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Guilhem Larigauderie; Christophe Furman; Michael Jaye; Catherine Lasselin; Corinne Copin; Jean-Charles Fruchart; Graciela Castro; Mustapha Rouis
From the Department of Atherosclerosis (G.L., C.F., C.L., C.C., J.-C.F., G.C., M.R.), SERLIA-INSERM UR545, Institut Pasteur de Lille, France; and GlaxoSmithKline (M.J.), King of Prussia, PA.
Correspondence to Mustapha Rouis, Department of Atherosclerosis, SERLIA-INSERM UR545, Institut Pasteur de Lille, 1 rue du Professeur Calmette, 59019 Lille, France. E-mail mustapha.rouis@pasteur-lille.fr
Abstract
Objective— Uptake of modified low-density lipoprotein (LDL) by macrophages through scavenger receptors results in lipid droplets accumulation and foam cell formation. Excess lipid deposition in macrophages has been reported to modulate expression of several genes including adipophilin. In this study, we investigated the function of adipophilin in lipid accumulation and cholesterol efflux in THP-1 macrophages.
Methods and Results— Adipophilin mRNA expression was 3.5-fold higher in human atherosclerotic plaques compared with healthy areas of the same arteries. Moreover, in the presence of acetylated LDL (AcLDL), triglycerides and cholesteryl esters were increased in macrophages overexpressing adipophilin by 40% and 67%, respectively, whereas their accumulation was reduced when endogenous cellular adipophilin was depleted using siRNA approach. In addition, neither overexpression nor downregulation of adipophilin altered expression of genes involved in lipid efflux. However, the affinity and the number of AcLDL receptors were not affected. After 24-hour incubation of lipid-loaded macrophages with apolipoprotein A-I, cholesterol efflux was reduced by 47% in adipophilin transfected cells versus control cells.
Conclusion— Our results showed that stimulation of adipophilin expression in macrophages by modified LDL promotes triglycerides and cholesterol storage and reduces cholesterol efflux. Therefore, adipophilin might contribute, in vivo, to lipid accumulation in the intima of the arterial wall.
Key Words: adipophilin ? macrophage ? acetylated LDL ? cholesterol efflux ? atherosclerosis
Introduction
Atherosclerosis, a complex disease process, is initiated by an infiltration of low-density lipoproteins (LDL) into the subendothelial space where they accumulate and become modified mainly by oxidation. In parallel, chemokines elaborated by endothelial cells attract circulating monocytes into the intima, where they differentiate into macrophages.1 Intimal macrophages contribute to the formation of arterial lesions by accumulating large amounts of cholesteryl ester (CE) through the uptake of modified lipoproteins such as oxidized LDL (oxLDL) by a variety of mechanisms, including the scavenger pathway.2,3 Several studies indicated that oxLDL has a number of diverse effects on macrophage function including growth stimulation4 proinflammatory effects such as expression of inflammatory cytokines,5 increase in cytotoxicity and expression of metalloproteinases,6 inhibition of expression of inducible nitric oxide synthase,7 and effects on lipid metabolism and accumulation.8,9 Using either a DNA array approach10 or a subtractive approach,11 numerous genes have been shown to be regulated on exposure of THP-1 cells to modified LDL. Adipophilin, or adipose differentiation-related protein (ADRP), a 50-kDa protein initially described in adipocytes,12 which is considered a marker of lipid accumulation, is among those genes upregulated by modified LDL.
Adipophilin is found associated with intracellular lipid droplets in a variety of cells and tissues that store or synthesize lipids.13,14 In addition to oxLDL,15 acetylated LDL (AcLDL)11 or enzymatically modified LDL,16 the nuclear receptor PPAR, is involved in lipid accumulation in human macrophages and THP-1-derived macrophages, and also increases adipophilin expression.17 However, the precise role of adipophilin in macrophage foam cell formation and, in turn, in the development of atherosclerotic lesions remains unclear. In this article, we evaluate the expression of adipophilin in human atherosclerotic lesions and examine its function in lipid accumulation and cholesterol efflux in human macrophages.
Methods
Cell Culture
Human monocytic THP-1 cells (ATTC) were maintained in RPMI 1640 medium (BioWhittaker; Cambrex, Belgium) containing 25 mmol/L HEPES buffer and 10% fetal calf serum (FCS). Three days before transfection, cells were seeded in 6-well culture dishes (Falcon; Becton Dickinson Labware) at a density of 2x106 cells/well. Differentiation of THP-1 monocytes to macrophages occurred in presence of 160 nM of phorbol 12-myristate 13-acetate (Sigma, France) for 72 hours.18
Plasmids and Transient Transfection Assays
Human adipophilin cDNA was amplified from macrophage mRNA by PCR using oligonucleotides designed to create XhoI (5') and MluI (3') cutting sites. The digested fragments were cloned into the pCI mammalian expression vector (Promega, France). Thereafter, the integrity of the insert was verified by nucleotide sequencing. THP-1 cells, grown in 6-well culture dishes in RPMI 1640 supplemented with 10% FCS, were transiently transfected using Effectene Transfection Reagent (Qiagen, France). The transfection efficiency using 500 ng of the pEGFP-C3 expression vector (Clontech Laboratories) represented approximately 15%. The Effectene–DNA complexes were not significantly toxic, as determined by quantitating adherent and nonadherent cells and by the trypan blue exclusion method. Cell viability was >95%.
Lipoprotein Isolation and Acetylation
LDL (d=1.03 to 1.053) were isolated from freshly drawn blood from healthy normolipidemic volunteers as described.19 As previously described, 1 mg protein/mL of LDL was acetylated with acetic anhydride.20 The level of acetylation was assessed by electrophoresis in cellulose acetate gels (Cellogel, SEBIA, France) and by the evaluation of the percent of amino acid acetylation according to the procedure of Habeeb AF.21 These modified LDL particles had 83% acetylated lysine residues.
Cell Association, Degradation, and Binding of Iodine 125–AcLDL
AcLDL were labeled with 125I, using the method reported earlier.22 The specific activity of 125I– AcLDL was 429 cpm/ng. Lipoprotein uptake and degradation were determined after incubation of the cells with 125I–AcLDL at 37°C for 4 hours and lipoprotein binding was determined after incubation of the cells with 125I–AcLDL for 4 hours at 4°C. Cell-associated radioactivity was measured after digestion of the cells with 1 N NaOH for 1 hour at room temperature. Lipoprotein degradation was determined as TCA-soluble, noniodide 125I in the postincubation medium.22 To determine the specific uptake and degradation via the AcLDL receptors, incubations with 125I–AcLDL were performed in the absence or presence of a 30-fold excess of unlabeled AcLDL. The specific binding and degradation were calculated by subtracting the nonspecific value from the total value.
Cholesterol Efflux
AcLDL (3 mg protein) were labeled by incubating 100 μCi of [1, 2(n)-3H]cholesterol (38 Ci/mmol; Amersham Biosciences, France) overnight at 37°C. THP-1 cells were loaded by incubation with 100 μg/mL of 3H-cholesterol-AcLDL (specific activity 9400 cpm/μg AcLDL protein.) for 48 hours in base medium containing 1% FCS. Cholesterol efflux was performed as described earlier.23 The radioactivity present in pCI, pCI–adipophilin, and siRNA-GAPDH transfected cells before efflux was between 2246±111 and 2531±90 cpm/μg cell protein. The radioactivity present in siRNA–adipophilin transfected cells was 1420±69 cpm/μg cell protein. After the last time point, cells were washed twice with PBS, dissolved in 0.1 mol/L NaOH, and assayed for protein concentration.
Lipid Extraction
Cells were washed three times with cold PBS and incubated with hexane/isopropanol (3v/2v). Total lipid extracts were evaporated to dryness under nitrogen and the residue was dissolved in 300 μL of isopropanol. Cellular lipid concentrations were determined by enzymatic assays using kits from bioMérieux (SA, France) for triglycerides (TG) and total cholesterol or from Wako (Chemicals Gmbh, Germany) for free cholesterol (FC). Esterified cholesterol mass was calculated as the difference between total and FC. Protein concentration was measured by the method of Lowry et al.24
Carotid Endarterectomy
Carotid endarterectomy specimens were obtained from the Department of Cardiovascular Surgery (CHRU, Lyon, France) according to the protocol described by Legedz et al25 for patients with severe carotid occlusive disease and subjected to Stary classification. All samples were obtained by qualified hospital staff, and all procedures were approved by the local human ethics committee.
RNA Analysis
Total RNA from THP-1 macrophages and carotid endoterectomy specimens were extracted using the RNeasy kit (Qiagen, France). For RT-PCR analyses, 5 μg of total RNA was treated by DNAse I (Life Technologies, France), reverse transcribed using random hexamer primers (Clontech Laboratories), and 17 to 25 rounds of PCRs were performed with primer sets for human adipophilin (5'-CTG-CTCACGAGCTGCATCATC-3' and 5'-TGTGAGATGGCA-GAGAACGGT-3'), and human 18S RNA (5'-CGAAGACGATC-AGATACCGTCGTAG-3' and 5'-AAGGGCATCACAGACCTG-TTATTG-3') as control. The resulting products were separated on a 2% agarose gel and stained with ethidium bromide.
siRNA Preparation and Transfection
The sequences for siRNA were designed and transcribed according to the Silencer siRNA Construction Kit instructions (Ambion Europe). Human adipophilin-specific siRNAs were positioned at the 5', 3', or medial portions of adipophilin mRNA and were compared with sequences in the human genome database to confirm that no other genes were targeted. A sequence targeting human GAPDH, purchased from Ambion, was used as a siRNA control. On the day of transfection, THP-1 macrophage cells were at 50% to 70% confluency. Transfections of siRNA were performed using jetSI according to the manufacturer’s instructions (Qbiogene, France). The oligonucleotide sequences designed to construct siRNA-adipophilin used in this study were: 5'-AAGCTAGAGCCGCAAATTGCACTTG-TCTC-3' and 5'-AATGCAATTTGCGGCTCTAGCCCTGTCTC-3'.
Western Blot Analysis
Transfected cells were washed three times with PBS, lysed in lysis buffer (PBS containing 1% Triton X100, 0.5% deoxycholate, 10 mmol/L Na pyruvate phosphate, 2 mmol/L Na vanadate, 100 mmol/L NaF, aprotinin, 0.5 mmol/L PMSF, ICN protease inhibitor cocktail) and centrifuged for 30 minutes at 10 000g, 4°C. Western blot analysis was performed after SDS-PAGE of cell lysates in the Cuve Mini Protean system (Bio-Rad SA, France). Proteins were transferred to Hybond ECL nitrocellulose membranes (Amersham Biosciences), and after incubation with first and second antibodies, were detected by ECL (Amersham Biosciences). Bands were normalized to ?-actin and expressed as a percent of control. Primary antibodies were used in different dilutions as follows: anti-adipophilin 1:1 (mouse monoclonal; Progen), anti-perilipin 1:2000 (guinea pig polyclonal; Progen), anti-?-actin 1:500 (goat polyclonal; Santa Cruz), anti-ABCA1 (ATP-binding cassette transporter A1) 1:500 (goat polyclonal; Santa Cruz), anti hSR-BI (human scavenger receptor B1) 1:500 (rabbit polyclonal; a gift from Dr Fruchart), anti-SR-A (scavenger receptor A) 1:250 (rabbit polyclonal; Santa Cruz), anti-CD36 (membrane glycoprotein belonging to the class B scavenger receptor family) 1:250 (rabbit polyclonal; Santa Cruz), and anti-apoE (apolipoprotein E) 1:200 (mouse monoclonal; a gift from Dr Fruchart).
Statistical Analysis
Statistical analyses were evaluated by Student t tests. P<0.05 was considered significant.
Results
On treatment with phorbol esters, THP-1 cells take on the characteristics of mature macrophages, including decreased LDL receptors expression and increased expression of scavenger receptors.26 These changes in gene expression enable the cells to take-up modified lipoproteins and subsequently to develop a foam cell-like morphology. Thus, by using these cells, we have shown that adipophilin expression is dramatically enhanced in the presence of AcLDL (4-fold increase with 100 μg/mL) in comparison with nontreated control cells (Figure I, available online at http://atvb.ahajournals.org). In addition, we evaluated the expression of adipophilin in human atherosclerotic plaques in which lipid-rich macrophages are abundant. Our results indicated that adipophilin expression was 3.5-fold higher in atherosclerotic lesions (n=5; P<0.001) compared with healthy areas of the same artery (n=5) (Figure II, available online at http://atvb.ahajournals.org). Approximately 70% of the total adipophilin mRNA in atheromatous tissue can be attributed to lipid-rich macrophages (CD68+ cells) isolated by laser microdissection from the same arterial lesions (data not shown).
To study the function of adipophilin in human macrophages, we transfected THP-1 macrophages with mammalian expression vector encoding human adipophilin and the results indicated 2-fold increase of adipophilin expression in comparison to cells transfected with the empty vector (data not shown). Expression of ABCA1 and apoE, known to be involved in cholesterol efflux, and expression of hSR-BI, SR-A and CD36, implicated in macrophage lipid accumulation, were affected neither by the transfection procedure nor by the elevated expression of adipophilin (Figure 1A).
Figure 1. Effect of adipophilin overexpression and downregulation on ABCA1, perilipin, hSR-BI, CD36, SR-A, and apoE expression in THP-1 macrophages. Total proteins were isolated and samples of 15 to 30 μg protein were separated by SDS-PAGE (10%, 7.5%, or 4% to 20%) and blotted onto a nitrocellulose membrane. A, THP-1 cells transfected with the empty vector pCI (control) or with pCI-adipophilin (Ad). B, THP-1 macrophages transfected with siRNA-GAPDH (control) or siRNA-adipophilin (siRNA). C, THP-1 macrophages transfected with siRNA-GAPDH (control) or siRNA-adipophilin (siRNA) and then incubated with 100 μg/mL AcLDL in medium containing 1% FCS for 48 hours.
To determine whether the overexpression of adipophilin in THP-1 macrophages affected the affinity and/or the number of the AcLDL receptors, we studied the interaction of 125I–AcLDL, at 4°C, and its degradation, at 37°C, on control cells and on adipophilin-transfected cells. In both situations, the binding of 125I–AcLDL was saturable (not shown). The data calculated according to Scatchard et al27 were plotted as straight lines (data not shown) with a correlation coefficient of 0.97±0.10 for control and adipophilin-transfected cells. The apparent dissociation constants (Kd of 2.17±0.57 μg/mL for control cells versus 2.70±0.74 μg/mL for adipophilin-transfected cells) were not significantly different. Similarly, the number of 125I–AcLDL receptors (Bmax=35.17±4.78 ng/mg for control cells versus 40.09±3.13 ng/mg for adipophilin-transfected cells) were also not significantly different. 125I–AcLDL degradation after binding was measured at 37°C and, again, no significant difference was observed between adipophilin overexpressing cells (260±49 ng bound/mg cell protein and 443±88 ng degraded/mg cell protein) and control cells (362±30 ng bound/mg cell protein and 434±34 ng degraded/ mg cell protein).
Next, we studied the impact of adipophilin overexpression on lipid accumulation in human macrophages incubated with AcLDL, as a source of exogenous lipids, for 48 hours. Our data demonstrated that overexpression of adipophilin induced a significant increase of triglycerides (1.4-fold) in comparison with the control cells (Figure 2). Although total cholesterol is weakly increased (1.2-fold), adipophilin overexpression leads to a significant increase of the cholesteryl ester pool (1.7-fold). Moreover, the impact of adipophilin depletion on lipid accumulation in THP-1 macrophages was examined using the siRNA approach. Endogenous adipophilin expression in nontreated or in AcLDL-treated cells was dramatically reduced after siRNA-adipophilin transfection and represented 13% in comparison to control siRNA-GAPDH transfected cells (control=100%; P<0.05) (Figure 3). Adipophilin expression was not altered when THP-1 cells were transfected with control siRNA-GAPDH. In siRNA-adipophilin transfected cells, triglycerides and total and esterified cholesterol were significantly reduced by 50%, 44%, and 64% (P<0.05), respectively, in comparison to control cells (Figure 4). Expression of ABCA1, hSR-BI, and apoE were not affected by downregulation of adipophilin expression (Figure 1B). Interestingly, in siRNA-adipophilin transfected cells preloaded with AcLDL, apoE expression was increased 20-fold (Figure 1C).
Figure 2. Effect of adipophilin expression on triglycerides and cholesterol mass on THP-1 macrophages incubated with 100 μg/mL of AcLDL in medium containing 1% FCS for 48 hours. Control cells were transfected with pCI vector and adipophilin cells were transfected with pCI–adipophilin. The results are mean±SD of 3 independent experiments performed in quadruplicates. Significance indicated in the figure.
Figure 3. Western blot analysis of adipophilin expressed by human macrophages. THP-1 macrophages were transfected with the siRNA–GAPDH (control) or with the siRNA–adipophilin (siRNA–adip) and then incubated with or without 100 μg/mL AcLDL in medium containing 1% FCS for 48 hours. Total proteins were isolated and samples of 20 μg were separated by SDS-PAGE (10%) and blotted onto a nitrocellulose membrane. The results are mean±SD of 3 independent experiments performed in triplicates.
Figure 4. Effect of adipophilin downregulation on triglycerides and cholesterol mass when incubated with lipid loaded THP-1 macrophages. Cells were transfected with control GAPDH siRNA or with adipophilin siRNA and incubated with 100 μg/mL of AcLDL in medium containing 1% FCS for 48 hours. The results are mean±SD of 2 independent experiments performed in quadruplicates.
Another protein, perilipin, which has been identified in adipocytes and steroidogenic cells,28,29 is involved in the regulation of triglyceride hydrolysis and is located on the surface of lipid droplets. Therefore, we evaluated the expression of this protein by Western blot and found no variation in its expression when adipophilin is downregulated or overexpressed (Figure 1).
To further investigate the function of adipophilin in human THP-1 macrophages, we assessed the impact of adipophilin overexpression on cholesterol efflux in cells preloaded with 3H-cholesterol-AcLDL. In the presence of the cholesterol acceptor, apolipoprotein A-I (ApoA-I), cholesterol efflux from control cells increased over time, whereas in adipophilin transfected cells, cholesterol efflux to ApoA-I at 8, 12, and 24 hours was significantly reduced compared with control cells (Figure 5A). At the 24-hour time point, cholesterol efflux in adipophilin transfected cells was reduced by 47% in comparison to control cells (P<0.05). In the absence of ApoA-I, cholesterol efflux was low and unaffected by adipophilin overexpression. In contrast to the decrease in cholesterol efflux to ApoA-I in THP1 macrophages overexpressing adipophilin, siRNA-mediated reduction in adipophilin expression had no effect on ApoA-I–mediated cholesterol eflux (Figure 5B).
Figure 5. A, Effect of adipophilin expression on cholesterol efflux in lipid-loaded THP-1 macrophages. Cells were incubated with 100 μg/mL of 3H-cholesterol-AcLDL in medium containing 1% FCS for 48 hours and equilibrated in medium containing 1% BSA for 24 hours. After equilibration, cells were incubated for 24 hours in the presence or absence of ApoA-I (25 μg/mL). Control cells were transfected with pCI and adipophilin cells were transfected with pCI-adipophilin. Samples were taken at 2, 4, 8, 12, and 24 hours. B, Effect of adipophilin downregulation on cholesterol efflux in lipid loaded THP-1 macrophages. Cells were transfected with control GAPDH siRNA or with adipophilin siRNA and incubated with 100 μg/mL 3H-cholesterol-AcLDL in medium containing 1% FCS for 48 hours and equilibrated in medium containing 1% BSA for 24 hours. After equilibration, cells were incubated in the presence or absence of ApoA-I. The results are mean±SD of 3 independent experiments performed in sextuplets.
Discussion
Macrophages contribute to the formation of arterial lesions by accumulating excessive amounts of lipids, mainly CE, through the uptake of modified lipoproteins by a variety of mechanisms, including the scavenger receptor pathways such as scavenger receptors A (SR-A) and CD36.30 Thus, elimination of accumulated CE from macrophage foam cells represents a promising therapeutic approach to prevent atherosclerotic lesions. Indeed, different strategies have been reported to diminish cellular CE content, such as the inhibition of the activity of acyl-CoA:cholesterol acyltransferase 1 (ACAT1), the enzyme responsible for the esterification of intracellular FC,31 the overexpression of the hormone-sensitive lipase, a multifunctional enzyme that catalyzes triacylglycerol and CE hydrolysis,32 and the increase in expression of ABCA1, a transporter implicated in the efflux of cellular FC.33
Despite its observed overexpression in lipid-loaded macrophages,10,15,16 no data exist on the role of adipophilin in lipid storage and cholesterol efflux in these cells and its relevance to atherosclerosis. In this article, we report a high expression of adipophilin in human arterial lesions compared with normal artery segments (Figure II), which is mainly in macrophage foam cells (not shown). We showed also, for the first time to our knowledge, that overexpression of adipophilin in THP-1 macrophages enhanced the accumulation of TG and CE (Figure 2) and diminished cellular cholesterol efflux (Figure 5A). The affinity, the number of AcLDL receptors and their internalization, and the expression of genes involved in reverse cholesterol transport such as ABCA1 and hSR-BI, or genes involved in lipid accumulation such as CD36 and SR-A, were not affected by the adipophilin overexpression in THP-1 macrophages (Figure 1A). Nevertheless, we cannot exclude an oxidative modification, even partly, of AcLDL by macrophages,34 which supply the cells with oxysterols, endogenous agonists of the LXR nuclear receptors.35 In this case, LXR will increase transcription of lipogenic genes SREBP-1c36 and FAS,37 resulting in enhanced TG levels. The greater increase in TG (1.4) versus cholesterol (1.2) likely reflects the lower basal level of TG versus cholesterol in unloaded THP-1 cells.
Inhibition of endogenous adipophilin expression, using the siRNA approach, dramatically reduced lipid accumulation (Figure 4). This experimental approach did not affect the expression of genes known to be involving in cholesterol efflux such as ABCA1, hSR-B1, and apoE. However, the expression of apoE was greatly enhanced in siRNA adipophilin transfected cells if they are loaded with AcLDL (Figure 1C). One potential explanation is that AcLDL, an exogenous source of free cholesterol, can be internalized and, as a consequence, free cholesterol could induce apoE expression in macrophages as reported previously.38 It is also possible that the significant decrease in cholesterol ester in siRNA adipophilin transfected cells (Figure 4) may be largely mediated by this apoE increase or by other non-ABCA1–mediated mechanisms of cholesterol efflux.
Our results in human THP-1 cells are similar to those obtained recently in mouse fibroblasts overexpressing murine ADRP, the equivalent of human adipophilin. In these cells, ADRP was localized around cytosolic lipid droplets. ADRP overexpression stimulated lipid accumulation and lipid droplet formation without induction of other adipocyte-specific genes or other lipogenic genes.39 ADRP was suggested to facilitate the uptake of long-chain free fatty acids in transiently transfected COS-7 cells by functioning as a carrier protein.40 Human adipophilin might contribute to the increased lipid content in macrophages by the same mechanism. However, additional mechanisms by which adipophilin stimulates lipid accumulation in macrophages cannot be excluded. Indeed, our data in Figure 5A showed that adipophilin overexpression prevented cholesterol efflux to ApoA-I. One possible explanation is that adipophilin may be localized around cytosolic lipid droplets in the macrophages protecting them from the activity of cholesterol esterases such as hormone-sensitive lipase and thereby decreasing the availability of FC for efflux. By a similar mechanism, perilipins, lipid droplet-associated proteins present in adipocytes, increase the accumulation of triglycerides in adipocytes by protecting them from the action of lipases.41
Nevertheless, while we observed a dramatic reduction in cholesterol efflux in the presence of excess adipophilin, we did not observe an increase, as expected, in ApoA-I–mediated cholesterol efflux in the absence of adipophilin in THP-1 macrophages (Figure 5B). These results are similar to those obtained with L-cell fibroblasts overexpressing sterol carrier protein-2 (SCP-2), in which SCP-2 overexpression decreased the level of ADRP protein by 70% and also inhibited HDL-mediated sterol efflux from lipid droplets (an effect related to decreased ADRP protein).42 The inability of adipophilin depletion to increase cholesterol efflux might be caused by the decreased level of CE within the cells. The increased level of apoE in lipid-loaded cells could contribute to this decrease. No evidence was found for the substitution of adipophilin by perilipin in siRNA-adipophilin transfected cells, which might prevent hydrolysis of lipid droplets and, in turn, prevent cholesterol efflux (Figure 1B).
Our data indicated that macrophage adipophilin expression is a consequence of lipid accumulation and contributes to further accumulation of lipids by inhibiting cellular cholesterol efflux. It is worthy to note that, in addition to adipophilin/ADRP and perilipins,43,44 several other proteins have been identified on the surface of lipid droplets, such as caveolin45–47 and vimentin,48,49 and they may play similar roles in lipid mobilization in specific cell types. For example, perilipin-deficient mice, which are resistant to diet-induced obesity, possess adipocytes with smaller lipid droplets and a higher rate of lipolysis in adipocytes compared with wild-type mice.50 Thus, the generation of ADRP-deficient mice or adipophilin knock-in mice would contribute to the understanding of the role of adipophilin in atherosclerosis.
Acknowledgments
This work was supported by grants from Genfit and the Leducq Foundation.
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