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 Larigauderie1,2,3, Clarisse Cuaz-Perolin1,2,3, 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 Inserm, U545, Lille, F-59019 France ´ ´ ´ Institut Pasteur de Lille, Departement d’Atherosclerose, Lille, F-59019 France ´ ´ Universite de Lille 2, Faculte de Pharmacie, Lille, F-59019 France Inserm IFR-17, Laboratoire de Microscopie Electronique, Lille, France GlaxoSmithKline, King of Prussia, PA, USA Keywords adipophilin; macrophage; atherosclerosis; lipid droplet; triglycerides Correspondence M Rouis, INSERM UR545, Institut Pasteur de Lille, rue du Professeur Calmette, 59019 Lille, France Fax: +33 20 87 73 60 Tel: +33 20 87 73 79 E-mail: mustapha.rouis@pasteur-lille.fr (Received 19 April 2006, revised 30 May 2006, accepted 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 adenovirus 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) dramatically 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 adipophilin expression using siRNA prevented lipid-droplet formation and significantly 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 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 glycosaminoglycans or antibodies which bind macrophages 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 G Larigauderie et al and promote LDL uptake by endocytosis [1] In addition, 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 pathway [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 contributes to cholesterol and TG retention within the vessel wall, but also alters macrophage biology Indeed, several 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 apolipoprotein 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 atheroma 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 differentiation-related protein (ADRP), a 50 kDa protein initially described in adipocytes [14], is a marker of lipid accumulation and is among the lipid dropletassociated proteins present in a variety of cells such as hepatocytes, adipocytes, muscle cells, mammary epithelial cells, fibroblasts, endothelial cells and macrophages [15,16] Macrophage expression of adipophilin is upregulated by oxLDL [17], acetylated LDL (AcLDL) [18], enzymatically modified LDL [19] and by synthetic agonists of the peroxisome proliferator-activated nuclear receptors d (PPARd) [20,21] and c (PPARc) [22,23] However, 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 overexpression or downregulation on lipid accumulation and droplet formation in human THP-1 macrophages Adipophilin enhances triglyceride storage 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 differentiated into macrophages with phorbol esters were able to rapidly take up AcLDL and to subsequently develop a foam cell-like morphology Under these conditions, adipophilin expression was enhanced dramatically [18] To further study the function of adipophilin in human macrophages, we generated an adenovirus vector-expressing human adipophilin (Ad.CMV.adipophilin) Using the control Ad.CMV.GFP vector, we demonstrated nearly 100% infection of THP-1 macrophages (data not shown) We assessed adipophilin expression using both quantitative PCR and immunoblotting 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, compared with control cells (Fig 1) When cells were loaded with 100 lgỈmL)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 Expression of adipophilin protein and mRNA in adenovirusinfected 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 FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ê 2006 FEBS 3499 Concentration (àg TG/mg cell protein) Adipophilin enhances triglyceride storage G Larigauderie et al 200 non-infected cells 175 Ad.CMV.GFP 150 Ad.CMV.adipophilin 125 100 * 75 50 25 No added lipids 10 100 50 VLDL (µg/ml) 10 100 AcLDL (µg/ml) Fig 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 lgỈmL)1 VLDL or AcLDL (10, 100 lgỈmL)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 lgỈmL)1 AcLDL was significant at P < 0.05 Fig Expression of adipophilin protein levels in human THP-1 macrophages loaded with VLDL THP-1 macrophages were incubated with 0, 10 or 100 lgỈmL)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 independent experiments *The difference between control and cells incubated with VLDL was significant at P < 0.01 (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 lgỈmL)1 VLDL or 10 lgỈmL)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 lgỈmL)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 following incubation with VLDL is due to the dramatic 3500 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 immunoblotting (Fig 3) In the presence of 10 and 100 lgỈmL)1 VLDL, adipophilin increased  12 ± 2.8and  28 ± 5.2-fold relative to control macrophages cultured in lipid-free medium Thus, elevation of cellular 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- FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS G Larigauderie et al philin-infected macrophages with 10 and 100 lgỈmL)1 VLDL for 48 h and measured adipophilin levels by immunoblotting On top of the already elevated level of adipophilin expression in Ad.CMV.adipophilininfected cells, 10 and 100 lgỈmL)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 comparison, no significant difference in adipophilin expression was observed in Ad.CMV.adipophilin-infected macrophages loaded or not with 10 and 100 lgỈmL)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 lgỈmL)1 AcLDL for 24 h showed a significant increase in lipid-droplet formation (Fig 4F) compared with AcLDL-loaded control cells (Fig 4E) or adipophilinoverexpressing 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 cytoplasm following Oil Red O staining (Fig 5A,C,I) However, incubation of adipophilin-infected macrophages 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 lgỈmL)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 lgỈmL)1 VLDL for 24 h, there was a substantial reduction in the number and size of lipid drop- Adipophilin enhances triglyceride storage 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 lgỈmL)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 (ACAT-1) esterification 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 infection (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 synthesis, 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 triacsin C, adipophilin-overexpressing cells produced more TG from palmitate than control cells (133.6 ± 8.4 vs 95 ± 9.7 lgỈmg)1 cell protein, respectively) The addition 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 FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS 3501 Adipophilin enhances triglyceride storage G Larigauderie et al A B C D E F G Fig Immunocytochemical analysis of adipophilin in THP-1 macrophages (magnification, 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 lgỈmL)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 lgỈmL)1) (F) Cells infected with Ad.CMV.adipophilin, then incubated for 24 h with AcLDL (100 lgỈmL)1) (G) Cells infected with Ad.CMV.GFP, then treated for 24 h with VLDL (100 lgỈmL)1) (H) Cells infected with Ad.CMV.adipophilin, then incubated for 24 h with VLDL (100 lgỈmL)1) H levels (66.5 ± 11.2 and 61.5 ± 10.7 lgỈmg)1 cell protein, respectively) (Fig 8) These results suggest that increased TG in cells overexpressing adipophilin is at least partly due to acyl-coenzyme A synthetase elevated activity or to the downstream incorporation of acyl-CoAs into TG Lipid esterification was quantified in either THP-1 macrophages overexpressing adipophilin (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 3502 effect on ACAT-1 activity, given that similar amounts of [14C]oleate incorporation into cholesteryl oleate were measured in both cases (data not shown) To further probe this, we assessed the impact on TG accumulation of pharmacological inhibition of ACAT-1 in Ad.CMV.adipophilin-infected and control THP-1 macrophages Enhanced TG accumulation in adipophilin-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 FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS G Larigauderie et al Adipophilin enhances triglyceride storage A C D E Fig 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 supplemented with 10% fetal bovine serum and 100 lgỈmL)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 incubated in RPMI-1640 supplemented with 100 lgỈmL)1 VLDL (F) Cells infected with 500 m.o.i of Ad.CMV.adipophilin then incubated in RPMI-1640 supplemented with 100 lgỈ mL)1 VLDL (G) siRNA–GAPDH-transfected cells incubated in RPMI-1640 containing 100 lgỈmL)1 (H) siRNA–adipophilintransfected cells incubated in RPMI-1640 containing 100 lgỈmL)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 B F G H I 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 lgỈmg)1 cell protein, 1.5-fold) were similar when cells were coincubated with palmitate plus CAY10486 (150.2 ± 7.6 lgỈmg)1 cell protein, 1.6-fold) These results indi- FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS 3503 Adipophilin enhances triglyceride storage G Larigauderie et al NS * Triglycerides (µg/mg cell protein) 160 140 non-infected cells Ad.CMV.GFP Ad.CMV.adipophilin * 120 100 80 60 40 20 No added lipids Fig 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, incubated with 10 or 100 lgỈmL)1 VLDL or AcLDL (10, 100 lgỈmL)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 control, Ad.CMV.GFP or Ad.CMV.Adipophilin-infected macrophages (without VLDL) was significant at P < 0.05 Fig 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 adipophilinoverexpressing 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 adipophilin in THP-1 macrophages TG accumulation in Ad.CMV.GFP- and Ad.CMV.adipophilin-infected cells incubated in the presence of palmitate (positive con3504 Palmitate Palmitate + triacsin C CAY10486 Palmitate + CAY10486 Fig Triglycerides synthesis is inhibited by triacsin C but not by an ACAT-1 inhibitor in THP-1 macrophages overexpressing adipophilin 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 lM palmitate complexed to BSA in the presence or absence of 2.5 lM triacsin C or for 48 h at 37 °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 macrophages), palmitate + triacsin C, CAY10486 or palmitate + CAY10486 (only control THP-1 macrophages) was significant at P < 0.01 NS, non significant trol) was compared with TG accumulation in cells incubated with palmitate plus bromopalmitate, a nonmetabolized 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 loaded with 100 lm fatty acids for 48 h Adipophilininfected cells accumulated 1.4 times more TG than control cells (Fig 9) No differences in TG accumulation were observed between any of the cell-treatment groups when cells were incubated only with bromopalmitate, 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; inhibition 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 palmitate alone or with bromopalmitate plus palmitate This FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ê 2006 FEBS Triglycerides (àg/mg cell protein) G Larigauderie et al 160 140 Adipophilin enhances triglyceride storage appears to be dominant over the inhibition of fatty acid oxidation by adipophilin non-infected cells Ad.CMV.GFP Ad.CMV.adipophilin NS * 120 ## ## * 80 Discussion NS 100 NS NS # 60 # 40 20 Palmitate Bromopalmitate AICAR - + - + - + + - + + + Fig 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 °C with or without fatty acids and AICAR as indicated on the figure *The difference between Ad.CMV.adipophilin cells and controls in the absence of lipids or in the presence of palmitate or palmitate + bromopalmitate was significant at P < 0.05 # The difference 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 controls in the absence of lipids or in the presence of palmitate alone was significant at P < 0.05 NS, non significant suggests that the enhanced TG content in adipophilinoverexpressing 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 macrophages with 5’-phosphoribosyl-5-aminoimidazole-4carboxamide (AICAR), which stimulates fatty acid oxidation by activation of AMP-activated protein kinase 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 palmitate-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-overexpressing cells were similar to control cells (57.0 ± 6.2 and 63.0 ± 6.0 lgỈmg)1 cell protein, respectively) Thus, stimulation of fatty acid oxidation by AICAR We studied the impact of adenoviral-mediated overexpression of adipophilin in THP-1-derived macrophages on the accumulation of TG when the cells were incubated 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 macrophages 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 lipiddroplet 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 adipocyte-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 lgỈmL)1), adipophilin overexpression resulted in elevated 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 immunofluorescence microscopy of THP-1 macrophages Our results confirmed the presence of adipophilin surrounding all sizes of lipid droplet in THP-1 macrophages (Fig 4) Moreover, droplet formation was stimulated in cells overexpressing adipophilin and conversely, 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.adipophilin-infected macrophages The intracellular TG content accumulation was dependent on the amount of VLDL in the culture media, and no significant differences were observed between Ad.CMV.adipophilin and control macrophages incubated with 0–100 lgỈmL)1 VLDL (Fig 2) Because endogenous adipophilin expression is induced by VLDL loading of cells (Fig 3), these data not indicate whether the level of lipids or adipophilin is rate-limiting for TG accu- FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS 3505 Adipophilin enhances triglyceride storage G Larigauderie et al mulation The mechanism of adipophilin stimulation by VLDL has been described in murine macrophages and shown to be dependent on activation of the nuclear receptor PPARd [21] To further investigate whether 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, siRNA–adipophilin-transfected cells accumulated substantially less lipid in the presence of VLDL (Figs 5H and 6) These results suggested that adipophilin-depleted cells might take up less VLDL and clearly indicated 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 accumulated  50% less TG compared with control cells [18] An additional hypothesis to consider would be that because lipids could not be stored in lipid droplets in adipophilin-deficient macrophages; their distribution 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, ADRPdeficient 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 lgỈmL)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.adipophilinand 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 limited amount of adipophilin was retained by the nominal 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 degraded 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 dietinduced fatty liver [32] 3506 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 nonmetabolizable 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 adipophilin-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 adipophilin may increase cellular TGs by inhibition of fatty acid oxidation, experiments were performed with AICAR, which stimulates fatty acid oxidation by activation 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 elevate 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, suggesting that increased TG in adipophilin-overexpressing 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 esterification 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 acylcoenzyme A synthetase and hence fatty acid incorporation into TG Under these conditions, no difference FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS G Larigauderie et al in TG content was observed between control and adipophilin-overexpressing cells, which showed that adipophilin does not protect TG from lipolysis However, we did observe a significant decrease in HSL activity on exogenous substrate in lysates of Ad.CMV.adipophilin-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 interpretation is in agreement with results obtained in murine adipocytes and in vivo in ADRP-deficient mice showing 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 measured in adipophilin-overexpressing cells [18] Macrophages 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 incorporation 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 discrepancy may reflect differences between the roles of hepatic 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 measurements in cells treated with different pharmacological agents, whereas the disparate conclusions from adipophilin-deficient mice are based on radioisotopic measures 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 accumulate inside the macrophage, such as occurs in consequence 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 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 Cell culture and siRNA transfection assays Human monocytic THP-1 cells (ATTC TIB-202, LGC Promochem, Molsheim, France) were maintained in RPMI1640 (BioWhittaker-Cambrex, Emerainville, France) containing 25 mmolỈL)1 Hepes buffer and 10% fetal bovine serum (Eurobio, Courtaboeuf, France) Three days before transfection, cells were seeded in six-well culture dishes (FalconÒ, Becton-Dickinson Labware, Franklin Lakes, NJ) at a density of · 106 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 techniques [35] The full-length adipophilin cDNA was generated 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 recombination between shuttle plasmids and pAdEASY-1 [36] and plaque purified High titre stocks of Ad.CMV.adipophilin and Ad.CMV.GFP (2.7 · 1012 and 8.5 · 1012 viral particlesỈmL)1, respectively) were produced in 293 cells and purified on CsCl gradients THP-1 macrophages (1 · 106 cells ⁄ well) were infected by highly purified adenovirus vectors at a m.o.i of either 100 or 500 plaque-forming units ⁄ cell in RPMI-1640, and 24 h later the infected macrophages were ready for further studies RNA analysis Total RNA from THP-1 macrophages was extracted using the RNeasy kit (Qiagen) For RT-PCR analyses, lg of total RNA was treated by DNAseI (Invitrogen Life Technologies, Cergy-Pontoise, France) and reverse transcribed using random hexamer primers (Clontech Laboratories, Mountainville, NJ) and M-MLV reverse transcriptase (Invi- FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS 3507 Adipophilin enhances triglyceride storage G Larigauderie et al trogen Life Technologies) For quantitative PCR, reversetranscribed transcripts were quantified using real-time PCR on a MX4000 Multiplex Quantitative QPCR System (Stratagene), using specific oligonucleotide primers for human adipophilin (5¢-CTGCTCACGAGCTGCATCATC-3¢ and 5¢-TGTGAGATGGCAGAGAACGGT-3¢) PCR amplification 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 mm MgCl2 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 blotting as described previously [18] Anti-adipophilin (mouse monoclonal, Progen, Heidelberg, Germany) was used at a : dilution, whereas anti-(b-actin) (goat polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA) was diluted : 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 previously [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 hematoxylin (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 randomly selected microscopic fields from at least three different preparations for each condition Immunocytochemistry THP-1 macrophages were cultured in two-well chamber slides (LAB-TEKÒII Nalge Nunc International, Roskilde, 3508 Denmark) at a concentration of · 106 cellsỈmL)1 The chambers were rinsed with NaCl ⁄ Pi and fixed in NaCl ⁄ Pi containing 4% paraformaldehyde for 30 at room temperature Following several washes with NaCl ⁄ Pi, cells were first incubated in NaCl ⁄ Pi)0.1 m glycine for 15 at 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 For immunostaining, the slides were incubated h with guineapig polyclonal antibodies against adipophilin (GP40, Progen, Heidelberg, Germany) diluted : 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 h in the dark with Texas Red-conjugated affinity purified anti-(guinea-pig IgG) serum (Rockland, Gilbertsville, PA) diluted : 400 in NaCl ⁄ Pi–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 lm triacsin C (Sigma) Triglycerides were quantified and the mass of TG was expressed relative to cell protein as described 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 Organics, 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] HSL activity THP-1 macrophages infected with either Ad.CMV.adipophilin 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 lm EDTA The homogenates were sequentially centrifuged at 1500 g (10 min) and FEBS Journal 273 (2006) 3498–3510 ª 2006 The Authors Journal compilation ª 2006 FEBS G Larigauderie et al 43 000 g (15 min) at °C Clarified 43 000 g supernatants were used for measurement of HSL activity Protein content 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 from cholesteryl [1-14C]oleate as described by Nakamura et al [41] with minor modifications The incubation reaction in a final volume of 200 lL contained 100 nm potassium phosphate buffer, pH 7.4, 0.025% BSA, 1.25 nmol cholesteryl [1-14C]oleate ( · 104 dpm) added in lL acetone, and 10 lg cell supernatant After incubation at 37 °C (30 min), the reaction was terminated by addition of mL of borate ⁄ carbonate buffer (0.1 m, pH 10.5) followed by mL of chloroform ⁄ methanol ⁄ heptane (1.39 : 1.28 : v ⁄ v ⁄ v) The reaction tubes were vortexed vigorously for min, centrifuged (1500 g for 20 at °C), and the released [1-14C]oleate in the aqueous phase was determined by scintillation counting The results are expressed as picomoles [14C]oleate released ⁄ minute ⁄ milligram protein Adipophilin enhances triglyceride storage 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 Academie Nationale de Medicine (to ´ Clarisse Cuaz-Perolin) We thank Dr Duverger for the gift of triacsin C and the Vector Core of the University Hospital of Nantes supported by the Association Francaise contre les Myopathies (AFM) for providing ¸ the Adenovirus vectors 10 11 12 References 13 Kruth HS (2001) Macrophage foam cells and atherosclerosis Front Biosci 6, D429–D455 Steinberg D, Parthasarathy S, Carew TE, Khoo JC & Witztum JL 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cells), nominal diffuse cytoplasmic adipophilin staining was observed