Redistribution of macrophage cholesteryl ester hydrolase from cytoplasm to lipid droplets upon lipid loading

Hydrolysis of intracellular cholesteryl esters (CEs) represents the first step in the removal of cholesterol from lipid-laden foam cells associated with atherosclerotic lesions. Neutral cholesteryl ester hydrolase (CEH) catalyzes this reaction, and we recently cloned the cDNA for the human macrophage CEH and demonstrated increased mobilization of intracellular CE droplets by CEH overexpression. The present study was undertaken to test the hypothesis that for CE hydrolysis, CEH must become associated with the surface of the cytoplasmic lipid droplets. Our data show the redistribution of CEH from cytosol to lipid droplets upon lipid loading of human THP-1 macrophages. Depletion of triacylglycerol (TG) by incubation with the acyl-CoA synthetase inhibitor Triacsin D had no effect on CEH association with the lipid droplets, suggesting that CEH associates with mixed (CE + TG) as well as TG-depleted CE droplets. However, CEH had 2.5-fold higher activity when mixed droplets were used as substrate in an in vitro assay, consistent with the reported higher cholesterol efflux from cells containing mixed isotropic droplets. Perilipin as well as adipophilin, two lipid droplet-associated proteins, were also present on the lipid droplets in THP-1 macrophages.In conclusion, CEH associates with its intracellular substrate (lipid droplets) and hydrolyzes CE more efficiently from mixed droplets. Hydrolysis of intracellular cholesteryl esters (CEs) represents the first step in the removal of cholesterol from lipid-laden foam cells associated with atherosclerotic lesions. Neutral cholesteryl ester hydrolase (CEH) catalyzes this reaction, and we recently cloned the cDNA for the human macrophage CEH and demonstrated increased mobilization of intracellular CE droplets by CEH overexpression. The present study was undertaken to test the hypothesis that for CE hydrolysis, CEH must become associated with the surface of the cytoplasmic lipid droplets. Our data show the redistribution of CEH from cytosol to lipid droplets upon lipid loading of human THP-1 macrophages. Depletion of triacylglycerol (TG) by incubation with the acyl-CoA synthetase inhibitor Triacsin D had no effect on CEH association with the lipid droplets, suggesting that CEH associates with mixed (CE + TG) as well as TG-depleted CE droplets. However, CEH had 2.5-fold higher activity when mixed droplets were used as substrate in an in vitro assay, consistent with the reported higher cholesterol efflux from cells containing mixed isotropic droplets. Perilipin as well as adipophilin, two lipid droplet-associated proteins, were also present on the lipid droplets in THP-1 macrophages. In conclusion, CEH associates with its intracellular substrate (lipid droplets) and hydrolyzes CE more efficiently from mixed droplets. Unregulated uptake of modified LDL by blood monocyte-derived macrophages results in the cytoplasmic accumulation of cholesteryl ester (CE)-rich lipid droplets and the formation of foam cells. Continued generation of foam cells within the intimal wall of the artery results in fatty streak formation, one of the earliest morphological changes associated with atherosclerosis (1Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s.Nature. 1993; 362: 801-809Google Scholar). Efficient cholesterol efflux from macrophages is critical for the prevention of foam cell formation and subsequent protection against atherosclerosis. Because there is no significant release of CE from cells, including macrophages, for efflux to occur, CE must first be hydrolyzed to free cholesterol. The obligatory first step in reverse cholesterol transport, therefore, is the hydrolysis of CE to release free cholesterol, a reaction catalyzed by a neutral cholesteryl ester hydrolase (CEH). Because the rate of CE hydrolysis is often slower than free cholesterol movement to the extracellular acceptor, CE hydrolysis is increasingly being recognized as the rate-limiting step in cellular cholesterol efflux (2Rothblat G.H. Llera-Moya M.D.L. Favari E. Yancey P.G. Kellner-Wiebel G. Cellular cholesterol flux studies: methodological considerations.Atherosclerosis. 2002; 163: 1-8Google Scholar, 3Morel D.W. Edgerton M.E. Warner G.E. Johnson W.J. Phillips M.C. Rothblat G.H. Comparison of the intracellular metabolism and trafficking of 25-hydroxycholesterol and cholesterol in macrophages.J. Lipid Res. 1996; 37: 2041-2051Google Scholar). Thus, macrophages with high neutral CEH activity do not accumulate CE in the presence of atherogenic lipoproteins such as β-VLDL, compared with macrophages with low CEH activity (4Ishii I. Oka M. Katto N. Shirai K. Saito Y. Hirose S. Beta-VLDL-induced cholesterol ester deposition in macrophages may be regulated by neutral cholesterol esterase activity.Arterioscler. Thromb. 1992; 12: 1139-1145Google Scholar). In addition, animal models of atherosclerosis, such as the hypercholesterolemic rabbit and the White Carneau pigeon, appear to possess macrophages in which stored CE is resistant to hydrolysis and subsequent mobilization (5Mathur S.N. Field F.J. Megan M.B. Armstrong H.L. A defect in mobilization of cholesteryl esters in rabbit macrophages.Biochim. Biophys. Acta. 1985; 834: 48-57Google Scholar, 6Yancey P.G. Clair R.W. St. Mechanism of the defect in cholesteryl ester clearance from macrophages of atherosclerosis-susceptible White Carneau pigeons.J. Lipid Res. 1994; 35: 2114-2129Google Scholar).Ghosh (7Ghosh S. Cholesteryl ester hydrolase in human monocyte/macrophage: cloning, sequencing and expression of full-length cDNA.Physiol. Genomics. 2000; 2: 1-8Google Scholar) recently reported the cloning and characterization of the human macrophage CEH cDNA and demonstrated the expression of CEH mRNA in the human monocyte/macrophage cell line THP-1 as well as in human peripheral blood monocyte-derived macrophages. The rate-limiting role of this enzyme in intracellular CE mobilization was confirmed by the observed concentration-dependent decrease in intracellular lipid droplets and cellular CE mass after overexpression (8Ghosh S. Clair R.W. St. Rudel L.L. Mobilization of cytoplasmic CE droplets by overexpression of human macrophage cholesteryl ester hydrolase.J. Lipid Res. 2003; 44: 1833-1840Google Scholar). Although this enzyme hydrolyzes CEs presented as lipid droplets in vitro and effectively mobilizes intracellular CE droplets, a direct association of CEH with cytoplasmic lipid droplets has not been demonstrated.Lipid droplets are spherical organelles found in many types of eukaryotic cells; they are composed of a core of neutral lipids covered by a monolayer of phospholipids, free cholesterol, and proteins. The lipid droplets present in adipocytes are well characterized as containing a core of triglycerides and surface proteins. In preadipocytes and early differentiated adipocytes, adipose differentiation-related protein (ADRP) is found associated with small lipid droplets. With maturation, perilipin A becomes the most abundant protein on the surface of large lipid droplets; ADRP is undetectable in mature adipocytes (9Londos C. Brasaemle D.L. Schultz C.J. Segrest J.P. Kimmel A.R. Perilipins, ADRP and other proteins that associate with intracellular neutral lipid droplets in animal cells.Semin. Cell Dev. Biol. 1999; 10: 51-58Google Scholar). Perilipin A functions to increase cellular triglyceride storage by decreasing the rate of triglyceride hydrolysis (10Brasaemle D.L. Rubin B. Harten I.A. Gruia-Gray J. Kimmel A.R. Londos C. Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis.J. Biol. Chem. 2000; 275: 38486-38496Google Scholar, 11Souza S.C. de Vergas I.M. Yamamoto M.T. Lien P. Franciosa M.D. Moss L.G. Greenburg A.S. Overexpression of perilipin A and B blocks the ability of tumor necrosis factor alpha to increase lipolysis in 3T3-L1 adipocytes.J. Biol. Chem. 1998; 273: 24665-24669Google Scholar). Furthermore, perilipin A is phosphorylated by cAMP-dependent protein kinase and serves an additional role in controlling the hormone-stimulated lipolysis in adipose tissue (12Egan J.J. Greenburg A.S. Chang M.K. Londos C. Overexpression of perilipin A and B blocks the ability of tumor necrosis factor alpha to increase lipolysis in 3T3-L1 adipocytes.J. Biol. Chem. 1990; 265: 18769-18775Google Scholar, 13Tansey J.T. Sztalryd C. Gruia-Gray J. Roush D.L. Zee J.V. Gavrilova O. Reitman M.L. Deng C.X. Li C. Kimmel A.R. et al.Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity.Proc. Natl. Acad. Sci. USA. 2001; 11: 6494-6499Google Scholar, 14Souza S.C. Muliro K.V. Liscum L. Lien P. Yamamoto M.T. Schaffer J.E. Dallal G.E. Wang X. Kraemer F.B. Obin M. et al.Modulation of hormone-sensitive lipase and protein kinase A-mediated lipolysis by perilipin A in an adenoviral reconstituted system.J. Biol. Chem. 2002; 277: 8267-8272Google Scholar) by regulating the activity of hormone-sensitive lipase. Although perilipin A is most abundant, the other isoforms, perilipins B and C, occur primarily in adipose and steroidogenic cells, respectively (9Londos C. Brasaemle D.L. Schultz C.J. Segrest J.P. Kimmel A.R. Perilipins, ADRP and other proteins that associate with intracellular neutral lipid droplets in animal cells.Semin. Cell Dev. Biol. 1999; 10: 51-58Google Scholar).Little is known about the lipid droplet-associated proteins in macrophage foam cells and whether the hydrolysis of stored CEs is regulated by these lipid droplet-associated proteins. In murine macrophages (RAW264.7), Chen et al. (15Chen J.S. Greenberg A.S. Tseng Y.Z. Wang S.M. Possible involvement of protein kinase C in the induction of adipose differentiation-related protein by sterol ester in RAW264.7 macrophages.J. Cell. Biochem. 2001; 83: 187-199Google Scholar) demonstrated the association of ADRP with the cytoplasmic lipid droplets (both small and large) and the concomitant increased expression of ADRP with lipid droplet accumulation. However, the presence of neither perilipin nor adipophilin, the human ortholog of ADRP, is described in human blood monocyte-derived macrophages or in THP-1 cells. Furthermore, it is not known whether CEH associates with cytoplasmic lipid droplets in a manner similar to hormone-sensitive lipase association with lipid droplets in adipocytes, a process regulated by perilipin A (16Sztalryd C. Xu G. Dorward H. Tansey J.T. Contreras J.A. Kimmel A.R. Londos C. Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation.J. Cell Biol. 2003; 161: 1093-1103Google Scholar).The present study was undertaken to determine the subcellular localization of CEH in human macrophages and to gain insight into the association of CEH with its physiological substrate (CE stored as cytoplasmic lipid droplets). We show here the redistribution of CEH from cytoplasm to lipid droplets upon lipid loading, demonstrating the association of CEH with its intracellular substrate, an obligatory step for the subsequent hydrolysis of CE present in the lipid droplets. Furthermore, CEH activity is affected by the presence or absence of triacylglycerol (TG) associated with CE in the intracellular lipid droplets. In addition, these data also show, for the first time, the association of perilipin and adipophilin with lipid droplets in lipid-laden THP-1 cells.MATERIALS AND METHODSMaterialsTHP-1 and HEK293T cells were obtained from ATCC (Manassas, VA). Acetylated LDL (AcLDL) was purchased from Intracel, Inc. (Frederick, MD). 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-23,24-bisnor-5-cholen-3-ol (NBD-cholesterol), Alexa® 546 conjugated goat anti-rabbit IgG, and Alexa® 488 conjugated goat anti-guinea pig IgG were from Molecular Probes (Eugene, OR). Peroxidase-labeled goat anti-guinea pig IgG was obtained from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD), and HRP-conjugated goat anti-rabbit IgG was from Bio-Rad (Hercules, CA). Guinea pig polyclonal antibodies to adipophilin and perilipin that cross-react with human, mouse, and rat were purchased from Research Diagnostics, Inc. (Flanders, NJ), and mouse monoclonal antibodies to Na+K+ATPase were from Santa Cruz Biotechnology (Santa Cruz, CA). The TNT™ Quick Coupled transcription/translation system and canine microsomes were from Promega (Madison, WI). [35S]methionine, cholesteryl [1-14C]oleate, and Western Lightning Chemiluminescence Reagent Plus were from New England Nuclear (Boston, MA). Phorbol 13-myristate, 12-acetate (PMA) and BSA were obtained from Sigma-Aldrich (St. Louis, MO). Cell culture media and reagents were purchased from Invitrogen (Carlsbad, CA). All other reagents used were of analytical grade and obtained from Fisher Scientific (Pittsburgh, PA).Cell cultureTHP-1 cells were maintained in RPMI-1640 medium containing 10% FBS according to the instructions supplied. For induction of macrophages, PMA (100 nM) was added to the medium and the cells were seeded at a density of 0.1 × 106 cells/cm2 into tissue culture dishes and maintained in a humidified atmosphere of 95% air and 5% CO2. Media containing PMA were replaced every 2 days, and experiments were started after 5–7 days in culture, when the cells were phenotypically macrophage (7Ghosh S. Cholesteryl ester hydrolase in human monocyte/macrophage: cloning, sequencing and expression of full-length cDNA.Physiol. Genomics. 2000; 2: 1-8Google Scholar). THP-1 macrophage cells were lipid loaded by incubation with AcLDL (50 μg/ml) for 48 h. In some studies, the loading medium was also supplemented with NBD-cholesterol (5 μg/ml) to fluorescently label the intracellular lipid droplets. Nonloaded cells were maintained in lipoprotein-deficient serum for the same period. HEK293T cells were maintained and propagated in DMEM supplemented with 10% FBS according to the guidelines provided.In vitro translationThe human macrophage CEH expression vector, pCMV-CEH, described previously (8Ghosh S. Clair R.W. St. Rudel L.L. Mobilization of cytoplasmic CE droplets by overexpression of human macrophage cholesteryl ester hydrolase.J. Lipid Res. 2003; 44: 1833-1840Google Scholar), was used for in vitro transcription/translation using the TNT™ Quick Coupled system with or without added canine microsomes according to the manufacturer's protocol. [35S]methionine was included to label the newly synthesized proteins. At the end of the incubation, half of the reaction was subjected to proteinase K (200 μg/ml) digestion (30 min on ice) to assess the susceptibility of newly synthesized protein to proteolytic degradation (17Clark A.J. Lavender P.M. Coates P. Johnson M.R. Rees L.H. In vitro and in vivo analysis of the processing and fate of the peptide products of the short proopiomelanocortin mRNA.Mol. Endocrinol. 1990; 4: 1737-1743Google Scholar). Cytosolic proteins are susceptible to degradation, and proteins localized in the endoplasmic lumen are protected under these conditions. Core glycosylation control RNA (provided in the kit) was used as a positive control to assess the functionality of the added microsomes. The reaction products were separated on a 12.5% SDS-PAGE gel. The gel was dried and subjected to autoradiography.ImmunocytochemistryCells were plated on fibronectin-coated Lab-Tek Cover Glass chambers. Three to 5 days after plating and specified treatments, the culture media were aspirated and cells were fixed in 3.7% paraformaldehyde in PBS for 10 min at 4°C. This is the method of choice for fixation because the cells retain their lipid content and the lipid droplet structure is unaffected (18DiDonato D. Brasaemle D.L. Fixation methods for the study of lipid droplets by immunofluorescence microscopy.J. Histochem. Cytochem. 2003; 51: 773-780Google Scholar). Cells were permeabilized by exposure to 0.15% Triton X-100 in PBS for 3 min at 4°C, washed with PBS, and blocked with 0.5% BSA in PBS for at least 1 h at room temperature. Cells were then incubated with primary antibody overnight at 4°C. Cells incubated in the absence of primary antibody were used as negative controls. For detection, cells were subsequently incubated with either Alexa Fluor® 546 or Alexa Fluor® 488 conjugated secondary antibody in 0.5% BSA for 1 h at room temperature. Cells were washed three times with PBS and once with distilled water. Cells were imaged at 90× magnification using an Olympus model IX70 inverted phase microscope fitted with a MagnaFire™ digital camera using either a tetramethylrhodamine-5-(and-6)-isothiocyanate [5(6)] (TRITC) filter cube (530–560 nm excitation, 590–650 nm emission; Chroma Technology Corp.) or a FITC filter cube (460–500 nm excitation, 510–560 nm emission). Although highly cross-adsorbed secondary antibodies were used, additional negative controls included the use of cross-species secondary antibodies (e.g., the use of Alexa Fluor® 488 conjugated anti-guinea pig secondary antibody with rabbit polyclonal antibody to CEH and the use of Alexa Fluor® 546 conjugated anti-rabbit antibody with guinea pig polyclonal antibody to perilipin or adipophilin). No cross-species reactivity was observed.Isolation of lipid droplet-associated proteinsTHP-1 cells (8 × 106 cells) were plated in 100 mm tissue culture dishes in the presence of PMA. After 3 days of differentiation, one set was lipid loaded using AcLDL (50 μg/ml) for 48 h. After a wash with cold PBS, cells were harvested and lysed by sonication in 200 mM phosphate buffer containing 0.25 M sucrose, 80 mM KCl, 5 mM 2-mercaptoethanol, and protease inhibitors, as described previously (7Ghosh S. Cholesteryl ester hydrolase in human monocyte/macrophage: cloning, sequencing and expression of full-length cDNA.Physiol. Genomics. 2000; 2: 1-8Google Scholar). Cell lysates were centrifuged at 100,000 g for 1 h to separate the cytosolic and total membrane fractions. The intracellular lipids floating on top of the cytosolic fraction were carefully collected and washed twice with the homogenizing buffer by centrifuging at 100,000 g for 1 h. Total proteins associated with equal volumes of lipid fraction from nonloaded and AcLDL-loaded cells were precipitated with TCA (final concentration, 10%) at 4°C for 1 h instead of the 24 h acetone precipitation described earlier (19Garcia A. Sekowski A. Subramanian V. Brasaemle D.L. The central domain is required to target and anchor perilipin A to lipid droplets.J. Biol. Chem. 2003; 278: 625-635Google Scholar). The protein precipitates were dissolved in the harvesting buffer and immediately neutralized to pH 7.4. Protein concentration was determined using the Bio-Rad protein assay kit.Western blot analysesAn aliquot containing 10–20 μg of total protein was separated on a 4–20% SDS-PAGE gel, and proteins were transferred to a polyvinylidene difluoride membrane. After a brief wash in TBS and blocking in 5% nonfat dry milk, the blots were incubated with primary antibody appropriately diluted in 5% nonfat dry milk overnight at 4°C. After three to five washes in TBS, the blots were subsequently incubated with secondary antibody appropriately diluted in 5% nonfat dry milk for 1 h at room temperature. After three to five washes in TBS, the blots were developed using Lightning Chemiluminescence Reagent Plus and exposed to X-ray film. Densitometric analyses were performed using Kodak 1D Image Analysis Software.Triglyceride depletionTHP-1 macrophages were loaded with lipid using AcLDL as described above. Incubation with the acyl-CoA synthetase inhibitor Triacsin D and albumin was used to modulate the cellular triglyceride concentration, as described previously (20Lada A.T. Willingham M.C. Clair R.W. St. Triglyceride depletion in THP-1 cells alters cholesteryl ester physical state and cholesterol efflux.J. Lipid Res. 2002; 43: 618-628Google Scholar). In brief, after 48 h of loading, the AcLDL-containing medium was removed, and cells were washed with medium and then incubated with medium containing 12.5 μM Triacsin D and 400 μM BSA for 24 h. Under these conditions, >90% of cellular triglyceride was removed without any significant decrease in the levels of cellular CE content, and CE was present as anisotropic intracellular droplets (20Lada A.T. Willingham M.C. Clair R.W. St. Triglyceride depletion in THP-1 cells alters cholesteryl ester physical state and cholesterol efflux.J. Lipid Res. 2002; 43: 618-628Google Scholar). Lipid droplet-associated proteins were isolated as described above.CEH activity measurementsHEK 293T cells were plated in six-well tissue culture dishes (6 × 105 cells/well) and transfected with either the empty vector pCMV or the CEH expression vector pCMV-CEH, as described previously (8Ghosh S. Clair R.W. St. Rudel L.L. Mobilization of cytoplasmic CE droplets by overexpression of human macrophage cholesteryl ester hydrolase.J. Lipid Res. 2003; 44: 1833-1840Google Scholar). Cell lysates were prepared, and CEH activity was determined using a radiometric assay (21Ghosh S. Grogan W.M. Activation of rat liver cholesterol ester hydrolase by cAMP-dependent protein kinase and protein kinase C.Lipids. 1989; 24: 733-736Google Scholar). Cholesteryl [1-14C] oleate was used as the radioactive substrate at a final concentration of 75 μM and presented in the reaction tube as droplets in acetone, determined to be the most suitable mode of substrate presentation (22Deykin D. Goodman D.S. The hydrolysis of long chain fatty acid esters of cholesterol with rat liver enzymes.J. Biol. Chem. 1962; 237: 3649-3656Google Scholar). To measure CEH activity toward a mixed droplet substrate, triolein (60%) and cholesteryl oleate (40%), based on the composition determined by Lada, Willingham, and St. Clair (20Lada A.T. Willingham M.C. Clair R.W. St. Triglyceride depletion in THP-1 cells alters cholesteryl ester physical state and cholesterol efflux.J. Lipid Res. 2002; 43: 618-628Google Scholar) for isotropic droplets, were mixed, dried under nitrogen, dissolved in acetone, and used at the same final concentration in the assay (75 μM) as pure cholesteryl oleate.RESULTSReconfirmation of the cytosolic localization of CEHIntracellular CEs are stored as cytoplasmic lipid droplets and hydrolyzed by the neutral cytosolic CEH. To determine whether CEH encoded by the human macrophage cDNA is associated with the cytoplasm or lumen of the endoplasmic reticulum, as predicted by its high degree of homology with members of the carboxylesterase family, it was transcribed and translated in vitro in the absence or presence of microsomes. Resistance to proteinase K digestion was used as a measure of the movement of newly synthesized protein into the lumen of microsomes (17Clark A.J. Lavender P.M. Coates P. Johnson M.R. Rees L.H. In vitro and in vivo analysis of the processing and fate of the peptide products of the short proopiomelanocortin mRNA.Mol. Endocrinol. 1990; 4: 1737-1743Google Scholar). As seen in Fig. 1, newly synthesized CEH remained susceptible to proteinase K in the presence of microsomes (lane 4), demonstrating the lack of CEH movement into the lumen of the microsomes and reconfirming the cytosolic localization of neutral CEH encoded by cDNA isolated from human macrophages. Control plasmid expressing a protein that is glycosylated by the microsomes was used as a positive control for assessing the functionality of the microsomes. In the absence of canine microsomes, a single protein was synthesized (Fig. 1, lane 5). This precursor was processed into higher molecular weight glycosylated proteins in the presence of microsomes (Fig. 1, lane 6), confirming the functionality of the canine microsomes used.Redistribution of CEH upon lipid loading of THP-1 macrophages with AcLDLCEH was immunolocalized within THP-1 macrophages that were either nonloaded or lipid loaded with AcLDL. In both sets, NBD-cholesterol was included in the culture medium to fluorescently label the intracellular CE droplets. Indirect immunofluorescence imaging revealed that in nonloaded cells, CEH was uniformly distributed within the cytoplasm of the cell [Fig. 2, diffuse red fluorescence associated with the two large cells (marked with arrows) and two small cells (marked with S→)], with no apparent lipid droplets in the cytoplasm (diffuse green fluorescence attributable to NBD-cholesterol). In contrast, in the presence of AcLDL, THP-1 macrophage cells accumulated large amounts of cholesterol visible as fluorescent (green) intracellular CE droplets (marked with D→) in the cytoplasm (marked with C→). In these cells, CEH was not uniformly distributed in the cytoplasm but, instead, localized encircling the NBD-cholesterol-labeled cytoplasmic lipid droplets, indicating redistribution of CEH upon lipid loading in these cells. The movement of CEH from the cytosol to lipid droplets is clearly evident from the CEH overlay on the phase-contrast image, in which the peripheral cytoplasm (marked with C→) of the cell shows negligible staining for CEH. However, the association of CEH with very small cytoplasmic droplets in cells not loaded with AcLDL cannot be completely ruled out, and Western blot analyses were used to determine the distribution of CEH between the cytoplasm and lipid droplets.Fig. 2Redistribution of CEH upon lipid loading. THP-1 macrophages, with or without lipid loading using AcLDL, were fixed and processed for immunocytochemistry, as described in Materials and Methods. Representative images taken at 90× magnification are shown. The left panels show the cells without AcLDL, and the right panels show cells incubated with AcLDL. Two large cells (marked with arrows) and two small, rounded cells (marked with S→) are visible in the left panels (No Ac-LDL). A single cell is shown in the right panels (Ac-LDL) showing distinct large lipid droplets (marked with D→) in the cytoplasm (marked with C→). Intracellular lipid droplets were visualized by monitoring the NBD-cholesterol fluorescence (green). Intracellular CEH was stained using anti-CEH rabbit polyclonal antibodies and Alexa Fluor® 546 conjugated goat anti-rabbit IgG (red). The phase-contrast images were overlaid with CEH immunostaining (red) to show the redistribution of CEH upon lipid loading. Note the uniform distribution of the red stain in the cells in the left panel (No Ac-LDL) and the absence of CEH (red) staining from the peripheral cytoplasm with concomitant increased red staining surrounding the lipid droplets in the right panel (Ac-LDL).View Large Image Figure ViewerDownload (PPT)Confirmation of intracellular localization of CEH by Western blot analysesTo further confirm the localization of CEH in nonloaded and lipid-loaded cells, cytoplasmic and lipid droplet fractions were isolated and examined for CEH protein content by Western blot analyses using rabbit polyclonal antibody to CEH, as described previously (8Ghosh S. Clair R.W. St. Rudel L.L. Mobilization of cytoplasmic CE droplets by overexpression of human macrophage cholesteryl ester hydrolase.J. Lipid Res. 2003; 44: 1833-1840Google Scholar). As seen from the representative Western blots in Fig. 3B, CEH protein is primarily associated with the cytoplasmic fraction in nonloaded cells (No AcLDL), with only a minor amount associated with the lipid-droplet fraction (Fat). Upon lipid loading, this distribution is reversed, with CEH being predominantly associated with the lipid droplet fraction. The relative distribution of immunoreactive CEH protein in these fractions was determined by densitometric analyses of the Western blots and is shown in Fig. 3A. In nonloaded cells (No AcLDL), ∼72 ± 1.75% of CEH protein was associated with the cytosolic fraction and 28 ± 1.75% was associated with the lipid droplets. Upon lipid loading with AcLDL, this distribution was essentially reversed, and 24 ± 8.58% of CEH protein was associated with the cytosolic fraction and 76 ± 8.58% was associated with the lipid droplets. Together with the immunocytochemical detection of CEH in THP-1 cells described above, these data clearly demonstrate the redistribution of CEH from the cytoplasm to the lipid droplets upon lipid loading. Possible contamination of the lipid droplet fraction with plasma membrane vesicles was ruled out by analyzing for the presence of the plasma membrane marker Na+K+ATPase. As seen from the representative blots in Fig. 3C, no immunoreactivity was observed in the lipid droplet fraction (Fat), although a strong immunoreactive band was present in the membrane fraction from the same cells (Mem.).Fig. 3Association of CEH with isolated lipid droplets. For Western blot analyses, lipid droplet-associated proteins were isolated from the same volume of floating lipid layer from THP-1 macrophages (8 × 106 cells) cultured in the absence or presence of AcLDL and resuspended in 50 μl. Equivalent amounts of cytosol and lipid droplet-associated proteins (Fat) from cells not loaded with AcLDL (No Ac-LDL) and those loaded with AcLDL (+Ac-LDL) were separated by SDS-PAGE and processed for Western blot analysis using anti-CEH rabbit polyclonal antibodies. The immunoreactive band at ∼66 kDa was quantified densitometrically. A: Distribution of CEH in cells loaded with AcLDL and nonloaded cells (No Ac-LDL). Data represent means ± SD of three independent experiments. B: Representative Western blot. Lipid droplet-associated proteins were also analyzed for the presence of the plasma membrane marker Na+K+ATPase using mouse monoclonal antibodies. C: Representative Western blot. Total membrane fraction (Mem.) from the same cells was used as a positive control.View Large Image Figure ViewerDownload (PPT)Effect of TG depletion on CEH association with intracellular lipid dropletsIncubation of THP-1 cells with AcLDL results in the accumulation of both CEs and TG, and TG can greatly influence the physical state of CE. To de