-202 are similar in fractions 22-25, which represent the
lipid-free apoE form (Fig. 5). This indicates that similar steady-state
levels of lipid-free apoE4 and apoE4-202 exist in the plasma of mice
infected with 2 × 109 pfu AdGFP-E4 and
1010 pfu AdGFP-E4-202, respectively. The average total
plasma apoE levels, based on a pool of plasma of five mice, were 315 µg/ml for apoE4 and 13 µg/ml for apoE4-202. The apparent lower
concentration of apoE4-202 in plasma and in the lipoprotein-containing
fractions most likely reflects the efficient catabolism of the
apoE4-202-containing lipoproteins.
Wild-type ApoE4 Increases Significantly the Rate of Hepatic
VLDL-triglyceride Production as Opposed to the Truncated Form
ApoE4-202 and the Control AdGFP Virus--
The rate of
VLDL-triglyceride secretion in the plasma was determined following
injection of Triton WR1339 5 days after the infection with the
recombinant adenoviruses. It was found that the rate of triglyceride
secretion decreased ~50% in mice infected with 1 × 1010 pfu of AdGFP-E4-202 and increased 10-fold in mice
infected with 2 × 109 pfu of AdGFP-E4, as compared
with mice infected with 1 × 1010 pfu of the control
AdGFP adenovirus (Fig. 6). The findings
suggest that the carboxyl-terminal region of apoE influences the rate of VLDL-triglyceride secretion and contributes to apoE-induced hypertriglyceridemia.
ApoE promotes receptor-mediated catabolism of apoE-containing
lipoproteins by cell receptors (1-5). Early studies showed that
mutation in apoE within the vicinity of amino acids 130-160 affected
the recognition of this protein by the LDL receptor and resulted in
dominant or recessive forms of type III hyperlipoproteinemia and
premature atherosclerosis (6-13, 47-49). Two additional functions of
apoE pertinent to triglyceride homeostasis were suggested by the
analysis of human subjects and transgenic animals expressing different
levels of apoE. The first function of apoE is related to secretion of
VLDL-triglycerides (35, 39, 40, 50), and the second is related to the
inhibition of lipolysis (25, 27, 35-38, 51). Both processes are
expected to increase the plasma triglyceride levels in humans and in
experimental animals (25-27, 35-38, 51). To dissect these apoE
functions, we have used adenovirus-mediated gene transfer to express at
similar levels either the full-length apoE4 or the truncated apoE4-202
form in apoE-deficient mice. The mice were analyzed 4-8 days
post-infection for apoE expression, plasma lipid and lipoprotein
profiles, and mechanisms that impede the clearance of plasma
cholesterol and triglycerides when the full-length apoE4 is overexpressed.
The Amino-terminal Segment 1-202 of ApoE Can Associate with
Lipoproteins and Direct Their Catabolism in Vivo
Receptor-mediated catabolism of apoE requires association of apoE
with lipoprotein particles, whereas lipid-free apoE does not bind to
lipoprotein receptors (1-6). Heparan sulfate proteoglycans may also be
involved in apoE binding on the cell surface (52-55). Domains of apoE
involved in receptor binding (47-49, 56), heparin binding (57-58),
and lipid and lipoprotein binding (59, 60) have been identified
in vitro. The receptor binding domain is found between
residues 136 and 152, whereas neighboring residues may also indirectly
affect receptor binding (48, 49). The amino acids 142-147 of the
receptor binding domain are also involved in heparin binding. Two other
heparin binding domains were found between residues 211 and 218 and 243 and 272 (57, 58). It has been proposed that binding of apoE-containing
lipoproteins to heparan sulfate proteoglycans contributes to their
subsequent internalization with or without the participation of
LRP (2, 6, 55). Other studies suggested that the region of apoE
between residues 244 and 299 contributes to the binding of apoE to
lipids and lipoproteins, whereas the amino-terminal region of apoE
lacks the determinants required for association with lipoproteins (60). In addition, residues 267-299 were found to be important for the tetramerization of apoE (60).
The present study refines the domains of apoE required for its
association with lipoproteins, a process required for their in
vivo clearance. Our findings establish that the amino-terminal residues 1-202 of apoE contain the domains necessary for the clearance of cholesteryl ester-rich lipoprotein remnants in vivo. This
implies that the region 1-202 of apoE contains the necessary
determinants for the association of apoE with these lipoprotein
remnants in vitro. This conclusion is supported by in
vitro data in the present study, showing that apoE4 and apoE4-202
remain associated with VLDL and LDL particles following
ultracentrifugation. The clearance of lipoprotein remnants by
apoE4-202, which contains only the 142-147 heparin binding domains,
is extremely efficient. Previous studies showed that the 243-272
heparin binding domain of apoE is shielded when apoE is bound to
lipids and lipoproteins and thus is inactive as a ligand for heparan
sulfate proteoglycans (57). It is possible that the major route of
clearance of the lipoproteins containing apoE4-202, which contains
only the 142-147 heparin binding domain, is via the LDL receptor and
LRP pathways without the involvement of the heparan sulfate
proteoglycan pathway. The potential participation of the LRP and
the LDL receptor in the clearance of apoE4-202-containing remnants may
make their uptake and the subsequent cholesterol clearance more
efficient and thus may account for the observed efficiency of
apoE4-202 in cholesterol clearance. This hypothesis is currently under investigation.
The Carboxyl-terminal Domain of ApoE Contributes to
Hypertriglyceridemia
A very important finding of this study is that removal of the
carboxyl-terminal region 203-299 of apoE4 prevents the development of
hypertriglyceridemia following adenoviral infection. Several control
experiments were performed to confirm that the different properties of
apoE4 and apoE4-202 are not the result of differences in expression,
secretion, or association with VLDL. Northern blot analysis of total
RNA has established unequivocally that the steady-state apoE mRNA
levels in mice overexpressing apoE4 that result in hypertriglyceridemia and the mRNA levels in mice overexpressing apoE4-202 that do not cause hypertriglyceridemia are very similar (Fig. 3, A-D).
This analysis indicates that it is unlikely that decreased expression of the truncated apoE forms is responsible for this effect. In addition, cell culture experiments showed that C127 (mouse mammary tumor) cell lines stably transfected and expressing apoE4 or apoE4-202 (data not shown), or HTB-13 cell cultures infected with recombinant adenovirus, secrete similar amounts of apoE4 and apoE4-202. These observations indicate that the truncated apoE4-202 form is stable and
is secreted as efficiently as its wild-type apoE4 counterpart.
Mechanisms of ApoE-induced Hypertriglyceridemia
Aside from differences in synthesis, secretion, and VLDL
association between apoE4 and apoE4-202, other factors that could have
contributed to the apoE-induced hypertriglyceridemia are increased
hepatic VLDL-triglyceride synthesis, decreased catabolism of apoE4 as
compared with apoE4-202-containing particles, and a combination of
both processes.
Increased Hepatic VLDL-Triglyceride Secretion Promoted by the
Carboxyl-terminal 203-99 Residues of ApoE4--
The present study
shows that the hepatic VLDL-triglyceride secretion, following injection
of Triton WR1339, decreases by 50% in mice expressing apoE4-202 and
increases 10-fold in mice expressing apoE4, as compared with the mice
infected with the control virus AdGFP (Fig. 6). This implies that apoE4
contributes to hypertriglyceridemia, at least partially, by increasing
VLDL-triglyceride secretion. The hypertriglyceridemic VLDL may be a
poor ligand for apoE-recognizing receptors, despite the fact that it is
enriched with apoE. A previous study also showed that VLDL-triglyceride
secretion is reduced by 50% in E Increased VLDL Catabolism Is Mediated by Truncated ApoE Forms and
Impediment of Triglyceride Hydrolysis Is Caused by Overexpression of
ApoE--
Work by others (60) has shown that injection of two
125I-truncated apoE forms extending from residues 1 to 191 and 1 to 244, respectively, in rabbits, resulted in their fast and very
efficient removal from plasma. These observations are consistent with
the finding of this study, which shows that apoE4-202 contributes to
the efficient clearance of apoE-containing lipoprotein remnants in vivo. The efficiency of apoE4-202-mediated clearance of
lipoprotein remnants also results in the concomitant clearance of the
apoE molecules, thus resulting in lower levels of steady-state plasma apoE4-202 as observed in this study. The steady-state levels of apoE
in plasma are a function of apoE synthesis, secretion, and receptor-mediated clearance that requires association of apoE with
lipoproteins. As shown in Figs. 2 and 4, mice expressing WT apoE4 do
not clear VLDL particles, and this results in the accumulation of
VLDL-associated apoE in the plasma of those mice (Fig. 5). In contrast,
mice expressing the truncated forms apoE4-202 clear VLDL
particles very efficiently, and this results in decreased cholesterol
and triglyceride and apoE levels in the plasma of apoE4-202-expressing
mice (Figs. 2, A and B and 5).
Aside from the intracellular effect of apoE on VLDL-triglyceride
secretion that we observe, other studies (35, 36) have shown that
excess of secreted apoE may displace partially the lipoprotein lipase
or apoCII and thus reduce lipolysis. Preliminary analysis of the
apoprotein composition of VLDL particles obtained from AdGFP-E4- and
AdGFP-E4-202-infected mice by SDS-PAGE showed that other proteins
present in triglyceride-rich VLDL, such as apoA-IV and apoA-I, are also
displaced by full-length apoE4 but not by apoE-202 (data not shown).
Analysis of the apoE structure by x-ray crystallography and computer
modeling showed that the amino-terminal domains of apoE contain
antiparallel helices (61, 62). It is possible that these amino-terminal
helices extending from residues 23 to 185 bind to specific sites on the
lipoprotein surface along with other protein particles. At a critical
apoE concentration these apoE sites may be saturated, and the clearance of apoE-containing lipoproteins may be optimized. With further increases in plasma apoE concentration, specific displacement or
inhibition of the function of other critical protein components of the
triglyceride-rich lipoproteins may take place by the
carboxyl-terminal helices, found within the 203-299 region of apoE. To
the extent that lipoprotein lipase and apoCII are affected by the
excess of apoE, one may expect that the rate of lipolysis of these
particles will be reduced and the plasma triglyceride levels will rise.
Overall, the current study indicates that the amino-terminal 1-202
residues of apoE are sufficient for binding to lipoprotein remnants to
an extent that promotes their efficient clearance in vivo,
whereas the carboxyl-terminal 203-299 region of apoE contributes to
hypertriglyceridemia. At least part of the hypertriglyceridemic effect
of apoE results from increased VLDL-triglyceride secretion. This
hypertriglyceridemic VLDL may be recognized poorly by apoE receptors.
Hypertriglyceridemia may be further exacerbated by diminished lipolysis
of VLDL. The identification of amino acid residues within the
carboxyl-terminal region of apoE, which mediate the
hypertriglyceridemic effect of apoE, is the subject of ongoing research.
Therapeutic Potential of Truncated ApoE Forms
Expression of apoE within a physiological range clears lipoprotein
remnants, whereas overexpression results in hypertriglyceridemia. The
undesirable side effect resulting from apoE overexpression diminishes
significantly the therapeutic value of apoE. The inability of the
truncated apoE form that lacks the carboxyl-terminal 203-299 region to
induce hypertriglyceridemia, as described in this research, coupled
with its intact ability to clear cholesterol, makes it an attractive
candidate in future gene therapy approaches to correct remnant removal disorders.
/
) to define the domains of apoE
required for cholesterol and triglyceride homeostasis in
vivo. A dose of 2 × 109 plaque-forming units of
apoE4-expressing adenovirus reduced slightly the cholesterol levels of
E
/
mice and resulted in severe
hypertriglyceridemia, due to accumulation of cholesterol and
triglyceride-rich very low density lipoprotein particles in plasma.
In contrast, the truncated form apoE4-202 resulted in a 90% reduction
in the plasma cholesterol levels but did not alter plasma triglyceride
levels in the E
/
mice. ApoE secretion by
cell cultures, as well as the steady-state hepatic mRNA levels in
individual mice expressing apoE4 or apoE4-202, were similar. In
contrast, very low density lipoprotein-triglyceride secretion in mice
expressing apoE4, but not apoE4-202, was increased 10-fold, as
compared with mice infected with a control adenovirus. The findings
suggest that the amino-terminal 1-202 region of apoE4 contains
the domains required for the in vivo clearance of
lipoprotein remnants. Furthermore, the carboxyl-terminal 203-299
residues of apoE promote hepatic very low density
lipoprotein-triglyceride secretion and contribute to apoE-induced hypertriglyceridemia.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Oligonucleotides used in overlap extension PCR
cells. The recombinant vector was linearized with
PacI and used to infect 911 cells. The subsequent steps
involved in the generation and expansion of recombinant adenoviruses
were plaque identification/isolation followed by infection and
expansion in 911 cells (43). These steps were followed by a
purification process involving CsCl ultracentrifugation performed
twice, followed by dialysis and titration of the virus. Usually, titers
of ~5 × 1010 pfu/ml were obtained.
20 °C. One or more animals from each group was sacrificed
on each of the indicated days so that mRNA expression in the mouse
liver could be analyzed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (51K):
[in a new window]
Fig. 1.
A and B,
flotation properties of apoE4 and apoE4-202. A,
separation by density gradient ultracentrifugation of apoE-containing
lipoprotein secreted by HTB-13 cells infected at a multiplicity of
infection of 20 with apoE-expressing recombinant adenoviruses. The
figure shows autoradiograms of different density fractions analyzed by
SDS-PAGE, following density gradient ultracentrifugation in the
presence of purified lipoproteins as described under "Experimental
Procedures." (Mr indicates proteins of
known molecular mass purchased from New England Biolabs). The densities
of the different fractions are as indicated. B, Western blot
analysis of apoE4 and apoE4-202 fractions bound to VLDL particles from
apoE and LDLR double-deficient mice. Approximately 15 µg of apoE4 and
apoE4-202 secreted by adenovirus-infected HTB-13 cells was mixed with
VLDL isolated from plasma of apoE and LDLR double-deficient mice and
incubated at 37 °C for 30 min. Then the VLDL-associated apoE was
separated from free apoE by density gradient ultracentrifugation and
analyzed by Western blotting analysis as described under
"Experimental Procedures."
/
) were infected with
the recombinant adenoviruses AdGFP-E4 or AdGFP-E4-202, respectively.
To assess potential nonspecific effects of virus infection, some mice
were infected with the control AdGFP virus. Analysis of plasma lipid
levels showed that the infection of mice with 2 × 109
pfu of the apoE4-adenovirus did not result in a significant reduction in the plasma cholesterol levels, as compared with the cholesterol levels of mice infected with the control virus and non-infected mice
(Fig. 2A). In addition,
infection with 2 × 109 pfu of the apoE4 adenovirus
induced severe hypertriglyceridemia (Fig. 2B). When
expression of apoE4 on day 8 post-infection was reduced or when the
dose used for infection was decreased to 5 × 108 pfu,
hypertriglyceridemia was also reduced or eliminated (Fig. 2,
A and B, right side). In contrast to the mice
infected with the adenovirus expressing the wild-type E4, mice that
were infected with 2 × 109 or even 1010
pfu of the adGFP-E4-202 adenovirus that expresses the truncated form
apoE4-202 had normal cholesterol levels and did not develop hypertriglyceridemia (Fig. 2, A and B, middle
part). The control virus AdGFP did not appear to have significant
effects on the cholesterol and triglyceride levels as compared with the
levels of non-infected apoE-deficient mice, ruling out the possibility of nonspecific effects of the infection process (Fig. 2, A
and B, left side).
View larger version (15K):
[in a new window]
Fig. 2.
A and B,
cholesterol (A) and triglyceride
(B) levels of
E /
mice
infected with the control adenovirus AdGFP or recombinant adenoviruses
expressing apoE4 or apoE4-202. Mice were infected in triplicate
with the indicated doses of recombinant virus, and serum samples were
isolated and analyzed for cholesterol (A) and triglyceride
levels (B) on the indicated days after infection as
described under "Experimental Procedures."
/
mice without causing
hypertriglyceridemia, whereas the full-length apoE4 does not clear
cholesterol from the plasma of E
/
mice and
causes hypertriglyceridemia (Fig. 3, C and D).
Thus, the different effects of apoE4 and apoE4-202 on
hypertriglyceridemia most likely are not due to different levels of
expression of these two apoE forms.
View larger version (23K):
[in a new window]
Fig. 3.
A-D, correlation of hepatic apoE
mRNA levels with plasma cholesterol and triglyceride levels of
individual mice infected with apoE4- or apoE4-202-expressing
adenoviruses. Total RNA was isolated from livers of infected mice
five days after infection and analyzed by Northern blotting for the
expression of apoE and GAPDH mRNA. A shows
representative autoradiograms of Northern blot analysis of total RNA
isolated from livers of mice infected with the indicated dose of the
recombinant adenoviruses expressing apoE4 or apoE4-202. B
shows apoE mRNA levels quantified by PhosphorImager using the
ImageQuant program (version 4.2A), were normalized for GAPDH mRNA
levels and reported in the format of a bar graph for each
mouse. C shows cholesterol levels of the individual mice
expressed in mg/dl. D shows triglyceride levels of the
individual mice expressed in mg/dl.
View larger version (19K):
[in a new window]
Fig. 4.
A and B,
FPLC profiles of cholesterol (A) and
triglycerides (B) of mice infected with apoE4- or
apoE4-202-expressing adenoviruses. Serum samples obtained from
mice infected with 2 × 109 of the control virus
AdGFP, or the recombinant adenoviruses expressing AdGFP-E4 or
AdGFP-E4-202 on days 5 and 8 post-infection, were fractionated by
FPLC, and then the cholesterol and triglycerides levels of each FPLC
fraction were determined as described under "Experimental
Procedures."
View larger version (11K):
[in a new window]
Fig. 5.
FPLC profile of plasma apoE4 in mice infected
with apoE4- or apoE4-202-expressing adenoviruses. Serum samples
from mice infected with either 2 × 109 pfu
apoE4-expressing adenovirus or 1 × 1010 pfu
apoE4-202-expressing adenoviruses were obtained 5 days post-infection.
ApoE was quantified by sandwich ELISA as described under
"Experimental Procedures."
View larger version (9K):
[in a new window]
Fig. 6.
Hepatic VLDL-triglyceride production analysis
in mice infected with 1 × 1010 pfu AdGFP, or 2 × 1010 pfu AdGFP-E4, or 1 × 1010 pfu
AdGFP-E4-202. Triton WR1339 (500 mg/kg body weight) was injected
into three fasted mice per virus group. Serum samples were collected at
20, 40, and 60 min after the injection with the detergent. As control,
serum samples were isolated 1 min immediately after the injection with
the detergent. Serum triglyceride levels were determined, and a linear
graph of serum triglyceride concentration versus time was
generated. The rate of VLDL-triglyceride secretion expressed in
mg/dl/min was calculated from the slope of the linear graph for each
individual mouse. The bar graph represents the mean ± S.D. of the individual rates of VLDL-triglyceride production per virus
group.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice (40). The findings imply that the carboxyl-terminal
residues 203-299 of apoE may be directly involved in intracellular
assembly and secretion of VLDL-triglycerides.
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ACKNOWLEDGEMENTS |
---|
We thank Markella Zanni for editorial suggestions and comments, Anne Plunkett for secretarial assistance, and Pamela Morani and Hans van der Boom for technical assistance.
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FOOTNOTES |
---|
* This work was supported by Biomed Grants BMH4-CT98-3272 and BMH4-CT96-0898, by National Institutes of Health Grant AG12717, by Kos Pharmaceuticals (Miami, FL), and by Alzheimer Association Grant IRG 002220.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed: Whitaker
Cardiovascular Institute, Dept. of Medicine, Boston University School of Medicine, 715 Albany St., W509, Boston, MA 02118-2394. Fax: 617-638-5141; E-mail: vzannis@bu.edu.
Published, JBC Papers in Press, February 9, 2001, DOI 10.1074/jbc.M100418200
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ABBREVIATIONS |
---|
The abbreviations used are: apo, apolipoprotein; Ad, adenovirus; ELISA, enzyme-linked immunosorbent assay; FPLC, fast pressure liquid chromatography; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescence protein; HDL, high density lipoprotein; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; VLDL, very low density lipoprotein; pfu, plaque-forming units; PBS, phosphate-buffered saline; LDL, low density lipoprotein; LDLR, LDL receptor; LRP-LDL receptor-related protein, IDL, intermediate density lipoproteins; bp, base pair; WT, wild type.
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