Báo cáo khoa học: Fatty acid composition of chylomicron remnant-like particles influences their uptake and induction of lipid accumulation in macrophages pot

Fatty acid composition of chylomicron remnant-like
particles influences their uptake and induction of lipid
accumulation in macrophages
Clara De Pascale
1
, Michael Avella
1
, Javier S. Perona
2
, Valentina Ruiz-Gutierrez
2
,
Caroline P. D. Wheeler-Jones
1
and Kathleen M. Botham
1
1 Department of Veterinary Basic Sciences, Royal Veterinary College, London, UK
2 Instituto de la Grasa (CSIC), Seville, Spain
The first visible lesions in atherosclerosis development
are fatty streaks, which are formed when macrophages
that have invaded the artery wall take up lipid from
plasma lipoproteins in the subendothelial space and
become so engorged that they form foam cells [1]. It is
known that low-density lipoprotein (LDL) has a major
role in the induction of foam cell formation, but it is
also clear that oxidation of the LDL particles, a pro-
cess that can occur within the artery wall, is necessary
before extensive lipid accumulation occurs [2]. Recent
work in our laboratory and others, however, has pro-
vided strong evidence that chylomicron remnants, the
lipoproteins that carry fat and cholesterol from the

diet, are also able to induce macrophages to form
foam cells, and furthermore, that prior oxidation of
the particles is not required for their effect [3–5].
Keywords
chylomicron remnant-like particles; fatty acid
composition; lipid accumulation;
macrophages
Correspondence
K. M. Botham, Department of Veterinary
Basic Sciences, Royal Veterinary College,
Royal College St, London NW1 0TU, UK
Fax: +44 207468 5204
Tel: +44 207468 5274
E-mail:
(Received 30 August 2006, revised 10 Octo-
ber 2006, accepted 23 October 2006)
doi:10.1111/j.1742-4658.2006.05552.x
The influence of the fatty acid composition of chylomicron remnant-like
particles (CRLPs) on their uptake and induction of lipid accumulation in
macrophages was studied. CRLPs containing triacylglycerol enriched in
saturated, monounsaturated, n)6orn)3 polyunsaturated fatty acids
derived from palm, olive, corn or fish oil, respectively, and macrophages
derived from the human monocyte cell line THP-1 were used. Lipid accu-
mulation (triacylglycerol and cholesterol) in the cells was measured after
incubation with CRLPs for 5, 24 and 48 h, and uptake over 24 h was
determined using CRLPs radiolabelled with [
3
H]triolein. Total lipid accu-
mulation in the macrophages was significantly greater with palm CRLPs
than with the other three types of particle. This was mainly due to

increased triacylglycerol concentrations, whereas changes in cholesterol
concentrations did not reach significance. There were no significant differ-
ences in lipid accumulation after incubation with olive, corn or fish CRLPs.
Palm and olive CRLPs were taken up by the cells at a similar rate, which
was considerably faster than that observed with corn and fish CRLPs.
These findings demonstrate that CRLPs enriched in saturated or monoun-
saturated fatty acids are taken up more rapidly by macrophages than those
enriched in n)6orn)3 polunsaturated fatty acids, and that the faster
uptake rate results in greater lipid accumulation in the case of saturated
fatty acid-rich particles, but not monounsaturated fatty acid-rich particles.
Thus, dietary saturated fatty acids carried in chylomicron remnants may
enhance their propensity to induce macrophage foam cell formation.
Abbreviations
CRLP, chylomicron remnant-like particle; LDL, low-density lipoprotein; LRP, LDL receptor-like protein; MUFA, monounsaturated fatty acid;
PMA, phorbol 12-myristate 13-acetate; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid; TC, total cholesterol; TG, triacylglycerol.
5632 FEBS Journal 273 (2006) 5632–5640 ª 2006 The Authors Journal compilation ª 2006 FEBS
Fat and cholesterol consumed in the diet are taken
up by intestinal cells and secreted into lymph in chylo-
microns. These large triacylglycerol (TG)-rich lipopro-
teins then enter the blood via the thoracic duct and
are metabolized by lipoprotein lipase, a process that
removes some of the triacylglycerol and leaves smaller
chylomicron remnant particles, which deliver the
remaining lipid to the liver [6]. A number of lines of
evidence provide a powerful case to support the ath-
erogenicity of chylomicron remnants [7,8]. It has been
demonstrated that the particles enter the artery wall as
efficiently as LDL [9,10], and are retained within the
subendothelial space [7,11]; accumulation of remnants
in the plasma of apoE– ⁄ – mice is associated with the

development of severe atherosclerosis [12]; remnant-
like lipoproteins have been isolated from human aortic
intima and atherosclerotic plaque [13,14]; and delay in
remnant clearance from the circulation has been found
to be correlated with the development of atheroscler-
otic lesions in human patients [15,16].
In previous studies, chylomicron remnants or chylo-
micron remnant-like particles (CRLPs) have been
shown to induce extensive lipid accumulation in a
variety of macrophage types, including the murine
macrophage cell line J774 [17], mouse peritoneal macro-
phages [18], primary human monocyte-derived
macrophages [3,4] and macrophages derived from the
human monocyte cell line THP-1 [4,5,19,20]. Both cho-
lesterol and TG accumulate in response to remnant
particles, and the concentrations of TG found are
much greater than those observed on exposure of the
macrophages to an equivalent amount of cholesterol in
oxidized LDL (oxLDL) [4,20]. Moreover, once inside
the cells, the lipid taken up appears to be resistant to
efflux [20], as is the case with lipid originating from
oxLDL [21].
It has been known for many years that the type of
fat in the diet influences the development of athero-
sclerosis, with consumption of polyunsaturated
(PUFA) or monounsaturated (MUFA), as compared
with saturated (SFA), fatty acids decreasing the risk
[22]. Our earlier work has shown that the fatty acid
composition of chylomicron remnants reflects that of
the diet [23], and modulates their removal from the

blood by the liver, with particles derived from fish
(rich in n)3 PUFAs) or corn (rich in n)6 PUFAs) oil
being taken up more rapidly than those from olive
(rich in MUFAs) or palm (rich in SFAs) oil [24–26].
The finding that variations in the fatty acid composi-
tion of remnants modify their uptake by liver cells rai-
ses the possibility that such changes also affect the
interaction of the particles with macrophages to
promote or retard foam cell formation, providing a
mechanism by which dietary fats could directly influ-
ence atherosclerotic lesion development during their
transport from the gut to the liver. Little is known,
however, about how the fatty acid composition of
chylomicron remnants affects their uptake by macro-
phages and induction of excessive lipid accumulation
in the cells.
In this study, we have investigated the effects of
chylomicron remnants of different fatty acid composi-
tion on macrophage foam cell formation using CRLPs
[4,5,19,20] and macrophages derived from the human
monocyte cell line THP-1. CRLPs enriched in SFAs,
MUFAs, n)6 PUFAs and n)3 PUFAs, obtained by
incorporating TG from palm, olive, corn and fish oil,
respectively, into the particles were incubated with
THP-1 macrophages, and the concentrations of choles-
terol and TG accumulated were determined. In addi-
tion, the effects of varying fatty acid composition
on the uptake of the CRLPs by the cells was studied
using the four types of particle radiolabelled with
[

3
H]triolein.
Results
Characteristics of CRLPs
The content of TG and total cholesterol (TC) and the
TG ⁄ TC ratio in CRLPs containing TG from palm
(palm CRLPs), olive (olive CRLPs), corn (corn
CRLPs) or fish (fish CRLPs) oil are shown in Table 1.
Although the TG and TC concentrations varied some-
what in the different types of CRLP, with those in
palm CRLPs tending to be lower, these values are
dependent on the dilution of the preparations. More
importantly, there were no significant differences in the
TG ⁄ TC ratio between palm, olive, corn and fish
CRLPs. Thus, although the CRLPs were added to the
cell incubations according to the TG concentration,
the amount of cholesterol present was also similar in
experiments with the different types of particle. The
Table 1. Lipid content of CRLPs derived from different oils. CRLPs
containing TG from palm, olive, corn or fish oils were prepared as
described in Experimental procedures, and TG and TC contents
were measured. Data shown are the mean ± SEM from seven
separate preparations.
CRLP type
TG
(lmolÆmL
)1
)
TC
(lmolÆmL

)1
)TG⁄ TC
Palm 9.0 ± 1.4 0.7 ± 0.2 10.6 ± 1.1
Olive 13.0 ± 1.2 1.6 ± 0.6 12.1 ± 2.6
Corn 11.7 ± 1.7 1.2 ± 0.2 10.5 ± 1.8
Fish 15.8 ± 3.3 1.5 ± 0.3 8.8 ± 1.3
C. De Pascale et al. Lipid accumulation in macrophages
FEBS Journal 273 (2006) 5632–5640 ª 2006 The Authors Journal compilation ª 2006 FEBS 5633
fatty acid composition of the TG in the different types
of CRLP (Table 2) reflected that of the oils from
which they were obtained [23]. Thus CRLPs prepared
with TG from palm oil or olive oil contained relatively
high concentrations of SFAs [particularly palmitic acid
(16:0)] or MUFAs [particularly oleic acid (18:1, n)9)],
respectively, whereas those containing TG from corn
or fish oil were relatively enriched in n)6 [mainly lino-
leic acid (18:2, n)6)] or n)3 PUFAs [eicosapentaenoic
acid (20:5) and docosahexanoic acid (22:6)]. The
amounts of apoE in palm, olive, corn or fish CRLPs
were not signficantly different (one way analysis
of variance) as assessed by SDS ⁄ PAGE [optical density
unitsÆ(lmol TG)
)1
(n ¼ 3): palm CRLPs, 102 ± 25;
olive CRLPs, 122 ± 46; corn CRLPs, 135 ± 19; fish
CRLPs, 99 ± 36].
Assessment of the lipid peroxidation products,
4-hydroxy-2(E)-nonenal + malondialdehyde, in the
CRLPs (Table 3) showed that there were no significant
differences between the four different types of particle

(one way analysis of variance, with Tukey-Kramer’s
test post hoc), although the concentrations tended to
be more variable in the fish CRLPs, as might be expec-
ted from their relatively high content of long-chain
n)3 PUFAs such as eicosapentaenoic acid and docosa-
hexanoic acid. Lipid peroxidation products after incu-
bation of the four types of CRLP with CuSO
4
(10 lm)
for 6 h were significantly lower with olive CRLPs than
with the other three types, indicating that these parti-
cles are more resistant to oxidation, but there were no
significant differences between palm, corn and fish
CRLPs.
Effects of the fatty acid composition of CRLPs
on their induction of lipid accumulation
in THP-1 macrophages
THP-1 macrophages were incubated with palm, olive,
corn or fish CRLPs (0.3 lmol TGÆmL
)1
) for 5, 24 or
48 h, and the effects on lipid accumulation in the cells
were determined. Total lipid (TG + TC) concentra-
tions were similar in incubations with all four types of
particle after 5 h, but analysis by two-way analysis of
Table 2. Fatty acid composition of TG in CRLPs. CRLPs were prepared using TG from palm, olive, corn or fish oil as described in Experimen-
tal procedures, and the fatty acid composition was determined by gas chromatography. Data are expressed as gÆ(100 g total fatty acids)
)1
and are the mean ± SEM from three separate preparations. ND, none detected.
Fatty acid

CRLPs
Palm Olive Corn Fish
14:0 4.54 ± 3.04 0.47 ± 0.09 3.06 ± 1.24 8.29 ± 0.32
16:0 38.67 ± 1.58 12.40 ± 0.17 11.52 ± 0.04 23.59 ± 0.89
16:1 n)7 0.63 ± 0.18 1.21 ± 0.05 0.14 ± 0.02 12.79 ± 0.14
18:0 8.28 ± 4.00 2.94 ± 0.02 2.03 ± 0.13 3.37 ± 0.92
18:1 n)9 36.48 ± 2.93 72.74 ± 0.27 30.00 ± 0.62 20.05 ± 4.07
18:1 n)7 ND ND 0.43 ± 0.43 2.91 ± 1.47
18:2 n)6 10.58 ± 0.65 9.41 ± 0.48 51.87 ± 1.81 5.38 ± 1.02
18:3 n)3 0.82 ± 0.23 0.82 ± 0.00 0.93 ± 0.03 2.87 ± 0.14
20:4 n)6 ND ND ND 0.97 ± 0.07
20:4 n)3 ND ND ND 1.69 ± 0.07
20:5 n)3 ND ND ND 10.26 ± 0.44
22:5 n)3 ND ND ND 1.84 ± 0.10
22:6 n)3 ND ND ND 5.98 ± 2.99
Total SFA 51.49 ± 2.36 15.82 ± 0.18 16.62 ± 1.33 35.25 ± 0.47
Total MUFA 37.12 ± 2.75 73.95 ± 0.31 30.58 ± 1.00 35.75 ± 2.80
Total n)6 PUFA 10.58 ± 0.64 9.41 ± 0.48 51.87 ± 1.82 6.35 ± 1.05
Total n)3 PUFA 0.82 ± 0.23 0.82 ± 0.01 0.93 ± 0.03 22.65 ± 2.25
Table 3. Concentrations of malondialdehyde (MDA) and 4-hydroxy-
2(E)-nonenal (4-HNE) in CRLPs. MDA and 4-HNE concentrations in
CRLPs containing TG from palm, olive, corn or fish oil (10 l
M) were
determined before (0 h) and after (6 h) incubation with CuSO
4
(10 lM) for 6 h. Data shown are the mean ± SEM from three
separate preparations. *P < 0.05, **P < 0.01 versus olive CRLPs
(one-way analysis of variance, with Tukey-Kramer’s test multiple
comparison test post hoc).
CRLP type

4-HNE + MDA [pmolÆ(nmol TG)
)1
]
0h 6h
Palm 4.4 ± 1.2 46.1 ± 8.0*
Olive 5.8 ± 2.0 7.4 ± 0.7
Corn 2.6 ± 1.1 48.5 ± 10.0*
Fish 11.9 ± 7.2 88.9 ± 10.7**
Lipid accumulation in macrophages C. De Pascale et al.
5634 FEBS Journal 273 (2006) 5632–5640 ª 2006 The Authors Journal compilation ª 2006 FEBS
variance showed that there was significantly more
accumulation in cells exposed to palm CRLPs than in
those exposed to olive, corn or fish CRLPs, with signi-
ficant differences after 24 and 48 h (Fig. 1). Similar
analysis of the TG and TC content of the cells showed
that TG followed a similar pattern to that found for
total lipid (Fig. 2A), with palm CRLPs causing greater
accumulation than corn (significant at 24 and 48 h) or
olive or fish (significant at 48 h) CRLPs. However,
although there were similar trends in TC concentra-
tions, because of the lower cellular concentrations,
these changes did not reach significance.
Effects of the fatty acid composition of CRLPs
on their uptake by THP-1 macrophages
The uptake of CRLPs by THP-1 macrophages was
assessed using particles labelled in TG with [
3
H]triolein.
There were no significant differences (one-way analysis
of variance) between the specific activities of the

four different types of CRLP used [dpmÆ(nmol TG)
)1
(n ¼ 4): palm, 2601 ± 326; olive, 2041 ± 154; corn,
2290 ± 303; fish, 1764 ± 140). Cells were incubated
with radiolabelled palm, olive, corn or fish CRLPs for
periods up to 24 h, and the radioactivity associated
with the cells was measured (Fig. 3). Palm and olive
CRLPs were taken up by the cells significantly more
rapidly than corn and fish CRLPs (P < 0.01) with sig-
nificant differences at 13 h (P < 0.01) and 24 h
(P < 0.01). Corn and fish CRLPs were taken up at
very similar rates, and there were also no significant
differences in the rates of uptake of palm and olive
CRLPs.
Fig. 2. THP-1 macrophages were incubated with CRLPs contain-
ing TG from palm, olive, corn or fish oil, and the TG (A) and TC
(B) accumulated in the cells was measured after 5, 24 and
48 h. Data are the mean from three separate experiments, and
error bars show the SEM. *P < 0.05, **P < 0.01 versus palm
CRLPs.
Fig. 1. THP-1 macrophages were incubated with CRLPs containing
TG from palm, olive, corn or fish oil, and the total lipid (TG + TC)
accumulated in the cells was measured after 5, 24 and 48 h. Data
are the mean from three separate experiments, and error bars
show the SEM. *P < 0.05, **P < 0.01 versus palm CRLPs.
Fig. 3. THP-1 macrophages were incubated with CRLPs contain-
ing TG from palm, olive, corn or fish oil radiolabelled with
[
3
H]triolein, and the amount of radioactivity associated with the

cells after the time periods up to 24 h was measured. Each
point is the mean from three separate experiments, and error
bars show the SEM.
C. De Pascale et al. Lipid accumulation in macrophages
FEBS Journal 273 (2006) 5632–5640 ª 2006 The Authors Journal compilation ª 2006 FEBS 5635
Discussion
In previous work, we have used human monocyte-
derived macrophages and macrophages derived from
the human monocyte cell line THP-1 to investigate the
uptake of chylomicron remnants and their induction of
lipid accumulation in the cells [4,5,7,19,20]. As chylo-
micron remnants uncontaminated with lipoproteins of
a similar density such as chylomicrons and very-low-
density lipoprotein cannot be obtained easily from
human blood, for these studies we used model CRLPs.
We have reported previously [4] that these particles
have a diameter of about 130 nm, which is within the
range reported for human chylomicron remnants (50–
150 nm [27]), and that the lipid composition in terms
of the percentage lipid mass of the various lipid classes
and TG ⁄ TC molar ratio is also similar to that of
physiological remnants [6]. Although there is some
overlap in size between small chylomicrons and chylo-
micron remnants, the TG ⁄ TC molar ratio in
chylomicron-like particles would be expected to be
considerably higher (30–40 : 1 [6]) than that in our
CRLPs (9–12 : 1, Table 1). Thus, the CRLPs used
resemble physiological remnants in size, density and
lipid composition and also contain human apoE. They
differ from physiological chylomicron remnants, how-

ever, in that they lack apoB48. However, CRLPs of
this type without apoB48 have been shown to behave
like the physiological lipoprotein in their clearance
from the blood and metabolism in vivo, and in their
effects on cultured cells in vitro [28–30]. In addition, in
earlier work we have demonstrated that chylomicron
remnants prepared in rats in vivo cause comparable
lipid accumulation in J774 macrophages to that
observed in the present study with CRLPs and THP-1
macrophages [17]. As the composition of CRLPs can
be easily manipulated, they provide a suitable and con-
venient model for the present investigation.
We have demonstrated previously that the fatty acid
composition of chylomicron remnants reflects that of
the fatty meal from which they were derived. Thus,
remnants from rats given an oral dose of palm, olive,
corn or fish oil are enriched in SFAs, MUFAs, n)6
PUFAs and n)3 PUFAs [23], as well as containing a
range of other fatty acids, and these enrichments influ-
ence the uptake and metabolism of the particles by the
liver [24–26,31]. The CRLPs used in our previous work
with macrophages contained a single TG molecular
species, trilinolein, but to mimic the physiological situ-
ation more closely, for the current investigation of the
effects of the fatty acid composition of the particles on
macrophage foam cell formation, we used CRLPs con-
taining TG derived from natural dietary oils. Thus, the
particles were enriched in the particular type of fatty
acid predominating in the oil, but also contained a
complex mixture of other fatty acids, as occurs in vivo.

As might be expected, the fatty acid composition of
CRLPs containing TG from palm, olive, corn or fish
oil (Table 2) was similar to both that of the parent oils
and that of rat physiological remnants derived from
them [23], so that the CRLPs were enriched in SFAs,
MUFAs, n)6 PUFAs and n)3 PUFAs, respectively.
Earlier work in our laboratory and others has estab-
lished that chylomicron remnants are taken up by macro-
phages and induce the extensive lipid accumulation
associated with foam cell formation [3,4,7,17,18,32,33].
However, although the degree of unsaturation of diet-
ary fatty acids is believed to influence atherosclerotic
plaque development [22], it is not known whether the
propensity of the remnants to induce foam cell forma-
tion is affected by their fatty acid composition. The
results presented here indicate that CRLPs enriched in
SFAs are taken up more rapidly by macrophages than
those high in n)6orn)3 PUFAs and cause greater
lipid accumulation in the cells (Figs 1–3). The latter
effect was mainly due to increased concentrations of
TG, although the TC content showed similar trends
which did not reach significance. Interestingly,
although the rate of uptake of the olive particles was
comparable to that of palm CRLPs (Fig. 3), the
amount of lipid accumulated in the cells was signifi-
cantly lower. As the radiolabel in these experiments
was in the oleate moiety of triolein, these findings sug-
gest that TG taken up from CRLPs containing high
concentrations of oleate may be metabolized more
readily than TG from the other types of CRLP, caus-

ing an increase in the release of the radiolabel as the
free fatty acid for oxidation or in its transfer to phos-
pholipid, thus lowering the lipid accumulation
(TG + TC) despite the relatively high rate of uptake.
This conclusion is supported by findings of Peres and
coworkers [34,35] with
14
C-labelled fatty acids, show-
ing that oleate is oxidized more rapidly than palmitate
in macrophages and that  7.5 times more oleate than
palmitate is transferred from these cells to lymphocytes
in coculture, indicating that macrophage metabolism
of oleate is more active than that of palmitate.
Although it has been established in extensive studies
that LDL has a major role in foam cell formation, it is
also clear that oxidation of the particles greatly enhan-
ces their effects [2]. In striking contrast, chylomicron
remnants have been shown to cause extensive lipid
accumulation in macrophages without prior oxidation
of the particles [3,4,7]. Furthermore, work in our
laboratory has shown that protection of CRLPs from
oxidation by incorporation of antioxidants such as
Lipid accumulation in macrophages C. De Pascale et al.
5636 FEBS Journal 273 (2006) 5632–5640 ª 2006 The Authors Journal compilation ª 2006 FEBS
lycopene or probucol into the particles enhances,
rather than inhibits, their uptake and induction of lipid
accumulation in THP-1 macrophages [5,19]. These
findings suggest that, in sharp contrast with LDL, oxi-
dation of chylomicron remnants decreases their uptake
by the cells. It is possible therefore that the differential

rates of uptake of CRLPs of different fatty acid com-
positions by macrophages observed in the present
study is due to differences in the oxidative state of the
particles. Evaluation of the lipid peroxidation prod-
ucts, 4-hydroxy-2(E)-nonenal and malondialdehyde, in
the preparations indicates that there were no signifi-
cant differences between the four types of particle tes-
ted at the start of the experiments. Moreover, in
general, there was no obvious correlation between the
resistance to oxidation of the various particle types
during incubation and the rate of their uptake by the
THP-1 macrophages, suggesting that other factors are
mainly responsible for the different uptake rates
observed. The olive CRLPs, however, were signifi-
cantly more resistant to oxidation than the other types,
and this may provide part of the explanation for their
more rapid uptake compared with corn and fish
CRLPs, as the latter particles may become oxidized
during the incubation period.
It is known that the liver takes up chylomicron rem-
nants mainly via the LDL receptor and the LDL
receptor-like protein (LRP) [36], which both recognize
apoE. In contrast, the exact mechanisms by which
chylomicron remnants are taken up by macrophages
have not been definitively established. However, the
LDL receptor and the LRP are both expressed by
these cells, and a number of studies have demonstrated
that they play an important role [7,18,33], although
there may be other entry routes including phagocytosis
and scavenger receptors such as scavenger receptor A

(SR-A) and CD36 [7,36], which are known to play a
part in the uptake of oxidized or chemically modified
LDL [37]. Factors that influence the binding of apoE
to its receptors therefore are likely to affect the uptake
of chylomicron remnants by macrophages. It has been
demonstrated that ApoE does not bind to the LDL
receptor family in its lipid-free state. The receptor-
binding region is in an amphipathic a-helix which is
part of a four-helix bundle structure in the N-terminal
domain, and interaction with lipid is necessary to
induce a conformational change that promotes high
affinity for the receptors [38]. Because of the import-
ance of apoE conformation for receptor binding, it is
thought that not all apoE molecules on a particular
remnant particle are able to act as ligands. In addition,
apoE has been shown to adopt different conformations
when complexed to different lipids [39], and it has been
suggested that the lipid composition of lipoproteins
can alter the conformation of the protein [40]. Thus,
the differential uptake of CRLPs of different fatty acid
composition by macrophages demonstrated here may
be explained by effects on uptake via the LDL recep-
tor and the LRP due to differences in both the confor-
mation of apoE and the number of apoE molecules
able to bind to the receptors.
A receptor for apoB48 has been identified in endo-
thelial cells and has also been reported to be expressed
in macrophages, and it has been suggested that it may
be involved in the uptake of chylomicron remnants by
macrophages [41]. However, antibodies to apoB48

have been shown not to inhibit the uptake of chylomi-
cron remnants by rat macrophages [42], and Elsegood
et al. [33] were unable to detect binding of chylo-
micron remnants to a protein with a molecular mass
corresponding to the apoB48 receptor in THP-1 macro-
phages. Furthermore, we have found that the
expression of the apoB48 receptor is very low in the
THP-1 macrophages used in this study, although we
were able to demonstrate strong expression in human
umbilical vein endothelial cells using the same system
(F. Bejta and K. M. Botham, unpublished results). It
is unlikely therefore that the lack of apoB48 in our
CRLPs has any significant effect on the results
obtained.
In summary, this study shows that the fatty acid
composition of chylomicron remnants influences their
uptake and induction of lipid accumulation in macro-
phages, with those enriched in SFAs or MUFAs being
taken up more rapidly than those enriched in n)6or
n)3 PUFAs. The faster uptake rate results in greater
lipid accumulation in the case of SFAs, but not
MUFA-rich particles, possibly because of increased
intracellular metabolism of TG in the latter case. These
findings suggest that dietary SFAs carried in chylo-
micron remnants may enhance their ability to induce
macrophage foam cell formation, thus increasing their
atherogenicity.
Experimental procedures
Fetal bovine serum (heat inactivated), penicillin, strepto-
mycin and 2-mercaptoethanol were obtain from Gibco

(Paisley, UK). RPMI 1640, Trypan blue, fatty acid-free
albumin (BSA), phospholipids, cholesterol, cholesteryl
oleate, phorbol 12-myristate 13-acetate (PMA) and
Menhaden fish oil were supplied by Sigma (Poole, Dorset,
UK). Palm (KTC Edibles Ltd, Wednesbury, UK), extra
virgin olive (Bertolli; Unilever Foods UK, Crawley, Sur-
rey, UK) and corn (Mazola; Bestfoods UK Ltd, Esher,
UK) oil were purchased from domestic suppliers. The
C. De Pascale et al. Lipid accumulation in macrophages
FEBS Journal 273 (2006) 5632–5640 ª 2006 The Authors Journal compilation ª 2006 FEBS 5637
radioisotope [
3
H]triolein was supplied by Perkin–Elmer
(Beaconsfield, UK).
Preparation of CRLPs
TGs for CRLP preparation were isolated from palm,
olive, corn and fish oils as follows: 1.5 mL of each oil was
added to 30 mL chloroform ⁄ methanol (2 : 1, v ⁄ v) and
0.88% KCl (40% total volume), mixed and left at 4 °C
overnight. The upper aqueous phase was then removed,
and TGs were isolated from the chloroform phase by
TLC (hexane ⁄ diethyl ether ⁄ formic acid (80 : 20 : 2, v ⁄ v ⁄ v).
The band corresponding to TG was collected, resuspended
in chloroform, and centrifuged twice at 1200 g for 20 min
(4 °C) with an MSE Mistral 3000i centrifuge (MSE,
London, UK) with 43122-105 swingout rotor to remove
the silica gel. The chloroform supernatant was collected
after each centrifugation and kept under argon at )20 °C
until required.
CRLPs were prepared by sonication (power setting

22–24 lm, 20 min, at 56 °C) of a lipid mixture containing
70% TG extracted from oils as above, 2% cholesterol, 5%
cholesteryl ester and 25% phospholipids in 0.9% (w ⁄ v) NaCl
in Tricine buffer (20 mm, pH 7.4). The resulting emulsion
was brought to a density of 1.21 gÆmL
)1
with KBr, layered
under a stepwise density gradient as described previously [43],
and centrifuged at 17 000 g for 20 min at 20 °C with a Beck-
man Optima L-80 centrifuge (Beckman Coulter, High
Wgcombe, UK) with SW40Ti swingout rotor. The upper
layer of grossly emulsified lipids was then removed and
replaced with an equal volume of NaCl solution (d ¼
1.020 gÆmL
)1
), and tubes were centrifuged at 70 000 g for 1 h
(20 °C) with a Beckman Optima L-80 centrifuge with SW40Ti
swingout rotor. Lipid particles harvested from the top layer
were incubated with d >1.020 gÆmL
)1
fraction of human
plasma (National Blood Transfusion Service, North London
Centre, London, UK) prepared by ultracentrifugation and
dialysed before use as described previously [27] at 37 °Cwith
shaking for 4 h (1 volume of particles : 2 volumes plasma).
CRLPs were then isolated by ultracentrifugation at d ¼
1.006 gÆmL
)1
(120 000 g for 12 h at 4 °C), harvested from the
top layer, purified by a second centrifugation at the same den-

sity with a Beckman Optima L-80 centrifuge with SW40Ti
swingout rotor (202 000 g for 4 h at 4 °C) and stored at 4 °C
under argon until required. All preparations were used within
1 week. SDS ⁄ PAGE analysis showed that all four types of
particles contained similar amounts of apoE. For preparation
of CRLPs labelled with [
3
H]triolein, the radioisotope
(1.85 MBq) was added to the lipid mixture before sonication.
The level of oxidation of the CRLPs containing TG from
the four different oils was determined by measuring the
concentrations of 4-hydroxy-2(E)-nonenal and malondialde-
hyde using the commercially available Bioxytech LPO-586
assay (Oxis International Inc, Portland, OR, USA), follow-
ing the manufacturer’s instructions.
Culture of THP-1 cells and experimental protocol
THP-1 monocytes were maintained in suspension in
RPMI 1640 culture medium containing 10% fetal bovine
serum, 100 UÆmL
)1
penicillin, 100 mgÆmL
)1
streptomycin
and 50 lm 2-mercaptoethanol (culture medium) at a density
of (3–9) · 10
5
cellsÆmL
)1
at 37 °C in 5% air ⁄ 95% CO
2

. The
cells were induced to differentiate into macrophages by
incubation with PMA (200 ngÆmL
)1
) for 72 h. After this
time, the cells adhering to the culture dishes were washed
with warm NaCl ⁄ P
i
(3 · 2 mL) to remove any undifferenti-
ated cells and traces of PMA. The viability of the THP-1
macrophages, as assessed by Trypan blue exclusion, was
> 95% in all experiments.
For lipid-accumulation studies, CRLPs (0.3 lmol triacyl-
glycerolÆmL
)1
) were incubated with macrophages for 5, 24
or 48 h. The cells were then washed with NaCl⁄ P
i
(3 · 3 mL), harvested in 0.7 mL NaCl ⁄ P
i
, disrupted by
sonication for 5 s (22–24 lm, power setting), and a sample
was taken for protein determination. Lipids were extracted
with chloroform ⁄ methanol (2 : 1, v ⁄ v) and 0.88% KCl
(40% total volume). For uptake studies, THP-1 macroph-
ages were incubated with CRLPs labelled with [
3
H]triolein
(0.3 lmol TGÆmL
)1

) for times up to 24 h. After the incuba-
tion period, cells were washed with NaCl ⁄ P
i
(4 · 3 mL),
and resuspended in 0.5 mL NaOH (0.5 m). Radioactivity
was determined by liquid-scintillation counting using
Hydrofluor as the scintillant.
Analytical methods
TC (cholesterol + cholesteryl ester) and TG in CRLPs and
in cells was determined by enzymatic analysis using com-
mercial enzymatic reagent kits (Alpha Laboratories, East-
leigh, UK). For analysis of the fatty acid composition of
TG in the CRLPs, TG were transmethylated using sodium
methoxide in methanol (0.5%), and the resulting fatty acid
methyl esters were analysed by gas chromatography, using
a model 5890 series II gas chromatograph (Hewlett-Packard
Co, Avondale, PA, USA) equipped with a flame ionization
detector and a capillary silica column Supelcowax 10 (Sul-
pelco Co, Bellefonte, PA, USA) of 60 m length and
0.25 mm internal diameter. The relative apoE content of
the different types of CRLP was assessed using
SDS ⁄ PAGE. The gels were stained with Coomassie blue
and the bands were quantified by absorbance volume analy-
sis. Cell protein content was measured by the method of
Lowry et al. [44] with BSA as standard.
Statistical analysis
Data were analysed by two-way analysis of variance fol-
lowed by Bonferroni’s multiple comparison test except
where indicated otherwise.
Lipid accumulation in macrophages C. De Pascale et al.

5638 FEBS Journal 273 (2006) 5632–5640 ª 2006 The Authors Journal compilation ª 2006 FEBS
Acknowledgements
This work was supported by a grant from the British
Heart Foundation (PG ⁄ 04 ⁄ 021 ⁄ 16758).
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