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

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

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
[

)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

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

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

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.

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

(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

(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

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

) 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.

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