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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 18, 2001 as doi:10.1096/fj.00-0882fje. |
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2
* Whitaker Cardiovascular Institute, Department of Medicine, Boston University School of Medicine, Boston Massachusetts 02118-2394, USA;
TNO-PG Prevention and Health, Leiden University Medical Center, Leiden, The Netherlands;
Department of Human and Clinical Genetics, Gaubius Laboratory, Leiden, The Netherlands; and
University of Crete, Department of Biochemistry and Institute of Molecular Biology and Biotechnology, Heraklion, Crete, Greece
2Correspondence: Whitaker Cardiovascular Institute, Department of Medicine, Boston University School of Medicine, 700 Albany St., W-509, Boston MA 02118-2394, USA. E-mail: vzannis{at}bu.edu
SPECIFIC AIMS
In this study we sought to dissect the domains of apoE required for cholesterol and triglyceride homeostasis in vivo using adenovirus-mediated gene transfer in apoE-deficient (E-/-) mice. Mice were infected with replication-deficient recombinant adenoviruses expressing the full-length apoE4 or the truncated apoE4–229 and apoE4–259 forms containing the amino acids 1–229 and 1–259, respectively. The ability of the amino- and carboxyl-terminal regions of apoE to associate with lipoproteins and promote receptor-mediated cholesterol clearance or cholesterol and triglyceride accumulation was assessed. Mechanisms were sought to explain the apoE-mediated cholesterol and triglyceride clearance by the truncated apoE forms and the apoE-induced hypertriglyceridemia by the full-length apoE.
PRINCIPAL FINDINGS
1. The full-length apoE4 and the truncated apoE forms apoE4–229
and apoE4–259 are secreted efficiently by cells and associate
similarly with VLDL particles
HTB-13 cells that do not synthesize endogenous apoE were infected
with recombinant adenoviruses expressing apoE4, apoE4–229, or
apoE4–259, designated AdGFP-E4, AdGFP-E4–229, and AdGFP-E4–259,
respectively, at a multiplicity of infection of 20. Analysis of the
culture medium by SDS-PAGE and sandwich ELISA showed that apoE4,
apoE4–229, and apoE4–259 are secreted efficiently at comparable
levels in the range of 60 to 80 µg of apoE/ml, respectively, 24 h postinfection.
To establish the ability of apoE4, apoE4–229, and apoE4–259 to associate with VLDL, 15 µg of apoE4, apoE4–229, or apoE4–259 was mixed and incubated at 37°C for 30 min with VLDL fractions isolated from the plasma of apoE-deficient mice by ultracentrifugation. The mixtures were then subjected to density gradient ultracentrifugation. The density fractions thus obtained were analyzed by SDS-PAGE and Western blotting using anti-apoE antibodies. It was found that both the full-length apoE4 and the truncated apoE4–229 and apoE4–259 forms associate with particles with densities in the VLDL and IDL region (data not shown).
2. The carboxyl-terminal 260–299 segment of apoE contributes to
hypertriglyceridemia in apoE-deficient mice
To assess the effects of apoE2, apoE3, apoE4, apoE4–229, or
apoE4–259 on hyperlipidemia in vivo, E-/- mice
were infected with either the control adenovirus AdGFP or the
recombinant adenoviruses expressing the full-length apoE2, or apoE3, or
apoE4 or the truncated forms apoE4–229 and apoE4–259 forms. Lipid
analysis showed that the infection of mice with 2 x
109 pfu of the apoE2- or apoE3-, or
apoE4-expressing adenovirus did not cause a significant reduction in
the plasma cholesterol levels and induced severe hypertriglyceridemia
compared to the mice infected with the control virus and noninfected
mice (Fig. 1A
, B
). In contrast, mice infected with adenoviruses
expressing apoE4–229 and apoE4–259 at doses of 2 x
109 or 4 x 109
displayed low cholesterol and triglyceride levels and did not develop
hypertriglyceridemia (Fig. 1A
, B
).
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3. The hepatic apoE4 and apoE4–229 and apoE4–259 mRNA levels are
similar in infected mice
To assess the expression of apoE4, apoE4–229, and apoE4–259 in
infected mice, at least three infected mice from each group were killed
5 days postinfection. Total RNA was isolated from the livers of these
mice and analyzed for apoE mRNA expression by Northern blotting.
The apoE mRNA levels in individual mice infected with a dose of 2 x 109 pfu AdGFP-E4 are similar to those in mice
infected with 4 x 109 pfu AdGFP-E4–229 or
AdGFP-E4–259 (Fig. 1C
). However, apoE4–229 and
apoE4–259 efficiently clears the cholesterol of apoE-deficient mice
without causing hypertriglyceridemia, as opposed to the full-length
apoE4 (Fig. 1A
, B
). Thus, the different effects of apoE4 and
apoE4–229 or apoE4–259 on hypertriglyceridemia most likely are not
due to different levels of expression and secretion of the full-length
and the truncated apoE forms.
4. ApoE4 overexpression results in accumulation of
triglyceride-rich VLDL particles whereas overexpression of apoE4–229
and apoE4–259 clears VLDL
FPLC analysis of plasma from adenovirus-infected mice showed that
in mice expressing apoE4 5–8 days postinfection, cholesterol and
triglyceride levels were high and were distributed predominantly in the
VLDL region (data not shown). In contrast, in mice expressing
apoE4–229 and apoE4–259 cholesterol and triglycerides 5 or 8 days
postinfection were low and were distributed in the VLDL region (data
not shown). As an additional control, infection of mice with
2 x 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).
5. Wild-type ApoE4 increases significantly the rate of
hepatic VLDL triglyceride production as opposed to the truncated form
apoE4–259 and the control AdGFP virus
The rate of VLDL triglyceride secretion in the plasma was
determined after injection of Triton WR1339 5 days after the infection
with the recombinant adenoviruses. We found that the rate of
triglyceride secretion increased 1.4-fold in mice infected with
AdGFP-E4–259 and 3.8-fold in mice infected with AdGFP-E4 vs. mice
infected with the control AdGFP adenovirus. The rate of VLDL
triglyceride secretion was 2.7-fold higher in mice infected with
AdGFP-E4 as opposed to mice infected with AdGFP-E4–259. The findings
suggest that the carboxyl-terminal region of apoE influences the rate
of VLDL triglyceride secretion. A model is proposed to account for the
formation and normal catabolism of chylomicrons and VLDL in mice
expressing the truncated apoE forms apoE4–229 and apoE4–259, and the
defective clearance of triglyceride-rich lipoproteins in mice
overexpressing the full-length apoE4 (Fig. 2
). The model indicates that overexpression of apoE is associated with
formation of triglyceride-rich lipoproteins, which cannot be cleared by
cell receptors. In contrast, normal chylomicrons and VLDL particles
formed in mice overexpressing apoE-229 or apoE-259 and were removed
efficiently by cell receptors, resulting in low plasma cholesterol and
triglyceride levels in these mice.
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CONCLUSIONS
1. The carboxyl-terminal domain 260–299 of apoE is required for
apoE-induced hypertriglyceridemia: potential mechanisms of
hypertriglyceridemia
An important finding of the present study is that without its
carboxyl-terminal region, which contains residues 260–299, apoE cannot
induce hypertriglyceridemia. Hypertriglyceridemia is independent of
apoE phenotype and is a mere consequence of overexpression of the
full-length apoE forms. We considered several factors that could have
contributed to the inability of apoE4–229, apoE4–259 to cause
hypertriglyceridemia. These include 1) decreased expression
or secretion of the truncated apoE forms (apoE2–229, apoE2–259),
2) inability of the truncated apoE forms to associate with
lipoprotein remnants, 3) a specific effect of the
carboxyl-terminal region 260–299 of apoE on VLDL triglyceride
secretion or with the clearance of triglyceride-rich lipoprotein
particles, 4) a combination of some of the above processes.
Northern blot analysis of total RNA has established unequivocally that
the steady-state apoE mRNA levels in individual mice expressing apoE4,
which display hypertriglyceridemia, and the mRNA levels in mice
expressing apoE4–229 or apoE4–259, which do not display
hypertriglyceridemia, are very similar (Fig. 1C
). This
finding indicates that it is unlikely that reduced expression of the
truncated apoE forms is responsible for the hypertriglyceridemic effect
of the full-length apoE. In addition, cell culture experiments showed
that the truncated apoE4–229 and apoE4–259 forms expressed in HTB13
cells or in permanent C127 cell lines (data not shown) are secreted as
efficiently as its wild-type apoE4 counterpart. The ability of the
carboxyl-terminal domain of apoE to affect the association of apoE with
VLDL was tested by density gradient separation of mixtures of VLDL
obtained from E-/- mice with apoE4, apoE4–229,
and apoE4–259 secreted by HTB-13 cells. This analysis showed that
apoE4, apoE4–229, and apoE4–259 associate similarly with VLDL.
Association of apoE with lipoprotein remnants is a prerequisite for the
clearance of apoE via cell receptors. Another factor that may affect
plasma triglyceride levels is the difference in the effects of apoE4 or
the truncated apoE4–229 and apoE4–259 on VLDL triglyceride secretion
and triglyceride lipolysis. We found that the VLDL triglyceride
secretion after injection of Triton-WR1339 increases slightly (
40%)
in mice expressing apoE4–259 and 3.7-fold in mice expressing apoE4
compared with control mice infected with the control virus AdGFP. This
implies that the ability of apoE4 to trigger hypertriglyceridemia may
be at least partially due to the increased VLDL-triglyceride secretion
caused by apoE4 as compared to apoE4–259.
The findings suggest that the carboxyl-terminal residues 260–299 of apoE may be directly involved in VLDL-triglyceride assembly and secretion. Aside from the intracellular effect of apoE on VLDL triglyceride secretion we observed, other studies have shown that excess of secreted apoE may displace partially the lipoprotein lipase or apoCII and thus reduce lipolysis. The observation that expression of the truncated apoE forms apoE4–229 and apoE4–259 to levels similar to those of full-length apoE4 does not cause hypertriglyceridemia may suggest that the carboxyl-terminal region of apoE may compete for the binding site of apoCII and lipoprotein lipase on the chylomicron and VLDL particles.
2. The amino-terminal 1–229 domain of apoE can associate with
lipoprotein remnants and direct their catabolism by cell receptors
The present study shows that the amino-terminal region 1–229 of
apoE contains the domains necessary for the association of apoE with
VLDL in vivo. Such an association is a prerequisite for remnant
clearance by cell receptors. It is possible that clearance of the
lipoproteins containing apoE4–229 and apoE4–259, which is mediated by
the LDL receptor and LRP pathways with or without the involvement of
the heparan sulfate proteoglycans, is facilitated when the
carboxyl-terminal residues of apoE are removed. This may account for
the efficient clearance of cholesterol from the plasma of
apoE-deficient mice infected with apoE4–229 or apoE4–259.
3. Therapeutic potential of the truncated apoE forms
One major parameter in successful gene therapy approaches is gene
dosage and expression levels. Consistent with previous findings in
humans and animal models, the current study shows that overexpression
of the full-length apoE causes hypertriglyceridemia. This undesirable
side effect diminishes significantly the therapeutic value of apoE. The
inability of the truncated apoE forms that lack all or part of the
carboxyl-terminal 260 to 299 region to induce
hypertriglyceridemia, coupled with their intact ability to clear
cholesterol, makes them attractive candidates in future gene therapy
applications to correct remnant removal disorders.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0882fje ; to cite this
article, use FASEB J. (May 18, 2001) 10.1096/fj.00-0882fje 
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