Differential effects of oxidized LDL on apolipoprotein AI and B synthesis
in HepG2 cells
Emmanuel Bourdona,1, Nadine Loreaua, Laurent Lagrosta, Jean Davignonb,
Lise Bernierb, Denis Blachea,⁎
aINSERM U498, Dijon, France;–Faculté de Médecine, Université de Bourgogne, 21079 Dijon, France
bHyperlipidemia and Atherosclerosis Research Group, Clinical Research Institute of Montreal, Montreal, Quebec, Canada
Received 20 December 2005; revised 5 May 2006; accepted 23 May 2006
Available online 3 June 2006
Oxidized low-density lipoproteins (Ox-LDL) are key elements in atherogenesis. Apolipoprotein AI (apoAI) is an active component of the
antiatherogenic high-density lipoproteins (HDL). In contrast, plasma apolipoprotein B (apoB), the main component of LDL, is highly correlated
with coronary risk. Our results, obtained in HepG2 cells, show that Ox-LDL, unlike native LDL, leads to opposite effects on apoB and apoAI,
namely a decrease in apoAI and an increase in apoB secretion as evaluated by [3H]leucine incorporation and specific immunoprecipitation.
Parallel pulse–chase studies show that Ox-LDL impaired apoB degradation, whereas apoAI degradation was increased and mRNA levels were
decreased. We also found that enhanced lipid biosynthesis of both triglycerides and cholesterol esters was involved in the Ox-LDL-induced
increase in apoB secretion. Our data suggest that the increase in apoB and decrease in apoAI secretion may in part contribute to the known
atherogenicity of Ox-LDL through an elevated LDL/HDL ratio, a strong predictor of coronary risk in patients.
© 2006 Elsevier Inc. All rights reserved.
Keywords: LDL oxidation; Lipid biosynthesis; Atherosclerosis; ApoAI; ApoB; LDL/HDL ratio; HepG2 cells; Free radicals
Population studies carried out in atherosclerotic patients
have largely established that a positive continuous linear
relationship exists between both serum low-density lipoprotein
(LDL)-cholesterol and apolipoprotein B100 (apoB) and the
extent of cardiovascular disease . LDL particles interact
with cells through LDL receptors and apoB works as a ligand
in this process . ApoB synthesis occurs in the liver and it is
involved in triglyceride-rich lipoprotein assembly . It is
well known that apoB overproduction occurs in diabetes,
insulin resistance, and coronary artery disease. High levels of
apoB-containing lipoproteins may be the result of an increased
production and/or a diminished catabolism. The resulting
increased residence time of LDL may increase its oxidiz-
ability. Accumulating data from biochemical, animal, and
epidemiological studies strongly support the hypothesis that
oxidative modification of LDL plays a crucial and causative
role in the pathogenesis of atherosclerosis [4–6].
Conversely, serum high-density lipoprotein (HDL)-choles-
terol and apoAI levels are negatively correlated with coronary
heart disease [7,8]. It is now clearly demonstrated that one of the
main protective effects of high HDL concentrations is related to
its involvement in reverse cholesterol transport, a system by
Free Radical Biology & Medicine 41 (2006) 786–796
Abbreviations: ACAT, acyl-CoA:cholesterol acyltransferase; BCA, bicin-
choninic acid; CE, cholesteryl ester; CVD, cardiovascular disease; DMEM,
Dulbecco’s minimum essential medium; EDTA, ethylenediamine tetraacetate;
N-LDL, Ac-LDL, and Ox-LDL, native, acetylated, and oxidized low-density
lipoproteins, respectively; EPA, eicosapentaenoic acid; FCS, fetal calf serum;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, gas chromatogra-
phy; LDH, D-lactic dehydrogenase; LPC, lysophosphatidylcholine; LPDS,
lipoprotein-depleted serum; Mops, 3-(N-morpholino)propanesulfonic acid;
PBS, phosphate-buffered saline; PIM, protease inhibitor mix; SDS–PAGE,
sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SSPE buffer, 150
mM sodium chloride, 10 mM sodium hydrogen phosphate, 1 mM EDTA, pH
7.4; TBARS, thiobarbituric acid-reactive substances; TG, triglycerides; TLC,
⁎Corresponding author. Fax: +33 380 39 3300.
E-mail address: email@example.com (D. Blache).
1Present address: LBGM, Université de la Réunion, BP 7151, 97715 Saint
Denis, Ile de La Réunion, France.
0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
which excess cholesterol is transported from peripheral tissues
by HDL to the liver for excretion [8,9]. ApoAI, as a major
component of HDL, is central to this process. Although the
intestine can also produce significant amounts of this
apolipoprotein, the liver is the main production site for apoAI.
Abundant data indicate that overexpression of apoAI results in
elevated HDL levels in mice and rabbits and inhibition of
atherosclerotic lesions. In addition to its major role in reverse
cholesterol transport, HDL has been reported to inhibit cell-
mediated LDL modifications  and to reduce cellular uptake
and degradation of native and oxidized LDL . HDL is also
capable of protecting against LDL peroxidation in vitro and in
An elevated LDL/HDL ratio is recognized as being a strong
predictor of coronary risk in patients. Low plasma levels of
HDL may be the result of abnormal apoAI biosynthesis and/or
increased degradation. In this context, it has been demonstrated
that in several pathological conditions oxidant stress alters LDL
and HDL levels through apoB and apoAI plasma metabolism,
respectively. Such a situation characterized by both increased
LDL oxidizability and occurrence of plasma oxidized LDL (Ox-
LDL) has been particularly encountered in hypothyroidism,
metabolic syndrome, and diabetes, perturbations largely known
and in diabetes, data from apoAI kinetics studies, conducted in
animal models and in humans, have established whether the
conditions occur due to a reduced production of apoAI, an
increased degradation, or both .
Consequently, the purpose of the present study was to
examine whether Ox-LDL would influence the synthesis and
secretion of apoAI and apoB using HepG2 cells. In previous
studies, we established that the liver synthesis of albumin was
strongly decreased by Ox-LDL . We found that Ox-LDL
drastically reduced the synthesis and secretion of apoAI,
whereas opposite effects were found for apoB. By means of
pulse–chase studies and Northern blot analyses and lipid
biosynthesis, we provide evidence indicating that Ox-LDL-
mediated differential effects on apoAI and apoB synthesis are
explained by different mechanisms. ApoB synthesis is mainly
driven by cholesterol supply, whereas reduced apoAI synthesis
may be linked to oxidation.
HepG2 cells were grown in a CO2incubator (5% CO2, 95%
air) in 75-cm2flasks (Polylabo, France). Cultures were
maintained in 20 ml Dulbecco’s minimum essential medium
(DMEM; Gibco) containing 10% fetal calf serum (FCS), 1.25%
L-glutamine, and 2% penicillin/streptomycin. Some 5 or 6 days
before each experiment, approximately 1,000,000 cells were
seeded in a six-well tissue cluster (Costar) in 3 ml DMEM
containing 10% FCS. Incubations were performed in 10%
lipoprotein-deficient FCS (LPDS) and with the indicated
amounts of LDL preparations or various agents. Cell viability
was assayed using lactate dehydrogenase (LDH) released into
the medium (Sigma Procedure 500) and expressed as the
percentage of total LDH activity in lysed cells.
LDL preparation and modification
LDLs (1019–1055 g/ml) were isolated by sequential
ultracentrifugation using KBr (Beckman centrifuge) of pooled
plasma from normolipidemic subjects . After dialysis
against phosphate-buffered saline (PBS), pH 7.4, LDLs were
assayed for protein content by the BCA method . Ox-LDLs
were obtained by incubating 100 μg/ml protein LDL with
25 μM CuSO4at 37°C overnight, which resulted in a medium
level of oxidation measured as conjugated dienes (absorbance at
234 nm in the ranges 0.5–0.7 and 0.3–0.5 for Ox-LDL and N-
LDL, respectively). Ac-LDLs were prepared from LDLs by the
addition of acetic anhydride in the presence of cold saturated
sodium acetate . In order to prevent LDL oxidation,
acetylation was carried out in argon-enriched capped vials.
After extensive dialysis against PBS, pH 7.4, LDLs were
filtered and sterilized by passing through 0.22-μm Millipore
membranes. LDL oxidation and integrity were evaluated by
determining (1) the content of thiobarbituric acid reactive
substances (TBARS), (2) lipid peroxides using an iodine
reagent , and (3) the chromophore fluorescence at 430 nm
after excitation at 355 nm . Agarose gel electrophoresis
(0.5%) was performed with a Beckman’s Paragon lipoprotein
electrophoresis kit .
Sterols and oxysterols were obtained from Sigma or
Steraloids except for 7α- and 7β-cholesterol, which were
synthesized as in our previous paper . When necessary
(purity <95%), the oxysterols were repurified by thin-layer
chromatography (TLC) on silica gel-impregnated plates
(Merck) with hexane/ethyl acetate (70/30, v/v) and purity was
checked by gas chromatography . Total lipid extracts were
carried out according to Folch et al.  and analyzed according
to our previous gas chromatographic technique . Phos-
pholipids and lysophosphatidylcholine (LPC) were analyzed
by HPLC with a light-scattering detector according to Blache
et al. .
Studies on secretion of apoAI and apoB by HepG2 cells
Dose–response studies of modified LDLs on apoAI and
apoB secretions were performed by incubating HepG2 cells
with various amounts of LDLs (0 to 200 μg/ml) at 37°C for
14 h. The effects of incubation time on apoAI or apoB
secretions were examined in the presence of 150 μg/ml LDLs
for varying times (4 to 96 h). At the end of the incubations,
culture media were removed from flasks and cell monolayers
washed with PBS and collected for DNA measurement .
Because of exogenous addition of apoB-containing LDL and
obvious interference, apoB secretion was evaluated by
incubation in leucine-poor DMEM containing 10% LPDS and
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