Adipocyte differentiation-related protein reduces the lipid droplet association of adipose triglyceride lipase and slows triacylglycerol turnover
2007; Elsevier BV; Volume: 48; Issue: 12 Linguagem: Inglês
10.1194/jlr.m700359-jlr200
ISSN1539-7262
AutoresLaura L. Listenberger, Anne G. Ostermeyer‐Fay, Elysa B. Goldberg, William J. Brown, Deborah A. Brown,
Tópico(s)Pancreatic function and diabetes
ResumoAlthough neutral lipid storage droplets are ubiquitous in eukaryotic cells, very little is known about how their synthesis and turnover are controlled. Adipocyte differentiation-related protein (ADRP; also known as adipophilin) is found on the surface of lipid droplets in most mammalian cell types. To learn how ADRP affects lipid storage, we stably expressed the protein in human embryonic kidney 293 (HEK 293) cells, which express little endogenous ADRP. As expected, ADRP was targeted to the surface of lipid droplets and caused an increase in triacylglycerol (TAG) mass under both basal and oleate-supplemented conditions. At least part of the increased mass resulted from a 50% decrease in the rate of TAG hydrolysis in ADRP-expressing cells. Furthermore, ADRP expression increased the fraction of total cellular TAG that was stored in lipid droplets. ADRP expression induced a striking decrease in the association of adipose triglyceride lipase (ATGL) and mannose-6-phosphate receptor tail-interacting protein of 47 kDa with lipid droplets and also decreased the lipid droplet association of several other unknown proteins. Transient expression of ADRP in two other cell lines also reduced the lipid droplet association of catalytically inactive ATGL. We conclude that the reduced lipid droplet association of ATGL and/or other lipases may explain the decrease in TAG turnover observed in ADRP-expressing HEK 293 cells. Although neutral lipid storage droplets are ubiquitous in eukaryotic cells, very little is known about how their synthesis and turnover are controlled. Adipocyte differentiation-related protein (ADRP; also known as adipophilin) is found on the surface of lipid droplets in most mammalian cell types. To learn how ADRP affects lipid storage, we stably expressed the protein in human embryonic kidney 293 (HEK 293) cells, which express little endogenous ADRP. As expected, ADRP was targeted to the surface of lipid droplets and caused an increase in triacylglycerol (TAG) mass under both basal and oleate-supplemented conditions. At least part of the increased mass resulted from a 50% decrease in the rate of TAG hydrolysis in ADRP-expressing cells. Furthermore, ADRP expression increased the fraction of total cellular TAG that was stored in lipid droplets. ADRP expression induced a striking decrease in the association of adipose triglyceride lipase (ATGL) and mannose-6-phosphate receptor tail-interacting protein of 47 kDa with lipid droplets and also decreased the lipid droplet association of several other unknown proteins. Transient expression of ADRP in two other cell lines also reduced the lipid droplet association of catalytically inactive ATGL. We conclude that the reduced lipid droplet association of ATGL and/or other lipases may explain the decrease in TAG turnover observed in ADRP-expressing HEK 293 cells. Eukaryotes store lipid in cytosolic lipid droplets, which consist of neutral lipid cores surrounded by phospholipid monolayers (1Londos 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: 51-58Crossref PubMed Scopus (368) Google Scholar, 2Murphy D.J. Vance J. Mechanisms of lipid-body formation.Trends Biol. Sci. 1999; 24: 109-115Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, 3Zweytick D. Athenstaedt K. Daum G. Intracellular lipid particles of eukaryotic cells.Biochim. Biophys. Acta. 2000; 1469: 101-120Crossref PubMed Scopus (271) Google Scholar). 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Although HSL has long been considered the key regulator of lipid metabolism in adipocytes, HSL-deficient mice are not obese and accumulate diacylglycerol instead of TAG (19Haemmerle G. Zimmermann R. Hayn M. Theussl C. Waeg G. Wagner E. Sattler W. Magin T.M. Wagner E.F. Zechner R. Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis.J. Biol. Chem. 2002; 277: 4806-4815Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar). This surprising finding stimulated a search for other lipases important in TAG turnover. Several observations support a role for one such enzyme, adipocyte triglyceride lipase (ATGL; also known as desnutrin, patatin-like phospholipase domain-containing 2, and calcium-independent phospholipase A2-ζ (20Raben D.M. Baldassare J.J. A new lipase in regulating lipid mobilization: hormone-sensitive lipase is not alone.Trends Endocrinol. 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Garty N.B. Blanchette-Mackie E.J. Londos C. Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets.J. Biol. Chem. 1991; 266: 11341-11346Abstract Full Text PDF PubMed Google Scholar, 28Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein.J. Lipid Res. 1997; 38: 2249-2263Abstract Full Text PDF PubMed Google Scholar, 29Miura S. Gan J. Brzostowski J. Parisi M.J. Schultz C.J. Londos C. Oliver B. Kimmel A.R. Functional conservation for lipid storage droplet association among perilipin, ADRP, and TIP47 (PAT)-related proteins in mammals, DrosophilaDictyostelium.J. Biol. 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Guinea pig polyclonal antibodies against ADRP and TIP47 and mouse monoclonal anti-ADRP antibodies were from the RDI Division of Fitzgerald Industries (Concord, MA). Other mouse monoclonal antibodies were supplied as follows: anti-green fluorescent protein (GFP) antibodies were from Clontech (Mountain View, CA); anti-HSP90α/β antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-transferrin receptor antibodies were from Zymed (South San Francisco, CA); and anti-GM130 antibodies from were BD Transduction Laboratories (San Jose, CA). Rabbit polyclonal anti-ATGL antibodies were generated as described (37Smirnova E. Goldberg E.B. Makarova K.S. Lin L. Brown W.J. Jackson C.L. ATGL has a key role in lipid droplet/adiposome degradation in mammalian cells.EMBO Rep. 2006; 7: 106-113Crossref PubMed Scopus (246) Google Scholar). Rabbit polyclonal anti-calnexin antibodies (38Manganas L.N. Trimmer J.S. Calnexin regulates mammalian Kv1 channel trafficking.Biochem. Biophys. Res. Commun. 2004; 322: 577-584Crossref PubMed Scopus (21) Google Scholar) were a gift from J. Trimmer (University of California, Davis). All fluorescent dye- and peroxidase-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). HEK 293, baby hamster kidney (BHK), NIH 3T3, and SKBr3 human breast cancer cells were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% iron-supplemented calf serum (JRH Biosciences, Lenexa, KS), 100 U/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate (Invitrogen). For MCF7 human breast cancer cells and HeLa cells, calf serum was replaced with 10% FBS (Hyclone, Logan, UT). Where indicated, cell culture medium was supplemented with 400 μM oleic acid (Nu-Check Prep, Elysian, MN) complexed to BSA at a 6.6:1 M ratio [prepared as described previously (39Listenberger L.L. Ory D.S. Schaffer J.E. Palmitate-induced apoptosis can occur through a ceramide-independent pathway.J. Biol. Chem. 2001; 276: 14890-14895Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar)]. HEK 293 cells were transfected with murine ADRP in pcDNA3 (Invitrogen; a gift from J. McManaman, University of Colorado Health Sciences Center, Denver) (40McManaman J.L. Zabaronick W. Schaack J. Orlicky D.J. Lipid droplet targeting domains of adipophilin.J. Lipid Res. 2003; 44: 668-673Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) via calcium phosphate. Stable clones were selected by culturing transfected cells with 0.5 mg/ml G418 (Cellgro, Herndon, VA) and maintained with 0.2 mg/ml G418. To assess ADRP expression levels, cells were lysed in 1% Triton X-100, 150 mM NaCl, 50 mM Tris, pH 7.4, 2 mM EDTA, 1 μg/ml pepstatin, and 1 μg/ml leupeptin. Aliquots of postnuclear supernatants containing equal amounts of protein were subjected to SDS-PAGE and Western blotting. Cells were fixed with 3% paraformaldehyde in phosphate-buffered saline (150 mM NaCl and 20 mM phosphate buffer, pH 7.4) for 20 min at room temperature. ADRP or ATGL was detected essentially as described for ADRP (41DiDonato D. Brasaemle D.L. Fixation methods for the study of lipid droplets by immunofluorescence microscopy.J. Histochem. Cytochem. 2003; 51: 773-780Crossref PubMed Scopus (109) Google Scholar), except that 0.5 mg/ml goat IgG in antibody diluent and blocking buffer was replaced, respectively, with 0.1% and 3% BSA. To visualize neutral lipids, 1 μg/ml BODIPY 493/503 (Molecular Probes, Carlsbad, CA) was added to cells during staining with secondary antibody. Stained cells were visualized with a Zeiss Axiovision 200 fluorescence microscope. Out-of-focus fluorescence was removed with deconvolution software using the inverse filter algorithm (Axiovision version 4.4). Single sections of a Z-stack are shown. Cells were cultured in medium alone or with 400 μM oleate complexed to BSA for 15 h. Cells were washed with 150 mM NaCl and 50 mM Tris, pH 7.4, and lipids were extracted with hexane-isopropanol (3:2) as described (42Millard E.E. Srivastava K. Traub L.M. Schaffer J.E. Ory D.S. Niemann-Pick type C1 (NPC1) overexpression alters cellular cholesterol homeostasis.J. Biol. Chem. 2000; 275: 38445-38451Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Five micrograms of oleyl alcohol (Sigma, St. Louis, MO) was added to each sample as a recovery standard. Lipids were dried under nitrogen, resuspended in CHCl3/methanol (1:1), and separated by TLC using hexane-isopropyl ether-acetic acid (65:35:2) as the solvent. Lipids were visualized by cupric acetate staining and compared with a standard curve [1–8 μg of cholesterol, triolein, and cholesteryl oleate (43Macala L.J. Yu R.K. Ando S. Analysis of brain lipids by high performance thin-layer chromatography and densitometry.J. Lipid Res. 1983; 24: 1243-1250Abstract Full Text PDF PubMed Google Scholar)]. Proteins were extracted from cell residues after lipid extraction with 0.1 M NaOH and quantified using a protein assay kit from Bio-Rad (Hercules, CA). One million HEK 293 or ADRP-expressing HEK 293 (293/ADRP) B8 cells were plated in 10 cm dishes (triplicate plates for each time point) and incubated overnight with medium containing 400 μM oleate complexed to BSA to promote lipid accumulation. At time zero, oleate-supplemented medium was removed and cells were incubated with medium containing 6 μM triacsin C (Biomol, Plymouth Meeting, MA). At the indicated times, lipids were extracted, separated by TLC, and compared with a standard curve as described above. TAG mass was normalized to a recovery standard (oleyl alcohol) and expressed relative to protein levels at time zero. Eight subconfluent 35 mm plates each of HEK 293 and 293/ADRP B8 cells were transfected with GFP-tagged ATGL (a gift from C. Jackson, National Institutes of Health, Bethesda, MD) (37Smirnova E. Goldberg E.B. Makarova K.S. Lin L. Brown W.J. Jackson C.L. ATGL has a key role in lipid droplet/adiposome degradation in mammalian cells.EMBO Rep. 2006; 7: 106-113Crossref PubMed Scopus (246) Google Scholar) using Lipofectamine 2000 (Invitrogen). ATGL-transfected cells (and one confluent 10 cm plate of untransfected cells as a carrier to facilitate lipid droplet recovery) were cultured with medium containing 400 μM oleate overnight. Cells were scraped into cold phosphate-buffered saline, pelleted, and resuspended in HEPES buffer (10 mM HEPES, 5 mM EDTA, pH 7.4, 1 μg/ml pepstatin, and 1 μg/ml leupeptin). After incubating on ice for 10 min, cells were homogenized with a 25 gauge needle. The homogenate was adjusted to 10% sucrose, transferred to a Beckman centrifuge tube (number 344062), and overlaid with ∼3.1 ml of HEPES buffer to fill the tubes. Samples were spun in a Beckman L8-55 centrifuge (SW60 rotor) at 280,000 g, 4°C, for 3.5 h. Fractions of ∼0.6 ml were collected from the top, and volumes were adjusted to 0.7 ml with HEPES buffer. Pellets were washed three times with HEPES buffer before resuspending in gel-loading buffer. Equivalent volumes of each gradient fraction were subjected to SDS-PAGE and Western blotting using an antibody against the GFP tag. Additional Western blots using antibodies against ADRP, HSP90, and calnexin confirmed that lipid droplets mostly segregated in fraction 1, cytosolic proteins were largely in the void volume (fractions 6 and 7) or pellet, and membrane proteins were efficiently pelleted. For quantification of TIP47 in subcellular fractions, four 10 cm plates each of HEK 293 and 293/ADRP B8 cells were grown, processed, and subjected to SDS-PAGE and Western blotting as for ATGL localization. For quantification of TAG mass in subcellular fractions, four confluent plates each of HEK 293 or 293/ADRP B8 cells were grown, processed for sucrose gradient centrifugation, and fractionated as described for ATGL localization. A recovery standard (oleyl alcohol) was added to each fraction, and lipids were extracted according to Bligh and Dyer (44Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43133) Google Scholar). Lipids in fraction 1 (floatable lipid droplets), fractions 6 and 7 (cytosol), the membrane pellet, and whole cell lysate were dried under nitrogen, resuspended in CHCl3/methanol (1:1), and separated by TLC as described above for quantification of lipid mass. Eight
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