Báo cáo khoa học: Adipophilin increases triglyceride storage in human macrophages by stimulation of biosynthesis and inhibition of b-oxidation doc

Adipophilin increases triglyceride storage in human
macrophages by stimulation of biosynthesis and
inhibition of b-oxidation
Guilhem Larigauderie
1,2,3
, Clarisse Cuaz-Pe
´
rolin
1,2,3
, Amena B. Younes
4
, Christophe Furman
1,2,3
,
Catherine Lasselin
1,2,3
, Corinne Copin
1,2,3
, Michael Jaye
5
, Jean-Charles Fruchart
1,2,3
and
Mustapha Rouis
1,2,3
1 Inserm, U545, Lille, F-59019 France
2 Institut Pasteur de Lille, De
´
partement d’Athe
´
roscle

´
rose, Lille, F-59019 France
3 Universite
´
de Lille 2, Faculte
´
de Pharmacie, Lille, F-59019 France
4 Inserm IFR-17, Laboratoire de Microscopie Electronique, Lille, France
5 GlaxoSmithKline, King of Prussia, PA, USA
Lipid-enriched macrophage-derived foam cells are an
early and characteristic feature of atherosclerotic
lesions. Lipid loading of macrophages in vitro can be
achieved by chemical modification of the apolipopro-
tein B component of low-density lipoprotein (LDL),
aggregation of LDL induced by either vortexing or
treatment with lipases, or complexing of LDL with gly-
cosaminoglycans or antibodies which bind macrophages
Keywords
adipophilin; macrophage; atherosclerosis;
lipid droplet; triglycerides
Correspondence
M. Rouis, INSERM UR545, Institut Pasteur
de Lille, 1 rue du Professeur Calmette,
59019 Lille, France
Fax: +33 3 20 87 73 60
Tel: +33 3 20 87 73 79
E-mail:
(Received 19 April 2006, revised 30 May
2006, accepted 5 June 2006)
doi:10.1111/j.1742-4658.2006.05357.x

Lipid accumulation alters macrophage biology and contributes to lipid
retention within the vessel wall. In this study, we investigated the role of
adipophilin on triglyceride accumulation and lipid-droplet formation in
THP-1-derived macrophages (THP-1 macrophages). In the presence of
acetylated low-density lipoprotein, macrophages infected with an adeno-
virus expressing human adipophilin showed a 31% increase in triglyceride
content and a greater number of lipid droplets compared with control cells.
Incubation of macrophages with very low-density lipoprotein (VLDL) dra-
matically increased cellular triglyceride content similarly in control and
adipophilin-overexpressing cells. By itself, VLDL increased adipophilin
expression, which explains the lack of effect of adipophilin overexpression
on cellular triglyceride content in macrophages loaded with VLDL. The
lipid-droplet content of macrophages was increased by overexpression of
adipophilin and ⁄ or loading with VLDL. In contrast, inhibition of adipo-
philin expression using siRNA prevented lipid-droplet formation and signi-
ficantly reduced intracellular triglyceride content. Using inhibitors of
b-oxidation and acyl-coenzyme A synthetase, results were obtained which
suggest that adipophilin elevates cellular lipids by inhibition of b-oxidation
and stimulation of long-chain fatty acid incorporation into triglycerides.
Adipophilin expression in THP-1 macrophages altered the cellular content
of different lipids and enhanced the size of lipid droplets, consistent with a
role for adipophilin in human foam cell formation.
Abbreviations
ACAT-1, acetyl-coenzyme A acetyltransferase 1; AcLDL, acetylated LDL; ADRP, murine adipose differentiation-related protein; AICAR,
5’-phosphoribosyl-5-aminoimidazole-4-carboxamide; CE, cholesteryl ester; FC, free cholesterol; HSL, hormone-sensitive lipase; LDL,
low-density lipoprotein; m.o.i., multiplicity of infection; oxLDL, oxidized LDL; PPAR, peroxisome proliferator-activated receptors; TG,
triglycerides; THP-1 macrophages, THP-1-derived macrophages.
3498 FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS
and promote LDL uptake by endocytosis [1]. In addi-
tion, several studies have reported that macrophages

can accumulate large amounts of cholesteryl ester (CE)
through the uptake of oxidized LDL (oxLDL) by a
variety of mechanisms, including the scavenger path-
way [2], and that VLDL are capable of inducing CE
and triglyceride (TG) accumulation in macrophages
[3,4]. The mechanism of TG accumulation in human
monocyte–macrophages primarily involves the direct
uptake of free fatty acids generated by the extracellular
lipoprotein lipase-mediated hydrolysis of VLDL–TG
followed by intracellular reesterification into lipids,
however, receptor-mediated uptake of intact VLDL
particles is also implicated [4–6].
Lipid accumulation in macrophages not only contri-
butes to cholesterol and TG retention within the vessel
wall, but also alters macrophage biology. Indeed, sev-
eral studies have indicated that a diversity of effects on
macrophage function can be attributed to lipid loading.
These include the upregulation of the genes for apo-
lipoprotein E [7], elastase [8] and tissue factor [9], as
well as altered expression of several other genes [10].
Within cells, lipid is stored in spherical organelles
called lipid droplets [11] which have been reported to
play active and diverse roles in the cellular life cycle.
Indeed, lipid droplets are involved in the maintenance
of intracellular cholesterol balance in fibroblasts [12]
and appear to be the principal source of fatty acids in
adipose and liver [13]. Moreover, correlations between
lipid droplets and certain human diseases such as athe-
roma plaque, steatosis, obesity and cancers have been
reported [11].

Lipid droplets are composed of a CE and TG core
surrounded by a phospholipid monolayer and coated
with specific proteins [11]. Adipophilin, or adipose dif-
ferentiation-related protein (ADRP), a 50 kDa protein
initially described in adipocytes [14], is a marker of
lipid accumulation and is among the lipid droplet-
associated proteins present in a variety of cells such as
hepatocytes, adipocytes, muscle cells, mammary epithe-
lial cells, fibroblasts, endothelial cells and macrophages
[15,16].
Macrophage expression of adipophilin is upregulated
by oxLDL [17], acetylated LDL (AcLDL) [18], enzy-
matically modified LDL [19] and by synthetic agonists
of the peroxisome proliferator-activated nuclear recep-
tors d (PPARd) [20,21] and c (PPARc) [22,23]. How-
ever, the precise role of adipophilin in macrophage
foam cell formation and, in turn, in the development
of atherosclerotic lesions remains unclear. In this
study, we investigated the impact of adipophilin over-
expression or downregulation on lipid accumulation
and droplet formation in human THP-1 macrophages.
Results
We have previously shown that adipophilin expression
was greater in human atherosclerotic lesions than in
healthy areas of the same artery and that the majority
of adipophilin mRNA in atheromatous tissue was
attributed to lipid-rich macrophages (CD68+ cells)
[18]. We have also reported that THP-1 cells differenti-
ated into macrophages with phorbol esters were able to
rapidly take up AcLDL and to subsequently develop a

foam cell-like morphology. Under these conditions, adi-
pophilin expression was enhanced dramatically [18]. To
further study the function of adipophilin in human
macrophages, we generated an adenovirus vec-
tor-expressing human adipophilin (Ad.CMV.adipo-
philin). Using the control Ad.CMV.GFP vector, we
demonstrated nearly 100% infection of THP-1 macro-
phages (data not shown). We assessed adipophilin
expression using both quantitative PCR and immuno-
blotting in cells infected with two different amounts of
Ad.CMV.adipophilin. At multiplicity of infection
values (m.o.i.) of 100 and 500, adipophilin mRNA
was increased  14 ± 0.8- and  39 ± 7.8-fold,
respectively, and adipophilin protein was increased
 6.5 ± 1.7- and  38 ± 15-fold, respectively, com-
pared with control cells (Fig. 1).
When cells were loaded with 100 lgÆmL
)1
AcLDL,
adipophilin overexpression resulted in a modest but
significant increase in TG (1.3-fold, P < 0.05)
(Fig. 2) and altered cellular CE and free cholesterol
Fig. 1. Expression of adipophilin protein and mRNA in adenovirus-
infected THP-1 macrophages. THP-1 macrophages were infected
with Ad.CMV.GFP or Ad.CMV.adipophilin at 100 and 500 m.o.i.
Three days later, total proteins and total RNA were isolated. Total
protein was analysed by western blotting (upper) and RNA was
quantified using real-time qPCR (lower). *The difference between
Ad.CMV.GFP-infected cells and cells infected with Ad.CMV.
adipophilin was significant at P < 0.01.

G. Larigauderie et al. Adipophilin enhances triglyceride storage
FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS 3499
(FC) content (1.4-fold increase, P < 0.01 and more
than twofold decrease, P < 0.01, respectively, data
not shown). However, when cells were loaded with 10,
50 and 100 lgÆmL
)1
VLDL or 10 lgÆmL
)1
AcLDL, no
significant difference in TG content was seen between
control and adipophilin-overexpressing cells (Fig. 2),
whereas cellular CE and FC contents were altered
similarly to AcLDL-loaded cells in the presence of
100 lgÆmL
)1
VLDL (1.6-fold increase, P < 0.05 and
60% decrease P < 0.05, respectively, data not shown).
The fact that we did not observe increased TG content
in adipophilin-overexpressing vs. control cells follow-
ing incubation with VLDL is due to the dramatic
increase in the cellular TG content in all cells under
these conditions. To examine whether VLDL increased
adipophilin in human macrophages, we incubated
THP-1 macrophages with increasing concentrations of
VLDL and examined adipophilin levels by immuno-
blotting (Fig. 3). In the presence of 10 and
100 lgÆmL
)1
VLDL, adipophilin increased  12 ± 2.8-

and  28 ± 5.2-fold relative to control macrophages
cultured in lipid-free medium. Thus, elevation of cellu-
lar adipophilin by VLDL renders it impossible to
observe an effect of Ad.CMV.adipophilin-mediated
adipophilin overexpression on TG content (Fig. 2). To
confirm this hypothesis, we incubated Ad.CMV.adipo-
0
25
50
75
100
125
150
175
200
No added
lipids
100
Concentration (µg TG/mg cell protein)
non-infected cells
Ad.CMV.GFP
Ad.CMV.adipophilin
10 50
VLDL (µg/ml)
10 100
AcLDL (µg/ml)
*
Fig. 2. Effect of adipophilin overexpression on lipid mass in THP-1 macrophages incubated with VLDL. Cells were infected with 500 m.o.i.
of Ad.CMV.GFP (control) or Ad.CMV.adipophilin and incubated with 0, 10, 50 or 100 lgÆmL
)1

VLDL or AcLDL (10, 100 lgÆmL
)1
) in medium
containing 1% fetal bovine serum for 48 h. The results are the means ± SD of three independent experiments performed in quadruplicate.
*The difference between control and cells infected with Ad.CMV.adipophilin in the presence of 100 lgÆ mL
)1
AcLDL was significant at
P < 0.05.
Fig. 3. Expression of adipophilin protein
levels in human THP-1 macrophages loaded
with VLDL. THP-1 macrophages were incu-
bated with 0, 10 or 100 lgÆmL
)1
VLDL in
RPMI-1640 containing 0.4% BSA for 48 h.
Total proteins were isolated and samples of
20 lg were separated by SDS ⁄ PAGE (10%)
and blotted onto a nitrocellulose membrane.
The results are mean ± SD of three inde-
pendent experiments. *The difference bet-
ween control and cells incubated with VLDL
was significant at P < 0.01.
Adipophilin enhances triglyceride storage G. Larigauderie et al.
3500 FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS
philin-infected macrophages with 10 and 100 lgÆmL
)1
VLDL for 48 h and measured adipophilin levels by
immunoblotting. On top of the already elevated level
of adipophilin expression in Ad.CMV.adipophilin-
infected cells, 10 and 100 lgÆmL

)1
VLDL increased
adipophilin  1.8 ± 0.71- and  5.2 ± 1.91-fold,
respectively, relative to Ad.CMV.adipophilin-infected
macrophages cultured in lipid-free medium. In com-
parison, no significant difference in adipophilin expres-
sion was observed in Ad.CMV.adipophilin-infected
macrophages loaded or not with 10 and 100 lgÆmL
)1
of AcLDL (data not shown).
The induction of adipophilin expression and the
excessive lipid loading of THP-1 macrophages in
response to VLDL treatment was confirmed using
immunolocalization experiments which revealed the
presence of numerous large lipid droplets surrounded
by adipophilin (Fig. 4B) and by Oil Red O staining
(Fig. 5E,F). In cells cultured in the absence of VLDL
(control cells), nominal diffuse cytoplasmic adipophilin
staining was observed (Fig. 4A); in the presence of
VLDL, adipophilin staining was pronounced in both
Ad.CMV.adipophilin- and Ad.CMV.GFP-infected cells
(Fig. 4G,H). Adenoviral-mediated overexpression of
adipophilin followed by an incubation with 100
lgÆmL
)1
AcLDL for 24 h showed a significant increase
in lipid-droplet formation (Fig. 4F) compared with
AcLDL-loaded control cells (Fig. 4E) or adipophilin-
overexpressing cells incubated without VLDL or
AcLDL (Fig. 4D).

To further assess the impact of adipophilin levels on
lipid-droplet formation we manipulated adipophilin
levels in THP-1 macrophages by infection with
Ad.CMV.adipophilin or by transfection of cells
with adipophilin siRNA. Noninfected and control
Ad.CMV.GFP-infected THP-1 macrophages grown in
serum-free RPMI-1640 or in 10% fetal bovine serum
showed a quasi absence of lipid droplets in the cyto-
plasm following Oil Red O staining (Fig. 5A,C,I).
However, incubation of adipophilin-infected macro-
phages in RPMI-1640 supplemented with 10% fetal
bovine serum (Fig. 5D) followed by staining with Oil
Red O showed a significant increase in lipid droplets
in comparison with Ad.CMV.GFP-infected cells
(Fig. 5C). In agreement with our intracellular TG
measurements, cells incubated with 100 lgÆmL
)1
VLDL (Fig. 5B,E,F,I) contained a greater number of
lipid droplets than cells incubated with or without
10% fetal bovine serum (Fig. 5A,C,D,I). When THP-1
macrophages were transfected with siRNA–adipophilin
or siRNA–GAPDH (control) followed by incubation
with 100 lgÆmL
)1
VLDL for 24 h, there was a sub-
stantial reduction in the number and size of lipid drop-
lets in siRNA–adipophilin-transfected cells (Fig. 5H),
whereas control siRNA–GAPDH-transfected cells
accumulated a large number of lipid droplets
(Fig. 5G). To verify the implication of adipophilin in

lipid-droplet formation, we measured the intracellular
accumulation of TG in siRNA–adipophilin-transfected
macrophages following 48 h incubation with either
10 or 100 lgÆmL
)1
VLDL. Inhibition of adipophilin
expression decreased cellular TG content in both cases
by  30% compared with control cells (Fig. 6).
Potential mechanisms by which adipophilin
increased lipid content in THP-1 macrophages include
protection of lipid droplets against the activity of
intracellular lipases such as hormone-sensitive lipase
(HSL), inhibition of fatty acid oxidation (which would
favour the recycling of fatty acids), enhancement of
acetyl-coenzyme A acetyltransferase 1 (ACAT-1) este-
rification activity or stimulation of lipid synthesis. The
effect of adipophilin on intracellular lipase activity was
determined by adding the acyl-coenzyme A synthetase
inhibitor triacsin C to the medium of macrophages
preloaded with oleate, to inhibit the reutilization of
fatty acids released from hydrolysed TG [24,25]. No
significant differences could be observed between
triascin C-treated Ad.CMV.adipophilin, Ad.CMV.
GFP and noninfected cells, which all contained
 58 ± 9.9% of the initial TG mass at 24 h post infec-
tion (data not shown), suggesting that adipophilin does
not elevate cellular TG by protecting it from cellular
lipases. However, because these results were obtained
using an indirect method (triacsin C inhibition) affecting
total cellular lipase, we subsequently measured more

specifically HSL activity in lysate-infected macrophages
loaded with AcLDL as an exogenous source of lipids
(because VLDL strongly induced adipophilin expression
even in Ad.CMV.GFP infected macrophages). Our data
showed a significant twofold decrease (P<0.001) in
HSL activity in Ad.CMV.adipophilin-infected cells
compared with control cells (Fig. 7). This inhibitory
effect on HSL activity, seen in the presence of elevated
amounts of adipophilin, may explain to some extent the
increased storage of lipids.
To examine the effect of adipophilin on TG synthe-
sis, Ad.CMV.adipophilin- and Ad.CMV.GFP-infected
macrophages were loaded with 400 lm palmitate,
either alone or with 2.5 lm triacsin C, and the cellular
TG content was quantified. In the absence of triac-
sin C, adipophilin-overexpressing cells produced more
TG from palmitate than control cells (133.6 ± 8.4 vs.
95 ± 9.7 lgÆmg
)1
cell protein, respectively). The addi-
tion of triacsin C together with palmitate reduced the
TG mass in both Ad.CMV.GFP- and Ad.CMV.
adipophilin-infected cells to approximately the same
G. Larigauderie et al. Adipophilin enhances triglyceride storage
FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS 3501
levels (66.5 ± 11.2 and 61.5 ± 10.7 lgÆmg
)1
cell pro-
tein, respectively) (Fig. 8). These results suggest that
increased TG in cells overexpressing adipophilin is at

least partly due to acyl-coenzyme A synthetase eleva-
ted activity or to the downstream incorporation of
acyl-CoAs into TG. Lipid esterification was quantified
in either THP-1 macrophages overexpressing adipophi-
lin (following infection with Ad.CMV.adipophilin)
or after downregulation of adipophilin expression
by transfection with siRNA–adipophilin. Neither
enhanced nor reduced adipophilin expression had an
effect on ACAT-1 activity, given that similar amounts
of [
14
C]oleate incorporation into cholesteryl oleate
were measured in both cases (data not shown). To fur-
ther probe this, we assessed the impact on TG accu-
mulation of pharmacological inhibition of ACAT-1
in Ad.CMV.adipophilin-infected and control THP-1
macrophages. Enhanced TG accumulation in adipophi-
lin-overexpressing cells was dependent on the addition
of palmitate, whereupon adipophilin-overexpressing
cells accumulated 1.6 times more TG than control
cells. In the absence of palmitate, TG accumulation
AB
CD
E
F
G
H
Fig. 4. Immunocytochemical analysis of adi-
pophilin in THP-1 macrophages (magnifica-
tion, 63·). Cells were cultured in RPMI-1640

supplemented with 10% fetal bovine serum.
Adipophilin was immunolocalized using a
specific polyclonal antibody as described in
Experimental procedures. (A) Control cells
grown without added lipids for 24 h. (B)
Cells incubated with VLDL (100 l gÆmL
)1
) for
24 h. (C) Cells infected with Ad.CMV.GFP.
(D) Cells infected with Ad.CMV.adipophilin.
(E) Cells infected with Ad.CMV.GFP, then
treated for 24 h with AcLDL (100 lgÆmL
)1
).
(F) Cells infected with Ad.CMV.adipophilin,
then incubated for 24 h with AcLDL
(100 lgÆmL
)1
). (G) Cells infected with
Ad.CMV.GFP, then treated for 24 h with
VLDL (100 lgÆmL
)1
). (H) Cells infected with
Ad.CMV.adipophilin, then incubated for 24 h
with VLDL (100 lgÆmL
)1
).
Adipophilin enhances triglyceride storage G. Larigauderie et al.
3502 FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS
in adipophilin-overexpressing and control cells was

similar and unaffected by addition of the ACAT-1
inhibitor CAY10486. The absolute amounts and fold
stimulation of TG accumulation in palmitate-loaded,
adipophilin-overexpressing cells (133.6 ± 6.7 lgÆmg
)1
cell protein, 1.5-fold) were similar when cells were co-
incubated with palmitate plus CAY10486 (150.2 ±
7.6 lgÆmg
)1
cell protein, 1.6-fold). These results indi-
AB
CD
EF
GH
I
Fig. 5. Lipid-droplet staining with Oil Red O
in THP-1 macrophages (magnification, 63·).
(A) Cells cultured in serum-free RPMI-1640.
(B) Cells cultured in RPMI-1640 supplemen-
ted with 10% fetal bovine serum and
100 lgÆmL
)1
VLDL. (C) Cells infected with
500 m.o.i. of Ad.CMV.GFP then maintained
in culture in RPMI-1640 supplemented with
10% fetal bovine serum. (D) Cells infected
with 500 m.o.i. of Ad.CMV.adipophilin then
incubated in RPMI-1640 supplemented with
10% fetal bovine serum. (E) Cells infected
with 500 m.o.i. of Ad.CMV.GFP then incuba-

ted in RPMI-1640 supplemented with 100
lgÆmL
)1
VLDL. (F) Cells infected with 500
m.o.i. of Ad.CMV.adipophilin then incubated
in RPMI-1640 supplemented with 100 lgÆ
mL
)1
VLDL. (G) siRNA–GAPDH-transfected
cells incubated in RPMI-1640 containing
100 lgÆmL
)1
. (H) siRNA–adipophilin-
transfected cells incubated in RPMI-1640
containing 100 lgÆmL
)1
VLDL. (I) Average
number of lipid droplets in THP-1
macrophages cultured and treated as
described in the preceding legend (A–H)
expressed as fold change from control cells
(described in A). * The difference was
significant at P < 0.05. #, non significant.
G. Larigauderie et al. Adipophilin enhances triglyceride storage
FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS 3503
cate that enhanced accumulation of TG in adipophilin-
overexpressing cells does not involve ACAT-1, because
specific inhibition of ACAT-1 was without effect.
Next, we examined whether inhibition of fatty acid
oxidation may also contribute to TG elevation by adi-

pophilin in THP-1 macrophages. TG accumulation in
Ad.CMV.GFP- and Ad.CMV.adipophilin-infected cells
incubated in the presence of palmitate (positive con-
trol) was compared with TG accumulation in cells
incubated with palmitate plus bromopalmitate, a non-
metabolized inhibitor of fatty acid oxidation [26]. For
these experiments, the concentrations of palmitate and
bromopalmitate used were 100 lm, because higher
bromopalmitate concentrations were toxic for THP-1
macrophages. Cells were infected or not with
Ad.CMV.GFP or Ad.CMV.adipophilin and then loa-
ded with 100 lm fatty acids for 48 h. Adipophilin-
infected cells accumulated 1.4 times more TG than
control cells (Fig. 9). No differences in TG accumula-
tion were observed between any of the cell-treatment
groups when cells were incubated only with bromo-
palmitate, which is poorly incorporated into TG. The
addition of both palmitate and bromopalmitate to
noninfected cells, as well as to cells infected with the
control vector (Ad.CMV.GFP) showed an increase in
TG mass in comparison with cells incubated with
palmitate only. This indicates that fatty acid oxidation
is an ongoing process in THP-1 macrophages; inhibi-
tion of fatty acid oxidation by bromopalmitate results
in elevated cellular TG content. In contrast to control
cells, no significant differences were observed between
adipophilin-overexpressing cells incubated with palmi-
tate alone or with bromopalmitate plus palmitate. This
Fig. 6. Effect of adipophilin downregulation on lipid mass in THP-1
macrophages incubated with VLDL. Cells were transfected with

siRNA–GAPDH (control) or siRNA–adipophilin and 24 h later, incuba-
ted with 10 or 100 lgÆmL
)1
VLDL or AcLDL (10, 100 lgÆmL
)1
)in
medium containing 1% fetal bovine serum for 48 h. The results are
the means ± SD of three independent experiments performed in
triplicates. *The difference between control and cells transfected
with siRNA-adipophilin was significant at P < 0.05. # The difference
between macrophages incubated in the presence of VLDL and con-
trol, Ad.CMV.GFP or Ad.CMV.Adipophilin-infected macrophages
(without VLDL) was significant at P < 0.05.
Fig. 7. Effect of adipophilin overexpression on HSL activity in
THP-1 macrophages. THP-1 cells infected with either Ad.CMV.
adipophilin or Ad.CMV.GFP and incubated for 48 h with AcLDL.
HSL activity was assayed as neutral CE by following the release of
[1-
14
C] oleic acid from cholesteryl [1-
14
C]oleate as described in
Experimental procedures. *The difference between control and
Ad.CMV.adipophilin cells was significant at P < 0.001.
0
20
40
60
80
100

120
140
160
No added
lipids
Palmitate Palmitate +
triacsin C
CAY10486 Palmitate +
CAY10486
Triglyceri eds( µ/gc gmellrptoein)
non-infected cells
Ad.CMV.GFP
Ad.CMV.adipophilin
*
*
NS
Fig. 8. Triglycerides synthesis is inhibited by triacsin C but not by
an ACAT-1 inhibitor in THP-1 macrophages overexpressing adipo-
philin. THP-1 macrophages were infected or not with 500 m.o.i. of
Ad.CMV.GFP and Ad.CMV.adipophilin and then incubated either for
16 h at 37 °C with 400 l
M palmitate complexed to BSA in the pres-
ence or absence of 2.5 l
M triacsin C or for 48 h at 37 °C with
400 l
M palmitate complexed to BSA in the presence or absence of
60 l
M CAY10486. TG content was quantified on lipid extracts.
*The difference between Ad.CMV.adipophilin cells in the presence
of palmitate or palmitate + CAY10486 and cells in the absence of

lipids or in the presence of palmitate (only control THP-1 macroph-
ages), palmitate + triacsin C, CAY10486 or palmitate + CAY10486
(only control THP-1 macrophages) was significant at P < 0.01. NS,
non significant.
Adipophilin enhances triglyceride storage G. Larigauderie et al.
3504 FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS
suggests that the enhanced TG content in adipophilin-
overexpressing cells may be partly due to inhibition of
fatty acid oxidation, because it cannot be further
inhibited by bromopalmitate.
To confirm this hypothesis, we subsequently treated
adipophilin-overexpressing or control THP-1 macro-
phages with 5’-phosphoribosyl-5-aminoimidazole-4-
carboxamide (AICAR), which stimulates fatty acid
oxidation by activation of AMP-activated protein kin-
ase. Cells were incubated with or without palmitate
and TG levels were assessed. Addition of AICAR
alone did not significantly change the intracellular TG
content, however, there was a trend for increased TG
in cells treated with both AICAR and palmitate
(Fig. 9). The rather modest effects of AICAR in palmi-
tate-loaded cells is not surprising, because AICAR
treatment did not affect adipophilin expression (data
not shown) and AICAR induces fatty acid oxidation
and therefore the degradation of palmitate. In the
presence of AICAR, TG levels in adipophilin-overex-
pressing cells were similar to control cells (57.0 ± 6.2
and 63.0 ± 6.0 lgÆmg
)1
cell protein, respectively).

Thus, stimulation of fatty acid oxidation by AICAR
appears to be dominant over the inhibition of fatty
acid oxidation by adipophilin.
Discussion
We studied the impact of adenoviral-mediated overex-
pression of adipophilin in THP-1-derived macrophages
on the accumulation of TG when the cells were incu-
bated in the presence of VLDL, AcLDL or palmitate.
Adipophilin is a lipid droplet-associated protein which
is expressed in a wide range of lipid-accumulating cells
including macrophages [10,16,27]. However, little is
known about the function of adipophilin in macro-
phages. By analogy with adipocytes, which share certain
common features [28,29], and preadipocytes, which
may convert to macrophages [30], stimulation of
human adipophilin expression might induce lipid-
droplet formation in macrophages. The function of
ADRP, the murine equivalent of human adipophilin,
has been analysed in murine fibroblasts and the results
showed that ADRP stimulated lipid accumulation and
lipid-droplet formation without induction of other adi-
pocyte-specific genes or other lipogenic genes [31].
More recently, ADRP-deficient mice were created
which showed reduced hepatic TG content as well as
protection from diet-induced fatty liver compared with
wild-type mice [32].
In macrophages incubated with AcLDL (100
lgÆmL
)1
), adipophilin overexpression resulted in eleva-

ted cellular TG content. Incubation of macrophages
with VLDL dramatically elevated the TG content of
both adipophilin-overexpressing and control cells. To
strengthen these results, we investigated the presence
of adipophilin around these lipid droplets using immu-
nofluorescence microscopy of THP-1 macrophages.
Our results confirmed the presence of adipophilin sur-
rounding all sizes of lipid droplet in THP-1 macro-
phages (Fig. 4). Moreover, droplet formation was
stimulated in cells overexpressing adipophilin and con-
versely, adipophilin expression was strongly increased
in macrophages loaded with VLDL (Fig. 3).
To determine whether exogenous lipid or cellular
adipophilin content was rate-limiting for cellular TG
accumulation, we quantitated TG in Ad.CMV.adipo-
philin-infected macrophages. The intracellular TG
content accumulation was dependent on the amount
of VLDL in the culture media, and no significant dif-
ferences were observed between Ad.CMV.adipophilin
and control macrophages incubated with 0–100
lgÆmL
)1
VLDL (Fig. 2). Because endogenous adipo-
philin expression is induced by VLDL loading of cells
(Fig. 3), these data do not indicate whether the level
of lipids or adipophilin is rate-limiting for TG accu-
0
20
40
60

80
100
120
140
160
Palmitate
Bromopalmitate
AICAR
lgirTy ( sedirecµ )nietorp llec gm/g
non-infected cells
Ad.CMV.GFP
Ad.CMV.adipophilin
-+ -
-
-
-
-
+
-
+
+
-
-
-
+
-
+
+
*
*

#
#
NS
NS
NS
##
##
NS
Fig. 9. Effect of adipophilin on TG content in THP-1 macrophages
grown in culture medium without or with 100 l
M palmitate, 100 l M
bromopalmitate and 500 lM AICAR. THP-1 macrophages were
infected with 500 m.o.i. of Ad.CMV.GFP and Ad.CMV.adipophilin.
Infected and noninfected cells were then incubated for 48 h at
37 °C with or without fatty acids and AICAR as indicated on the fig-
ure. *The difference between Ad.CMV.adipophilin cells and con-
trols in the absence of lipids or in the presence of palmitate or
palmitate + bromopalmitate was significant at P < 0.05. # The dif-
ference between controls in the presence of bromopalmitate and
controls in the presence of palmitate alone or bromopalmitate +
palmitate was significant at P < 0.05. ## The difference between
controls in the presence of bromopalmitate + palmitate and con-
trols in the absence of lipids or in the presence of palmitate alone
was significant at P < 0.05. NS, non significant.
G. Larigauderie et al. Adipophilin enhances triglyceride storage
FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS 3505
mulation. The mechanism of adipophilin stimulation
by VLDL has been described in murine macrophages
and shown to be dependent on activation of the nuc-
lear receptor PPARd [21]. To further investigate whe-

ther adipophilin or lipids were rate-limiting for lipid
accumulation, we incubated adipophilin-infected
macrophages with lipids and showed an increase in
the size of lipid droplets (Fig. 5D,I). In contrast, si-
RNA–adipophilin-transfected cells accumulated sub-
stantially less lipid in the presence of VLDL (Figs 5H
and 6). These results suggested that adipophilin-deple-
ted cells might take up less VLDL and clearly indica-
ted that adipophilin was rate-limiting for lipid
accumulation in human macrophages. This conclusion
is strengthened by our previous data showing that
siRNA–adipophilin-transfected macrophages accumu-
lated  50% less TG compared with control cells
[18]. An additional hypothesis to consider would be
that because lipids could not be stored in lipid drop-
lets in adipophilin-deficient macrophages; their distri-
bution may also be different under these conditions.
This latter hypothesis is strengthened by the fact that
although livers from ADRP-deficient and wild-type
mice showed similar total lipid abundance, ADRP-
deficient mice contained significantly less TG. In these
mice, subcellular distribution analyses revealed that
TG was reduced in the cytosolic fraction but
increased twofold in the microsomal fraction [32].
As shown in Fig. 3, human THP-1 macrophages
incubated for 48 h with 100 lgÆmL
)1
VLDL contained
 30 times more adipophilin protein level than control
cells. Adipophilin levels in VLDL-loaded control cells

were similar to those measured in VLDL-loaded
Ad.CMV.adipophilin-infected macrophages (data not
shown), and no differences in adipophilin staining was
observed between VLDL-loaded Ad.CMV.adipophilin-
and Ad.CMV.GFP-infected macrophages (Fig. 4G,H).
The data suggest that adipophilin in excess of the level
induced by lipid loading may be degraded. Consistent
with this, when adipophilin was overexpressed simply
with the adenovirus without added lipids, only a lim-
ited amount of adipophilin was retained by the nom-
inal amount of intracellular lipids, as observed by Oil
Red O staining (Fig. 5D). It appears that macrophages
adjust their adipophilin content depending on the
presence of cellular lipids and the ectopic expression of
adipophilin following by adenoviral infection is degra-
ded. Thus, adipophilin is rate-limiting for lipid
accumulation in human macrophages, and both its
expression and stability appear to be regulated by
lipids. This hypothesis is supported by the fact that in
the absence of ADRP, mice were resistant to diet-
induced fatty liver [32].
Because Ad.CMV.adipophilin-infected macrophages
contained significantly more lipid droplets than control
macrophages, we investigated the possible impact of
adipophilin on fatty acid oxidation by using the non-
metabolizable fatty acid bromopalmitate, an inhibitor
of fatty acid oxidation. In palmitate-loaded control
cells, bromopalmitate elevated cellular TG content,
which indicates that fatty acid oxidation is an ongoing
process in THP-1 macrophages. In contrast, in adipo-

philin-overexpressing cells loaded with palmitate,
bromopalmitate failed to increase the already elevated
level of TG. These results suggest that the presence of
an elevated pool of adipophilin is sufficient to protect
fatty acids from b-oxidation. To examine whether adi-
pophilin may increase cellular TGs by inhibition of
fatty acid oxidation, experiments were performed with
AICAR, which stimulates fatty acid oxidation by acti-
vation of AMP-activated protein kinase. Incubation of
cells with AICAR resulted in loss of enhanced TG
accumulation in adipophilin-overexpressing cells. The
data suggest that stimulation of fatty acid oxidation by
AICAR is dominant over the inhibition of fatty acid
oxidation by adipophilin. The mechanism by which
this occurs is unknown, but may be complex, because
adipophilin is not known to be phosphorylated by
AMP-activated kinase.
Another mechanism by which adipophilin might ele-
vate cellular TG is by stimulation of acyl-coenzyme A
synthetase and ⁄ or the incorporation of acyl-CoA into
TG. To address this, triacsin C was used to inhibit
acyl-coenzyme A synthetase, a key enzyme whose fatty
acyl-CoA products may be incorporated into TG or
become substrates for fatty acid oxidation. Inhibition
of acyl-coenzyme A synthetase abrogated the elevated
level of TG in adipophilin-overexpressing cells, sug-
gesting that increased TG in adipophilin-overexpress-
ing cells is due, at least in part, to elevated activity of
acyl-coenzyme A synthetase or to the downstream
incorporation of acyl-CoAs into TG.

However, neither enhanced adipophilin expression
nor its inhibition had an effect on whole-cell esterifica-
tion activity (data not shown). The elevated TG
pool in Ad.CMV.adipophilin-infected cells remained
increased after specific ACAT-1 inhibition, suggesting
that ACAT-1 was not implicated in the TG increase
and that the fatty acid pool utilized by ACAT-1 was
either very small compared with or not the same as
that used to generate intracellular TG. We also
assessed whether adipophilin protected TG from
hydrolysis. For this, cells were preloaded with oleate
followed by treatment with triacsin C to block acyl-
coenzyme A synthetase and hence fatty acid incorpor-
ation into TG. Under these conditions, no difference
Adipophilin enhances triglyceride storage G. Larigauderie et al.
3506 FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS
in TG content was observed between control and adi-
pophilin-overexpressing cells, which showed that adi-
pophilin does not protect TG from lipolysis. However,
we did observe a significant decrease in HSL activity
on exogenous substrate in lysates of Ad.CMV.adipo-
philin-infected cells compared with controls. This result
can be reconciled with the conclusion from the
triacsin C experiment that adipophilin does not protect
TG from lipolysis by the proposition that TG in lipid
droplets in adipophilin-overexpressing cells is relatively
inaccessible to HSL and other lipases. This interpret-
ation is in agreement with results obtained in murine
adipocytes and in vivo in ADRP-deficient mice show-
ing that there was no significant effect of adipophilin

on basal and isoproterenol-stimulated lipolysis [32].
Because HSL also hydrolyses CE, the reduced activity
of HSL may explain the elevated levels of CE meas-
ured in adipophilin-overexpressing cells [18]. Macro-
phages also contain CE hydrolase [33] and in future
studies it will be of interest to compare the effects of
adipophilin overexpression on macrophage expression
of these two lipases.
In summary, our results suggest that adipophilin
increases TG in macrophages by stimulating incorpor-
ation of acyl-CoA into TG as well as by inhibition of
fatty acid oxidation. This contrasts with findings in
ADRP-deficient mice, in which no difference in the
rates of fatty acid oxidation were observed between
ADRP-deficient and wild-type mice [32]. This discrep-
ancy may reflect differences between the roles of hep-
atic vs. nonhepatic adipophilin, species differences
(mouse vs. human) or methodological differences.
Concerning the latter, our conclusions regarding the
effects of adipophilin on fatty acid oxidation and TG
biosynthesis are largely based on TG mass measure-
ments in cells treated with different pharmacological
agents, whereas the disparate conclusions from adipo-
philin-deficient mice are based on radioisotopic meas-
ures in primary hepatocytes. We note that in
adipophilin knockout mice, TG and nonesterified fatty
acids accumulated in the microsomal compartment
where TG is synthesized [32]. These findings are in
agreement with our suggestion that adipophilin might
associate with intracellular fatty acids, which then

escape from b-oxidation pathways and are redirected
for esterification and storage. Thus, when lipids accu-
mulate inside the macrophage, such as occurs in conse-
quence to VLDL loading, adipophilin expression is
stimulated, and lipid transport or incorporation into
nascent or ongoing lipid droplets ensues.
In conclusion, we provided clear evidence that
adenoviral-mediated overexpression of human
adipophilin enhanced lipid-droplet formation in human
macrophages. Our results indicated that adipophilin
contributes to TG accumulation by stimulating the
generation and ⁄ or incorporation of fatty acyl CoAs
into TG and ⁄ or by inhibiting fatty acid oxidation.
Additional experiments are required to more precisely
define the mode of action of adipophilin in human
macrophages and its relevance in atherosclerosis.
Experimental procedures
Cell culture and siRNA transfection assays
Human monocytic THP-1 cells (ATTC TIB-202, LGC
Promochem, Molsheim, France) were maintained in RPMI-
1640 (BioWhittaker-Cambrex, Emerainville, France) con-
taining 25 mmolÆL
)1
Hepes buffer and 10% fetal bovine
serum (Eurobio, Courtaboeuf, France). Three days before
transfection, cells were seeded in six-well culture dishes
(Falcon
Ò
, Becton-Dickinson Labware, Franklin Lakes, NJ)
at a density of 1 · 10

6
cells ⁄ well. Differentiation of THP-1
monocytes to macrophages occurred in the presence of
160 nm phorbol 12-myristate 13-acetate (Sigma, Saint
Quentin, France) for 72 h [34]. Transfections of siRNA
were carried out as described previously [18]. About 80%
inhibition of adipophilin expression was obtained.
Recombinant adenovirus expression
Recombinant vectors were constructed using standard tech-
niques [35]. The full-length adipophilin cDNA was gener-
ated by RT-PCR from total RNA of THP-1 cells using
oligonucleotides designed to create XhoI(5¢) and MluI(3¢)
cutting sites. The digested fragment was cloned under the
control of the CMV promoter in the pShuttle-CMV vector
(Stratagene, La Jolla, CA). The recombinant adenovirus
was constructed in 293 cells by in vivo homologous recom-
bination between shuttle plasmids and pAdEASY-1 [36]
and plaque purified. High titre stocks of Ad.CMV.adipo-
philin and Ad.CMV.GFP (2.7 · 10
12
and 8.5 · 10
12
viral
particlesÆmL
)1
, respectively) were produced in 293 cells
and purified on CsCl gradients. THP-1 macrophages
(1 · 10
6
cells ⁄ well) were infected by highly purified adeno-

virus vectors at a m.o.i. of either 100 or 500 plaque-forming
units ⁄ cell in RPMI-1640, and 24 h later the infected macro-
phages were ready for further studies.
RNA analysis
Total RNA from THP-1 macrophages was extracted using
the RNeasy kit (Qiagen). For RT-PCR analyses, 5 lgof
total RNA was treated by DNAseI (Invitrogen Life Tech-
nologies, Cergy-Pontoise, France) and reverse transcribed
using random hexamer primers (Clontech Laboratories,
Mountainville, NJ) and M-MLV reverse transcriptase (Invi-
G. Larigauderie et al. Adipophilin enhances triglyceride storage
FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS 3507
trogen Life Technologies). For quantitative PCR, reverse-
transcribed transcripts were quantified using real-time PCR
on a MX4000 Multiplex Quantitative QPCR System (Strat-
agene), using specific oligonucleotide primers for human
adipophilin (5¢-CTGCTCACGAGCTGCATCATC-3¢ and
5¢-TGTGAGATGGCAGAGAACGGT-3¢). PCR amplifica-
tion was performed with the Brilliant Quantitative PCR
Core reagent Kit mix (Stratagene) in a volume of 25 lL
containing 100 nm of each primer and 4 mm MgCl
2
as
recommended by the manufacturer. The PCR conditions
were 95 °C for 10 min, followed by 40 cycles of 30 s at
95 °C, 30 s at 55 °C, and 30 s at 72 °C. Adipophilin
mRNA levels were normalized to 28S rRNA (5¢-AAA
CTCTGGTGGAGGTCCGT-3¢ and 5¢-CTTACCAAAAG
TGGCCCACTA-3¢).
Western blot analysis

Human THP-1 macrophages were infected with
Ad.CMV.adipophilin or Ad.CMV.GFP and adipophilin
and b-actin were identified in cell lysates by western blot-
ting as described previously [18]. Anti-adipophilin (mouse
monoclonal, Progen, Heidelberg, Germany) was used at a
1 : 1 dilution, whereas anti-(b-actin) (goat polyclonal, Santa
Cruz Biotechnology, Santa Cruz, CA) was diluted 1 : 500.
Lipoprotein isolation and acetylation
LDL (d ¼ 1.03–1.053) and VLDL (d ¼ 1.006–1.019) were
isolated from freshly drawn blood from healthy
normolipidaemic volunteers as described [37,38]. One mg
protein ⁄ mL sample of LDL was acetylated with acetic
anhydride as described previously [39].
Lipid analysis and Oil Red O staining
Cellular lipid content was determined as described previ-
ously [18]. For Oil Red O staining, THP-1 macrophages
were infected with adenovirus or transfected with siRNA as
described above. Thereafter, cells were fixed and stained
with Oil Red O (0.3% in 60% isopropanol) and hematoxy-
lin (Merck, Darmstadt, Germany), followed by extensive
washes with water. Cells were examined using a computer
supported Leica Leitz DMRB image analysis system (Leica,
Cambridge, UK). Images were captured using a CoolSnap
camera (Photometrics, Tucson, AZ). Lipid-droplet number
was quantified manually from digital images of 10 ran-
domly selected microscopic fields from at least three differ-
ent preparations for each condition.
Immunocytochemistry
THP-1 macrophages were cultured in two-well chamber
slides (LAB-TEK

Ò
II Nalge Nunc International, Roskilde,
Denmark) at a concentration of 1 · 10
6
cellsÆmL
)1
. The
chambers were rinsed with NaCl ⁄ P
i
and fixed in NaCl ⁄ P
i
containing 4% paraformaldehyde for 30 min at room tem-
perature. Following several washes with NaCl ⁄ P
i
, cells were
first incubated in NaCl ⁄ P
i
)0.1 m glycine for 15 min at
room temperature and then with a solution of 50 mm
NH
4
Cl in NaCl ⁄ P
i
. Slides were washed with NaCl ⁄ P
i
and
incubated in NaCl ⁄ P
i
containing 5% BSA for 30 min. For
immunostaining, the slides were incubated 1 h with guinea-

pig polyclonal antibodies against adipophilin (GP40, Pro-
gen, Heidelberg, Germany) diluted 1 : 200 in NaCl ⁄ P
i
–BSA
containing 0.05% Triton X-100. Slides used as negative
controls were incubated either with NaCl ⁄ P
i
–BSA–Triton
or with normal guinea-pig serum (Tebu-Bio, Le Perray en
Yvelines, France). After extensive washing, the slides were
incubated for 1 h in the dark with Texas Red-conjugated
affinity purified anti-(guinea-pig IgG) serum (Rockland,
Gilbertsville, PA) diluted 1 : 400 in NaCl ⁄ P
i
–BSA–Triton
containing 2% human normal serum. Cells were examined
using a computer-supported image analysis system Leica
Q500MC (Leica).
Analysis of fatty acid and TG metabolism
To assess the effect of inhibition of fatty acid synthesis on
TG accumulation, THP-1 macrophages infected with either
Ad.CMV.adipophilin or Ad.CMV.GFP were incubated for
16 h with 400 lm palmitic acid (Sigma) complexed to BSA,
to increase the storage of TG, either alone or with 2.5 l m
triacsin C (Sigma). Triglycerides were quantified and the
mass of TG was expressed relative to cell protein as des-
cribed previously [18]. To assess the effect of inhibition or
stimulation of b-oxidation on TG accumulation, THP-1
macrophages infected with Ad.CMV.adipophilin or
Ad.CMV.GFP were incubated for 48 h with 100 lm oleic

acid, palmitic acid (Sigma), bromopalmitate (Acros Organ-
ics, Noisy Le Grand, France) or AICAR complexed to
BSA. To assess the impact of inhibition of ACAT-1 on
TG accumulation, THP-1 macrophages infected with
Ad.CMV.adipophilin or Ad.CMV.GFP were incubated for
48 h with 400 lm palmitic acid and CAY10486 (SPI-BIO,
Montigny le Bretonneux, France) complexed to BSA. Cells
were rinsed sequentially with NaCl ⁄ P
i
–BSA and NaCl ⁄ P
i
.
Lipids were then extracted and TG was quantified and TG
mass was expressed relative to cell protein as described
previously [18].
HSL activity
THP-1 macrophages infected with either Ad.CMV.adipo-
philin or Ad.CMV.GFP and incubated for 48 h with
AcLDL were homogenized in 50 mm Tris ⁄ HCl buffer
(pH 7), 250 mm sucrose, and 5 lm EDTA. The homogen-
ates were sequentially centrifuged at 1500 g (10 min) and
Adipophilin enhances triglyceride storage G. Larigauderie et al.
3508 FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS
43 000 g (15 min) at 4 °C. Clarified 43 000 g supernatants
were used for measurement of HSL activity. Protein con-
tent of supernatants was determined using the technique
described by Peterson [40]. HSL activity was assayed as
neutral CE by following the release of [1-
14
C] oleic acid

from cholesteryl [1-
14
C]oleate as described by Nakamura
et al. [41] with minor modifications. The incubation reac-
tion in a final volume of 200 lL contained 100 nm potas-
sium phosphate buffer, pH 7.4, 0.025% BSA, 1.25 nmol
cholesteryl [1-
14
C]oleate ( 3 · 10
4
dpm) added in 4 lL
acetone, and 10 lg cell supernatant. After incubation at
37 °C (30 min), the reaction was terminated by addition of
1 mL of borate ⁄ carbonate buffer (0.1 m, pH 10.5) followed
by 3 mL of chloroform ⁄ methanol ⁄ heptane (1.39 : 1.28 : 1
v ⁄ v ⁄ v). The reaction tubes were vortexed vigorously for
1 min, centrifuged (1500 g for 20 min at 4 °C), and the
released [1-
14
C]oleate in the aqueous phase was determined
by scintillation counting. The results are expressed as pico-
moles [
14
C]oleate released ⁄ minute ⁄ milligram protein.
Statistical analysis
Statistical analyses were evaluated by Student’s t-tests and
probability values < 0.05 were considered significant.
Acknowledgements
This work was supported by grants from Genfit (Lille,
France), Fondation de France and the Fondation

Leducq and Acade
´
mie Nationale de Me
´
dicine (to
Clarisse Cuaz-Pe
´
rolin). We thank Dr Duverger for the
gift of triacsin C and the Vector Core of the University
Hospital of Nantes supported by the Association
Franc¸ aise contre les Myopathies (AFM) for providing
the Adenovirus vectors.
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