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

Adipophilin increases triglyceride storage in human macrophages by stimulation of biosynthesis and inhibition of b-oxidation Guilhem Larigauderie1,2,3, Clarisse Cuaz-Pe´ rolin1,2,3, Amena B. Younes4, Christophe Furman1,2,3, Catherine Lasselin1,2,3, Corinne Copin1,2,3, Michael Jaye5, Jean-Charles Fruchart1,2,3 and Mustapha Rouis1,2,3

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

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: mustapha.rouis@pasteur-lille.fr

(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- triglyceride content. Using inhibitors of ﬁcantly reduced intracellular 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.

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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 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-

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Results

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]. generated an adenovirus

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, increased (cid:1) 14 ± 0.8-

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 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 and (cid:1) 39 ± 7.8-fold, respectively, and adipophilin protein was increased (cid:1) 6.5 ± 1.7- and (cid:1) 38 ± 15-fold, respectively, com- pared with control cells (Fig. 1).

increase When cells were loaded with 100 lgÆmL)1 AcLDL, adipophilin overexpression resulted in a modest but in TG ((cid:1)1.3-fold, P < 0.05) signiﬁcant (Fig. 2) and altered cellular CE and free cholesterol

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].

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.

remains unclear.

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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 In this study, we investigated the impact of adipophilin over- expression or downregulation on lipid accumulation and droplet formation in human THP-1 macrophages.

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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 lgÆmL)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.

(Fig. 3). presence 10 In of

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(FC) content ((cid:1)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 and the 100 lgÆmL)1 VLDL, adipophilin increased (cid:1) 12 ± 2.8- and (cid:1) 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-

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control siRNA–GAPDH-transfected

lets in siRNA–adipophilin-transfected cells (Fig. 5H), whereas cells accumulated a lipid droplets large number of (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 (cid:1) 30% compared with control cells (Fig. 6). by which Potential mechanisms 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 (cid:1) 1.8 ± 0.71- and (cid:1) 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).

transfection of

adipophilin increased lipid content in THP-1 macrophages include the activity of lipid droplets against protection 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. contained GFP and noninfected cells, which all (cid:1) 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.

cells

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To further assess the impact of adipophilin levels on formation we manipulated adipophilin lipid-droplet in THP-1 macrophages by infection with levels cells Ad.CMV.adipophilin or by 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 cells comparison with Ad.CMV.GFP-infected (Fig. 5C). In agreement with our intracellular TG incubated with 100 lgÆmL)1 measurements, 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- 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

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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 lgÆmL)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).

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effect on ACAT-1 activity, given that similar amounts [14C]oleate incorporation into cholesteryl oleate of were measured in both cases (data not shown). To fur- ther probe this, we assessed the impact on TG accu- inhibition of ACAT-1 mulation of pharmacological 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 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

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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.

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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- 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,

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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. 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 (cid:1)C with 400 lM palmitate complexed to BSA in the pres- ence or absence of 2.5 lM triacsin C or for 48 h at 37 (cid:1)C with 400 lM palmitate complexed to BSA in the presence or absence of 60 lM 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.

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 [1-14C] oleic acid from cholesteryl [1-14C]oleate as described in Experimental procedures. *The difference between control and Ad.CMV.adipophilin cells was signiﬁcant at P < 0.001.

cate that enhanced accumulation of TG in adipophilin- overexpressing cells does not involve ACAT-1, because speciﬁc inhibition of ACAT-1 was without effect.

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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

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appears to be dominant over the inhibition of fatty acid oxidation by adipophilin.

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Fig. 9. Effect of adipophilin on TG content in THP-1 macrophages grown in culture medium without or with 100 lM palmitate, 100 lM 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 (cid:1)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.

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 stimulation of may convert induce lipid- human adipophilin expression might 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

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.

(Fig. 4). Moreover, droplet

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- formation was phages stimulated in cells overexpressing adipophilin and con- versely, adipophilin expression was strongly increased in macrophages loaded with VLDL (Fig. 3).

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cell protein, 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 respectively). Thus, stimulation of fatty acid oxidation by AICAR 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-

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cells

adipophilin was rate-limiting for

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 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. reduced in the cytosolic 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 adipophilin-infected incubated 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- lipid ted that accumulation in human macrophages. This conclusion is strengthened by our previous data showing that siRNA–adipophilin-transfected macrophages accumu- lated (cid:1) 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 fraction but increased twofold in the microsomal fraction [32].

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 to the downstream acyl-coenzyme A synthetase or incorporation of acyl-CoAs into TG.

rate-limiting for

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As shown in Fig. 3, human THP-1 macrophages incubated for 48 h with 100 lgÆmL)1 VLDL contained (cid:1) 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 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]. However, neither enhanced adipophilin expression nor its inhibition had an effect on whole-cell esteriﬁca- tion activity (data not shown). The elevated TG in Ad.CMV.adipophilin-infected cells remained pool 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

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Adipophilin enhances triglyceride storage

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

13-acetate

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(cid:2), Becton-Dickinson Labware, Franklin Lakes, NJ) at a density of 1 · 106 cells ⁄ well. Differentiation of THP-1 monocytes to macrophages occurred in the presence of 160 nm phorbol 12-myristate (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.

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.

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 · 1012 and 8.5 · 1012 viral particlesÆmL)1, respectively) were produced in 293 cells and puriﬁed on CsCl gradients. THP-1 macrophages (1 · 106 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.

in primary hepatocytes. We note that

RNA analysis

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 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.

Total RNA from THP-1 macrophages was extracted using the RNeasy kit (Qiagen). For RT-PCR analyses, 5 lg of 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-

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overexpression of In conclusion, we provided clear evidence that adenoviral-mediated human adipophilin enhanced lipid-droplet formation in human

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Adipophilin enhances triglyceride storage

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 MgCl2 as recommended by the manufacturer. The PCR conditions were 95 (cid:1)C for 10 min, followed by 40 cycles of 30 s at 95 (cid:1)C, 30 s at 55 (cid:1)C, and 30 s at 72 (cid:1)C. Adipophilin mRNA levels were normalized to 28S rRNA (5¢-AAA CTCTGGTGGAGGTCCGT-3¢ and 5¢-CTTACCAAAAG TGGCCCACTA-3¢).

THP-1 macrophages were

Denmark) at a concentration of 1 · 106 cellsÆmL)1. The chambers were rinsed with NaCl ⁄ Pi and ﬁxed in NaCl ⁄ Pi containing 4% paraformaldehyde for 30 min at room tem- perature. Following several washes with NaCl ⁄ Pi, cells were incubated in NaCl ⁄ Pi)0.1 m glycine for 15 min at ﬁrst room temperature and then with a solution of 50 mm NH4Cl in NaCl ⁄ Pi. Slides were washed with NaCl ⁄ Pi and incubated in NaCl ⁄ Pi 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 ⁄ Pi–BSA containing 0.05% Triton X-100. Slides used as negative controls were incubated either with NaCl ⁄ Pi–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 ⁄ Pi–BSA–Triton containing 2% human normal serum. Cells were examined using a computer-supported image analysis system Leica Q500MC (Leica).

Western blot analysis

Human 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.

Analysis of fatty acid and TG metabolism

from freshly

blood

LDL (d ¼ 1.03–1.053) and VLDL (d ¼ 1.006–1.019) were isolated from healthy drawn normolipidaemic volunteers as described [37,38]. One mg protein ⁄ mL sample of LDL was acetylated with acetic anhydride as described previously [39].

infected with Ad.CMV.adipophilin

Lipoprotein isolation and acetylation

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 lm 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 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 ⁄ Pi–BSA and NaCl ⁄ Pi. Lipids were then extracted and TG was quantiﬁed and TG mass was expressed relative to cell protein as described previously [18].

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.

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

THP-1 macrophages were cultured in two-well chamber slides (LAB-TEK(cid:2)II Nalge Nunc International, Roskilde,

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Immunocytochemistry

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Adipophilin enhances triglyceride storage

the absence of change in LPL mass. Biochim Biophys Acta 1631, 51–60.

5 Lindqvist P, Ostlund-Lindqvist AM, Witztum JL,

Steinberg D & Little JA (1983) The role of lipoprotein lipase in the metabolism of triglyceride-rich lipoproteins by macrophages. J Biol Chem 258, 9086–9092. 6 Evans AJ, Sawyez CG, Wolfe BM, Connelly PW,

Maguire GF & Huff MW (1993) Evidence that choles- teryl ester and triglyceride accumulation in J774 macro- phages induced by very low density lipoprotein subfractions occurs by different mechanisms. J Lipid Res 34, 703–717.

7 Rouis M, Nigon F, Eggerman TL, Brewer HB Jr &

Chapman MJ (1990) Apolipoprotein E expression by human monocyte-derived macrophages. Modulation by opsonised zymosan and cholesterol. Eur J Biochem 189, 447–453.

8 Rouis M, Nigon F, Lafuma C, Hornebeck W & Chap-

43 000 g (15 min) at 4 (cid:1)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 [1-14C] oleic acid [1-14C]oleate as described by Nakamura from cholesteryl 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 [1-14C]oleate ((cid:1) 3 · 104 dpm) added in 4 lL cholesteryl acetone, and 10 lg cell supernatant. After incubation at 37 (cid:1)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 (cid:1)C), and the released [1-14C]oleate in the aqueous phase was determined by scintillation counting. The results are expressed as pico- moles [14C]oleate released ⁄ minute ⁄ milligram protein.

man MJ (1990) Expression of elastase activity by human monocyte–macrophages is modulated by cellular choles- terol content, inﬂammatory mediators, and phorbol myristate acetate. Arteriosclerosis 10, 246–255.

Statistical analyses were evaluated by Student’s t-tests and probability values < 0.05 were considered signiﬁcant.

Statistical analysis

Acknowledgements

9 Lesnik P, Rouis M, Skarlatos S, Kruth HS & Chapman MJ (1992) Uptake of exogenous free cholesterol induces upregulation of tissue factor expression in human monocyte-derived macrophages. Proc Natl Acad Sci USA 89, 10370–10374.

10 Shiffman D, Mikita T, Tai JT, Wade DP, Porter JG,

Seilhamer JJ, Somogyi R, Liang S & Lawn RM (2000) Large scale gene expression analysis of cholesterol- loaded macrophages. J Biol Chem 275, 37324–37332. 11 Murphy DJ (2001) The biogenesis and functions of lipid bodies in animals, plants and microorganisms. Prog Lipid Res 40, 325–438.

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.

References

1 Kruth HS (2001) Macrophage foam cells and athero-

sclerosis. Front Biosci 6, D429–D455.

12 Pol A, Luetterforst R, Lindsay M, Heino S, Ikonen E & Parton RG (2001) A caveolin dominant negative mutant associates with lipid bodies and induces intracel- lular cholesterol imbalance. J Cell Biol 152, 1057–1070. 13 Gibbons GF, Islam K & Pease RJ (2000) Mobilisation of triacylglycerol stores. Biochim Biophys Acta 1483, 37–57.

2 Steinberg D, Parthasarathy S, Carew TE, Khoo JC &

Witztum JL (1989) Beyond cholesterol. Modiﬁcations of low-density lipoprotein that increase its atherogenicity. N Engl J Med 320, 915–924.

14 Jiang HP & Serrero G (1992) Isolation and characteri- zation of a full-length cDNA coding for an adipose differentiation-related protein. Proc Natl Acad Sci USA 89, 7856–7860.

15 Brasaemle DL, Barber T, Wolins NE, Serrero G,

Blanchette-Mackie EJ & Londos C (1997) Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein. J Lipid Res 38, 2249–2263.

3 Huff MW, Evans AJ, Sawyez CG, Wolfe BM & Nestel PJ (1991) Cholesterol accumulation in J774 macro- phages induced by triglyceride-rich lipoproteins. Com- parison of very low density lipoprotein from subjects with type III, IV, and V hyperlipoproteinemias. Arter- ioscler Thromb 11, 221–233.

16 Heid HW, Moll R, Schwetlick I, Rackwitz HR &

4 Milosavljevic D, Kontush A, Griglio S, Le Naour G,

Keenan TW (1998) Adipophilin is a speciﬁc marker of lipid accumulation in diverse cell types and diseases. Cell Tissue Res 294, 309–321.

Thillet J & Chapman MJ (2003) VLDL-induced trigly- ceride accumulation in human macrophages is mediated by modulation of LPL lipolytic activity in

FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS

3509

G. Larigauderie et al.

Adipophilin enhances triglyceride storage

17 Wang X, Reape TJ, Li X, Rayner K, Webb CL,

in the development of obesity-related insulin resistance. J Clin Invest 112, 1821–1830.

Burnand KG & Lysko PG (1999) Induced expression of adipophilin mRNA in human macrophages stimulated with oxidized low-density lipoprotein and in athero- sclerotic lesions. FEBS Lett 462, 145–150.

30 Charriere G, Cousin B, Arnaud E, Andre M, Bacou F, Penicaud L & Casteilla L (2003) Preadipocyte conver- sion to macrophage. Evidence of plasticity. J Biol Chem 278, 9850–9855.

18 Larigauderie G, Furman C, Jaye M, Lasselin C, Copin C, Fruchart JC, Castro G & Rouis M (2004) Adipophi- lin enhances lipid accumulation and prevents lipid efﬂux from THP-1 macrophages: potential role in atherogen- esis. Arterioscler Thromb Vasc Biol 24, 504–510.

31 Imamura M, Inoguchi T, Ikuyama S, Taniguchi S, Kobayashi K, Nakashima N & Nawata H (2002) ADRP stimulates lipid accumulation and lipid-droplet formation in murine ﬁbroblasts. Am J Physiol Endocri- nol Metab 283, E775–E783.

19 Buechler C, Ritter M, Duong CH, Orso E, Kapinsky M & Schmitz G (2001) Adipophilin is a sensitive marker for lipid loading in human blood monocytes. Biochim Biophys Acta 1532, 97–104.

32 Chang BH, Li L, Paul A, Taniguchi S, Nannegari V, Heird WC & Chan L (2006) Protection against fatty liver but normal adipogenesis in mice lacking adipose differentiation-related protein. Mol Cell Biol 26, 1063– 1076.

33 Ghosh S (2000) Cholesteryl ester hydrolase in human

20 Vosper H, Patel L, Graham TL, Khoudoli GA, Hill A, Macphee CH, Pinto I, Smith SA, Suckling KE, Wolf CR et al. (2001) The peroxisome proliferator-activated receptor delta promotes lipid accumulation in human macrophages. J Biol Chem 276, 44258–44265.

21 Chawla A, Lee CH, Barak Y, He W, Rosenfeld J, Liao D, Han J, Kang H & Evans RM (2003) PPARdelta is a very low-density lipoprotein sensor in macrophages. Proc Natl Acad Sci USA 100, 1268–1273.

monocyte ⁄ macrophage: cloning, sequencing, and expres- sion of full-length cDNA. Physiol Genomics 2, 1–8. 34 Lada AT, Rudel LL & Clair RW (2003) Effects of LDL enriched with different dietary fatty acids on cholesteryl ester accumulation and turnover in THP-1 macro- phages. J Lipid Res 44, 770–779.

22 Hodgkinson CPS (2003) Microarray analysis of peroxi- some proliferator-activated receptor-gamma induced changes in gene expression in macrophages. Biochem Biophys Res Commun 308, 505–510.

35 Rouis M, Adamy C, Duverger N, Lesnik P, Horellou P, Moreau M, Emmanuel F, Caillaud JM, Laplaud PM, Dachet C et al. (1999) Adenovirus-mediated overexpres- sion of tissue inhibitor of metalloproteinase-1 reduces atherosclerotic lesions in apolipoprotein E-deﬁcient mice. Circulation 100, 533–540.

23 Hirakata M, Tozawa R, Imura Y & Sugiyama Y (2004) Comparison of the effects of pioglitazone and rosiglita- zone on macrophage foam cell formation. Biochem Biophys Res Commun 323, 782–788.

36 He TC, Zhou S & da Costa LT, Yu J, Kinzler KW & Vogelstein B (1998) A simpliﬁed system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 95, 2509–2514.

24 Tomoda H, Igarashi K & Omura S (1987) Inhibition of acyl-CoA synthetase by triacsins. Biochim Biophys Acta 921, 595–598.

37 Havel RJ, Eder HA & Bragdon JH (1955) The distribu- tion and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 34, 1345–1353.

25 Tomoda H, Igarashi K, Cyong JC & Omura S (1991) Evidence for an essential role of long chain acyl-CoA synthetase in animal cell proliferation. Inhibition of long chain acyl-CoA synthetase by triacsins caused inhibition of Raji cell proliferation. J Biol Chem 266, 4214–4219.

26 Grimaldi PA, Knobel SM, Whitesell RR & Abumrad

38 Magret V, Elkhalil L, Nazih-Sanderson F, Martin F, Bourre JM, Fruchart JC & Delbart C (1996) Entry of polyunsaturated fatty acids into the brain: evidence that high-density lipoprotein-induced methylation of phos- phatidylethanolamine and phospholipase A2 are involved. Biochem J 316, 805–811.

39 Basu SK, Goldstein JL, Anderson GW & Brown MS

NA (1992) Induction of aP2 gene expression by nonme- tabolized long-chain fatty acids. Proc Natl Acad Sci USA 89, 10930–10934.

27 Buechler C, Ritter M, Duong CQ, Orso E, Kapinsky M & Schmitz G (2001) Adipophilin is a sensitive marker for lipid loading in human blood monocytes. Biochim Biophys Acta 1532, 97–104.

(1976) Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozy- gous familial hypercholesterolemia ﬁbroblasts. Proc Natl Acad Sci USA 73, 3178–3182.

40 Peterson GL (1977) A simpliﬁcation of the protein assay method of Lowry et al. which is more generally applic- able. Anal Biochem 83, 346–356.

41 Nakamura K, Inoue Y, Watanabe N & Tomita T

28 Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL & Ferrante AW Jr (2003) Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 112, 1796–1808.

29 Xu H, Barnes GT, Yang Q, Tan G, Yang D, Chou CJ, Sole J, Nichols A, Ross JS, Tartaglia LA & Chen H (2003) Chronic inﬂammation in fat plays a crucial role

(1988) Studies on cholesterol esterase in rat adipose tis- sue: comparison of substrates and regulation of the activity. Biochim Biophys Acta 963, 320–328.

FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS

3510