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• W2021004639 abstract "Previous in vitro studies have established that hormone sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP) form a physical complex that presumably positions the FABP to accept a product fatty acid generated during catalysis. To assess AFABP-HSL interaction within a cellular context, we have used lipocytes derived from 293 cells (C8PA cells) and examined physical association using fluorescence resonance energy transfer. Transfection of C8PA cells with cyan fluorescent protein (CFP)-HSL, yellow fluorescent protein (YFP)-adipocyte FABP, or YFP-liver FABP revealed that under basal conditions each protein was cytoplasmic. In the presence of 20 μm forskolin, CFP-HSL translocated to the triacylglycerol droplet, coincident with BODIPY-FA labeled depots. Fluorescence resonance energy transfer analysis demonstrated that CFP-HSL associated with YFP-adipocyte FABP in both basal and forskolin-treated cells. In contrast, little if any fluorescence resonance energy transfer could be detected between CFP-HSL and YFP-liver FABP. These results suggest that a pre-lipolysis complex containing at least AFABP and HSL exists and that the complex translocates to the surface of the lipid droplet. Previous in vitro studies have established that hormone sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP) form a physical complex that presumably positions the FABP to accept a product fatty acid generated during catalysis. To assess AFABP-HSL interaction within a cellular context, we have used lipocytes derived from 293 cells (C8PA cells) and examined physical association using fluorescence resonance energy transfer. Transfection of C8PA cells with cyan fluorescent protein (CFP)-HSL, yellow fluorescent protein (YFP)-adipocyte FABP, or YFP-liver FABP revealed that under basal conditions each protein was cytoplasmic. In the presence of 20 μm forskolin, CFP-HSL translocated to the triacylglycerol droplet, coincident with BODIPY-FA labeled depots. Fluorescence resonance energy transfer analysis demonstrated that CFP-HSL associated with YFP-adipocyte FABP in both basal and forskolin-treated cells. In contrast, little if any fluorescence resonance energy transfer could be detected between CFP-HSL and YFP-liver FABP. These results suggest that a pre-lipolysis complex containing at least AFABP and HSL exists and that the complex translocates to the surface of the lipid droplet. Lipolysis in adipocytes is a complex biochemical process brought about by a combination of hormonal and metabolic determinants (1Londos C. Brasaemle D.L. Schultz C.J. Adler-Wailes S.C. Levin D.M. Kimmel A.R. Rondinone C.M. Ann. N. Y. Acad. Sci. 1999; 892: 155-168Crossref PubMed Scopus (219) Google Scholar, 2Holm C. Osterlund T. Laurell H. Contreras J.A. Annu. Rev. Nutr. 2000; 20: 365-393Crossref PubMed Scopus (343) Google Scholar, 3Collins S. Surwit R.S. Recent Prog. Horm. Res. 2001; 56: 309-328Crossref PubMed Scopus (207) Google Scholar, 4Greenberg A.S. Shen W. Muliro K. Patel S. Souza S. Roth R. Kraemer F.B. J. Biol. Chem. 2001; 276: 45456-45461Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 5Kraemer F.B. Shen W.J. J. Lipid Res. 2002; 43: 1585-1594Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 6Yin W. Mu J. Birnbaum M.J. J. Biol. Chem. 2003; 278: 43074-43080Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Generally described, adipocyte lipolysis involves hormonal activation of protein kinase A that results in the phosphorylation of at least two key proteins, perilipin A, a droplet-associated protein implicated in controlling access of hydrolytic enzymes to the triacylglycerol, and the hormone-sensitive lipase (HSL), 1The abbreviations used are: HSL, hormone-sensitive lipase; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; FABP, fatty acid-binding protein; AFABP, adipocyte FABP; LFABP, liver FABP; FRET, fluorescence resonance energy transfer; PA, perilipin A; BSA, bovine serum albumin; PBS, phosphate-buffered saline.1The abbreviations used are: HSL, hormone-sensitive lipase; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; FABP, fatty acid-binding protein; AFABP, adipocyte FABP; LFABP, liver FABP; FRET, fluorescence resonance energy transfer; PA, perilipin A; BSA, bovine serum albumin; PBS, phosphate-buffered saline. believed to be the primary lipase in fat cells responsible for hydrolysis of stored lipid (7Tansey J. Huml A. Vogt R. Davis K. Johns J. Fraser K. Brasaemle D. Kimmel A. Londos C. J. Biol. Chem. 2003; 278: 8401-8406Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Phosphorylation of perilipin A results in the dynamic restructuring of the lipid droplet, thereby allowing access of the phosphorylated hormone-sensitive lipase to the triacylglycerol substrate (8Tornqvist H. Krabisch L. Belfrage P. J. Lipid Res. 1972; 13: 424-426Abstract Full Text PDF PubMed Google Scholar, 9Sztalryd C. Xu G. Dorward H. Tansey J.T. Contreras J.A. Kimmel A.R. Londos C. J. Cell Biol. 2003; 161: 1093-1103Crossref PubMed Scopus (414) Google Scholar). Although these two proteins are appreciated as central to lipid mobilization, other droplet-associated proteins, including other lipases or accessory factors, may also participate in the process and add additional levels of complexity and control (10Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. J. Lipid Res. 1997; 38: 2249-2263Abstract Full Text PDF PubMed Google Scholar, 11Souza S.C. de Vargas L.M. Yamamote M.T. Lien P. Franciosa M.D. Moss L.G. Greenberg A.S. J. Biol. Chem. 1998; 273: 24665-24669Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 12Syu L.-J. Saltiel A.R. Mol. Cell. 1999; 4: 109-115Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 13Nakamura N. Fujimoto T. Biochem. Biophys. Res. Commun. 2003; 306: 333-338Crossref PubMed Scopus (63) Google Scholar).Recently another participant in the lipolysis process has been identified as the intracellular fatty acid binding protein (FABP). The adipocyte FABP forms a physical complex with the hormone-sensitive lipase, activating the enzyme by sequestering fatty acids and relieving product inhibition (14Shen W.-J. Sridhar K. Bernlohr D.A. Kraemer F.B. Proc. Natl. Acad Sci. U. S. A. 1999; 96: 5528-5553Crossref PubMed Scopus (178) Google Scholar). Association between the two proteins has been evaluated in vitro using a combination of yeast two-hybrid analysis, glutathione S-transferase pull downs, and co-immune precipitation as well as deletion and point mutation analysis (14Shen W.-J. Sridhar K. Bernlohr D.A. Kraemer F.B. Proc. Natl. Acad Sci. U. S. A. 1999; 96: 5528-5553Crossref PubMed Scopus (178) Google Scholar, 16Shen W.-J. Liang Y. Hong R. Patel S. Natu V. Jenkins A. Bernlohr D.A. Kraemer F.B. J. Biol. Chem. 2001; 276: 49443-49448Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The results of these studies indicated that the adipocyte FABP forms a complex with the amino-terminal domain of HSL (referred to as a docking domain) in a region bounded by amino acids 190–200 of the lipase (16Shen W.-J. Liang Y. Hong R. Patel S. Natu V. Jenkins A. Bernlohr D.A. Kraemer F.B. J. Biol. Chem. 2001; 276: 49443-49448Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Docking of AFABP onto the amino-terminal domain of HSL juxtaposes the fatty acid-binding protein adjacent to the catalytic carboxyl-terminal region so as to accept a product fatty acid during triacylglycerol hydrolysis.To explore the specificity of HSL-FABP interaction, isothermal titration microcalorimetry has recently been used (17Jenkins-Kruchten A.E. Bennaars-Eiden A. Ross J.R. Shen W.-J. Kraemer F.B. Bernlohr D.A. J. Biol. Chem. 2003; 278: 47636-47643Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Using isothermal titration microcalorimetry, both the adipocyte and keratinocyte FABPs were shown to associate with HSL in a 1:1 stoichiometry and an affinity in the low nanomolar range (17Jenkins-Kruchten A.E. Bennaars-Eiden A. Ross J.R. Shen W.-J. Kraemer F.B. Bernlohr D.A. J. Biol. Chem. 2003; 278: 47636-47643Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In contrast, the intestinal and liver FABP forms, proteins that exhibit the same α-carbon fold as does AFABP but have grossly different amino acid sequences, did not associate to any measurable extent. Moreover, the physical association of adipocyte FABP with HSL required the presence of fatty acids, but due to the experimental design, it was not clear if such fatty acids associate with the FABP or the lipase.The in vitro analysis of FABP association with HSL using purified components does not address many of the regulatory features linked to lipolysis in vivo. For example, it is not clear if the FABP and HSL form a basal complex that co-translocates to the surface of the droplet or if HSL translocates to the droplet, at which time the FABP docks onto the enzyme. Also, it is not clear what role hormone-stimulated phosphorylation of HSL has to play in FABP association; all studies to date have been carried out using non-phosphorylated proteins. To address the first of these questions, we have turned our attention to the use of lipocytes derived from 293 cells overexpressing a combination of fatty acid transport protein 1 and perilipin A as a simple vehicle for assessing FABP-HSL interaction within the cellular context. Because of the high endogenous level of FABP in adipocytes, the surrogate cell line lacking HSL or FABP but having lipid droplets affords the opportunity to study protein-protein interaction as well as translocation in a hormonally sensitive system. From perilipin studies carried out by Londos et al. (7Tansey J. Huml A. Vogt R. Davis K. Johns J. Fraser K. Brasaemle D. Kimmel A. Londos C. J. Biol. Chem. 2003; 278: 8401-8406Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) as well as studies in the Greenberg laboratory (18Souza S.C. Muliro K. Liscum L. Lien P. Yamamoto M.T. Schaffer J.E. Dalla G.E. Wang X. Kraeme F.B. Obin M. Greenberg A.S. J. Biol. Chem. 2002; 277: 8267-8272Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) with cells expressing combinations of the fatty acid transport protein 1 and perilipin A, it is evident that cells expressing perilipin A exhibit increased hormone-stimulated lipolysis. Additionally, Hatch et al. (19Hatch G. Smith A.J. Xu F. Hall A. Bernlohr D.A. J. Lipid Res. 2002; 43: 1380-1389Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) found that 293 fibroblasts expressing FATP1 exhibit increased oleate and lignocerate influx and that such lipid is preferentially channeled into triacylglycerol. To that end, we stably co-express perilipin A and FATP1 in 293 cells producing lipocytes that accumulate triacylglycerol droplets in defined structures. Into such cells we have transiently expressed fluorescent fusion proteins of CFP-HSL and YFP-FABP and evaluated not only translocation of HSL in response to forskolin stimulation but also physical association between the lipase and FABP by FRET under basal and lipolytic conditions. In this report we find that the adipocyte FABP, but not liver FABP, forms a complex with HSL under basal conditions and that the pre-lipolysis complex co-translocates to the surface of the lipid droplet in the presence of forskolin.EXPERIMENTAL PROCEDURESMaterials—Fatty acids were obtained from Nu-Chek Prep, Inc., Elysian, MN. pcDNA3.1/Zeo, Zeocin, Geneticin, Lipofectamine, and tissue culture reagents were obtained from Invitrogen. BODIPY D 3835 (4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid) and Alexa Fluor 488 were obtained from Molecular Probes, Inc., Eugene, OR. pEYFP-C1 and pECFP-C1 were obtained from Clontech Laboratories, Inc., Palo Alto, CA. Gold Seal glass coverslips for imaging were purchased from Thomas Scientific, Swedesboro, NJ. The non-esterified fatty acid (NEFA) assay system was purchased from WAKO Chemicals, Richmond, VA. Cholesteryl [1-14C]oleate was purchased from Amersham Biosciences. All other reagents were purchased from Sigma-Aldrich.Cloning and Cell Biology—An A206K mutation was introduced into each of the green fluorescent protein derivatives to reduce intrinsic dimerization (20Zacharias D. Violin J. Newton A. Tsien R. Science. 2002; 296: 913-916Crossref PubMed Scopus (1759) Google Scholar) using the QuikChange™ site-directed mutagenesis technique of Stratagene Cloning Systems, La Jolla, CA. HSL was then subcloned in-frame into the A206K pECFP-C1 expression vector, whereas AFABP and LFABP were subcloned into A206K pEYFP-C1. Expression of the fusion proteins was confirmed by both the expression of fluorescence in 293 cells and by Western blot analysis. Perilipin A was subcloned into pCDNA3.1/Zeo and expression in 293 cells confirmed by Western blot analysis. All cloning was verified by DNA sequencing.Generation of C8PA Lipocytes—Perilipin A, subcloned into pcDNA3.1/Zeo, was linearized and introduced into 293 cells stably expressing FATP1 (C8 cells) by electroporation according to the manufacturer's instructions (BTX division of Genetronics, Inc., San Diego, CA). Dilutions of the electroporated cells were plated in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum onto surfaces pretreated with 25 μg/ml of poly-l-lysine to enhance attachment and incubated at 37 °C, 5% CO2. After 48 h, selection was initiated with the addition of 300 μg/ml Zeocin for expression of perilipin A and 400 μg/ml of Geneticin for expression of FATP1. Individual lines were developed and analyzed immunochemically for perilipin A expression using a goat anti-perilipin A antibody (9Sztalryd C. Xu G. Dorward H. Tansey J.T. Contreras J.A. Kimmel A.R. Londos C. J. Cell Biol. 2003; 161: 1093-1103Crossref PubMed Scopus (414) Google Scholar) and/or FATP1 expression using an anti-FATP1 antibody (19Hatch G. Smith A.J. Xu F. Hall A. Bernlohr D.A. J. Lipid Res. 2002; 43: 1380-1389Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). A stable cell line expressing perilipin A and FATP1 (referred to as C8PA) was selected and maintained on 300 μg/ml Zeocin and 400 μg/ml Geneticin during the course of all studies. C8PA cells for all experiments were plated onto surfaces pretreated with 25 μg/ml of poly-l-lysine.Immunocytochemistry—C8PA cells plated on 13-mm glass coverslips and incubated in 300 μm oleic acid, 100 μm fatty acid-free BSA were fixed in 3% paraformaldehyde for 15 min at 25 °C. The cells were rinsed in phosphate-buffered saline (PBS, pH 7.2) containing 0.01% digitonin and incubated for 2 h at 25 °C in blocking buffer containing 0.01 m phosphate, 5% goat serum, 5% glycerol, 1.0% cold water fish gelatin, and 0.01% digitonin. The cells were subsequently incubated overnight at 4 °C in a 1:500 dilution of goat-anti-perilipin antibody (9Sztalryd C. Xu G. Dorward H. Tansey J.T. Contreras J.A. Kimmel A.R. Londos C. J. Cell Biol. 2003; 161: 1093-1103Crossref PubMed Scopus (414) Google Scholar) in PBS containing 0.01% digitonin. After washing in PBS with 0.01% digitonin, the coverslips were incubated for 1 h at 25 °C in a 1:400 dilution of Alexa Fluor 488 (2 mg/ml) donkey anti-goat IgG in PBS with 0.01% digitonin. After washing, the coverslips were incubated at 25 °C in Dulbecco's modified Eagle's medium with BODIPY D3835 (10 μg/ml), then washed with PBS, mounted, and visualized for perilipin or fat droplets by confocal microscopy.Confocal Microscopy—Preparations were viewed using a Bio-Rad MRC-1024 confocal microscope attached to a Nikon Diaphot inverted microscope (Bio-Rad) equipped with a 15-millwatt krypton/argon laser. Excitation filters allowing 488 and 568 nm were used sequentially to visualize the Alexa 488 and BODIPY probes, respectively. The samples were viewed using either a 20×, 0.75 n.a. plan apo or a 40×, 1.0 n.a. plan apo objective. Digital images were collected using LaserSharp version 3.2 software (Bio-Rad). Stored digital images were analyzed using Image Pro Plus Version 4.5 software (Media Cybernetics, Silver Spring, MD 20910).Lipolysis in C8PA Cells—For in situ lipolysis, C8PA cells were loaded with lipid to maximize droplet formation by a 48-h incubation with 300 μm oleic acid complexed to 100 μm fatty acid-free BSA. After loading with fatty acids, the cells were washed 2 times with phosphate-buffered saline and incubated either with or without 20 μm forskolin in Krebs-Ringers Hepes buffer (pH 7.4) with 2% BSA and 5% glucose for 4 h (18Souza S.C. Muliro K. Liscum L. Lien P. Yamamoto M.T. Schaffer J.E. Dalla G.E. Wang X. Kraeme F.B. Obin M. Greenberg A.S. J. Biol. Chem. 2002; 277: 8267-8272Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Media was withdrawn at 0, 1, 2, 3, and 4 h and assayed for non-esterified fatty acids.Fluorescence Resonance Energy Transfer—For FRET analysis C8PA cells were plated onto 13-mm coverslips placed into 12-well dishes. At ∼70% confluence wells were transfected with expression plasmids specific for CFP-HSL and/or YFP-FABP. 24 h later cells were lipid-loaded with 300 μm oleic acid, 100 μm BSA to maximize droplet formation. Lipolytic conditions were initiated by the addition of 20 μm forskolin, and digital images for FRET were captured after 4 h.During microscopy cells were kept in media plus 10% fetal bovine serum at room temperature. Digital images were collected using a Roper CoolSnap HQ 12 bit monochrome camera and captured to a Pentium IV 2.6 GHz personal computer using Image Pro Plus version 4.5 software (Media Cybernetics) for microscope automation and image analysis on a Nikon Eclipse E800 photomicroscope. Images were captured with a 40×, 0.75 n.a. plan fluor or a 60×, 1.40 n.a. plan apo objective. Cells with relatively equal levels of expression were selected for imaging. For detection of CFP-HSL, cells were viewed with an excitation filter of 436/20 nm, a dichroic beam splitter of 455 nm, and an emission filter of 480/40 nm. YFP-FABP was detected by using a filter set with an excitation filter of 500/25 nm, a dichroic beam splitter of 515 nm, and an emission filter of 535/30 nm. The filters for FRET were an excitation filter of 436/20 nm, a dichroic beam splitter of 455 nm, and an emission filter of 535/50 nm. Filters were obtained from the Chroma Technology (Brattleboro, VT). Images were acquired using 2 × 2 binning mode and 100–250-ms integration times on the camera. The exposure times were equal within each series of images and were chosen so that all pixel intensities were within the linear range of the camera. Images were first background-subtracted and registered to ensure accurate pixel alignment. The CFP-HSL image was then thresholded, changing the intensities of all pixels outside of the cell to zero. Thresholding was based on the CFP-HSL image because it had the largest signal-to-noise ratio, providing the clearest distinction between the cell and background. The thresholded CFP-HSL image was used to generate a binary image with all values within the cell = 1 and all outside = 0. The FRET and YFP-FABP images were multiplied by the binary image, ensuring that the same pixels were analyzed in all three images as described in detail (21Chamberlain C.E. Kraynov V.S. Hahn K.M. Methods Enzymol. 2000; 325: 389-400Crossref PubMed Google Scholar). Emission appearing in the FRET image because of emission from CFP-HSL or direct excitation of YFP-FABP was removed by subtracting a fraction of the CFP-HSL and YFP-FABP images from the FRET image. This fraction depended on the filter set and exposure condition used and was determined as described (21Chamberlain C.E. Kraynov V.S. Hahn K.M. Methods Enzymol. 2000; 325: 389-400Crossref PubMed Google Scholar). Corrected FRET was calculated on a pixel-by-pixel basis for the entire image using: corrected FRETC = FRET - (0.50 × CFP-HSL) - (0.02 × YFP-FABP), where FRET, CFP-HSL, and YFP-FABP correspond to background-subtracted images of cells co-expressing CFP-HSL and YFP-FABP acquired through the FRET, CFP, and YFP channels, respectively. 0.50 and 0.02 are the fractions of bleed-through of CFP and YFP fluorescence, respectively, through the FRET filter channel. Controls were performed in which images were obtained in different orders. The order in which images were obtained had no effect. For presentation, a low pass filter kernel was applied to the corrected FRET image to remove high frequency noise (22Castleman K. Digital Image Processing. Prentice-Hall, Upper Saddle River, NJ1996: 207-209Google Scholar). Images were contrast stretched, pseudocolored, and formatted for display using Adobe Photoshop 7.0 software (Adobe Systems, Mountain View, CA).HSL Activity Assay—Plasmids directing the expression of either HSL or CFP-HSL were transiently transfected into 293 cells, washed twice in phosphate-buffered saline, and harvested into 1 ml of 50 mm Tris-HCL, 0.1 mm EDTA (pH 7) with 20 μg/ml leupeptin, 2 μg/ml antipain, and 1 μg/ml pepstatin. The extract was sonicated at 30% power 3 times for 30 s each on ice using a Misonix Sonicator XL. The homogenate was centrifuged at 12,000 × g for 15 min, and the supernatant was used in the assays. Substrate preparation and assay conditions were as previously described (23Osterlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar). Briefly, for 4 ml of substrate 0.45 mm cholesteryl oleate, 1.4 mg of phosphatidylcholine/phosphatidylinositol (3:1, w/w), and 0.9 μCi/ml cholesteryl [14C]oleate were mixed and dried under nitrogen. The dried substrate was emulsified by adding 1 ml of 0.1 m potassium phosphate buffer (pH 7.0) and sonicated 2 × 1 min in a Branson 2510 water bath sonicator. The addition of 3 ml of buffer followed by sonicating 4 × 30 s emulsified the substrate. For reactions, 100 μl of substrate is mixed with 50 μl of enzyme at 37 °C and the production of fatty acids was measured after 30 min as described (23Osterlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar).RESULTSPreviously we have reported the development of fibroblastic cells stably expressing fatty acid transport protein 1 (19Hatch G. Smith A.J. Xu F. Hall A. Bernlohr D.A. J. Lipid Res. 2002; 43: 1380-1389Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Such cells, termed C8 cells, exhibit increased transport of long chain fatty acids and accumulate triacylglycerols. However, triacylglycerol accumulated by C8 cells was disorganized and appeared rather diffusely distributed within the cell based on oil red O staining. Greenberg and co-workers (18Souza S.C. Muliro K. Liscum L. Lien P. Yamamoto M.T. Schaffer J.E. Dalla G.E. Wang X. Kraeme F.B. Obin M. Greenberg A.S. J. Biol. Chem. 2002; 277: 8267-8272Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) as well as the Londos laboratory (7Tansey J. Huml A. Vogt R. Davis K. Johns J. Fraser K. Brasaemle D. Kimmel A. Londos C. J. Biol. Chem. 2003; 278: 8401-8406Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) have reported that perilipin A, the key droplet-associated protein in fat cells, organizes the lipid into well defined structures and provides hormonal competence. To that end we stably transfected C8 cells with perilipin A (PA) to produce a cell line termed C8PA that not only transported fatty acids and synthesized triacylglycerol but organized said lipid into well defined droplets. Fig. 1A shows the oil red O staining of C8PA cells after a 48 h “load” in which FA·BSA (3:1) was incubated in the medium to provide substrate for FATP1. We refer to such lipid filled C8PA cells as lipocytes.Adipose tissue in vivo as well as cultured adipocytes exhibits cAMP-dependent activation of lipolysis and increases free fatty acid release from cells (1Londos C. Brasaemle D.L. Schultz C.J. Adler-Wailes S.C. Levin D.M. Kimmel A.R. Rondinone C.M. Ann. N. Y. Acad. Sci. 1999; 892: 155-168Crossref PubMed Scopus (219) Google Scholar). To assess the lipolytic capacity of C8PA cells, we incubated cells with 20 μm forskolin for 4 h and evaluated fatty acid release from the cells. As shown in Fig. 1B, forskolin stimulated a 2-fold increase in fatty acid release from C8PA lipocytes in a process that was linear for at least 4 h. Although this 2-fold stimulation is significantly less than fatty acid release from cultured adipocytes (24Tansey 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. Londos C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6494-6499Crossref PubMed Scopus (601) Google Scholar), it does demonstrate that the cultured lipocytes are lipolytically competent at low levels albeit in the absence of HSL. Similar results were obtained when cells were treated with 1 mm dibutyryl-cAMP (results not shown), but the magnitude of the increase was not as robust. Therefore, forskolin stimulation was adopted as standard conditions.Lipolytic stimulation of cultured adipocytes results in a dramatic restructuring of the droplet surface and a reorganization of the local droplet environment (8Tornqvist H. Krabisch L. Belfrage P. J. Lipid Res. 1972; 13: 424-426Abstract Full Text PDF PubMed Google Scholar, 9Sztalryd C. Xu G. Dorward H. Tansey J.T. Contreras J.A. Kimmel A.R. Londos C. J. Cell Biol. 2003; 161: 1093-1103Crossref PubMed Scopus (414) Google Scholar). To assess the organizational structure of the lipid droplets in C8PA lipocytes, the cells were incubated for 2 h with BODIPY-oleate, and the fluorescent fatty acid was accumulated into droplets. As shown in Fig. 2, under basal conditions, BODIPY-oleate accumulated into small droplets organized into a central cluster. When visualized with transmitted light differential interference contrast (Fig. 2A), the droplets appear as a central tight cluster of droplets, whereas by fluorescence (Fig. 2C), the clustered nature of the organization of smaller droplets was clear. After forskolin stimulation, the clustered droplets were dramatically restructured, as revealed by both differential interference contrast (Fig. 2B) and BODIPY-oleate fluorescence (Fig. 2D). Moreover, consistent with previous studies (9Sztalryd C. Xu G. Dorward H. Tansey J.T. Contreras J.A. Kimmel A.R. Londos C. J. Cell Biol. 2003; 161: 1093-1103Crossref PubMed Scopus (414) Google Scholar) perilipin A was found on the droplets coincident with the fluorescent lipid localization (Figs. 2, E and F).Fig. 2Fluorescence analysis of C8PA cells. BODIPY-oleate-loaded C8PA cells under basal (A) or with 20 μm forskolin (B) are shown as analyzed using differential interference contrast imaging. Fluorescent images of the BODIPY-oleate-loaded C8PA cells are shown under basal (C) conditions or with 20 μm forskolin (D). Immunofluorescence localization is shown of perilipin A in C8PA cells (E) overlaid onto an image of the same field showing localization of the neutral lipid after incubation with BODIPY-oleate (F). The scale bar on panel D is 10 μm and represents the image size for panels A–D. The scale bar on the inset for panel D is 25 μm and represents the image size for each inset in panels A–D. The scale bar in panel F is 5 μm and represents the images in panels E and F.View Large Image Figure ViewerDownload (PPT)Using the C8PA lipocyte cell model, we addressed the location and translocation of adipocyte FABP and HSL by immunofluorescence using fluorescent fusion proteins. Previous studies show that fusion proteins with adipocyte FABP are fully active for fatty acid binding as well as association with HSL (14Shen W.-J. Sridhar K. Bernlohr D.A. Kraemer F.B. Proc. Natl. Acad Sci. U. S. A. 1999; 96: 5528-5553Crossref PubMed Scopus (178) Google Scholar, 16Shen W.-J. Liang Y. Hong R. Patel S. Natu V. Jenkins A. Bernlohr D.A. Kraemer F.B. J. Biol. Chem. 2001; 276: 49443-49448Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). To verify that CFP-HSL retains activity, we transiently transfected CFP-HSL and HSL into 293 cells. The cells were harvested and assayed for neutral cholesteryl-oleate activity. Control 293 cells transfected with vector alone exhibit no activity, verifying that these cells have little endogenous neutral cholesteryl esterase activity (Fig. 3). CFP-HSL and native HSL have significantly increased activity over vector-transfected cells and show similar levels of activity. CFP-HSL, therefore, maintains its neutral cholesteryl esterase activity as compared with HSL.Fig. 3Activity of CFP-HSL. Expression plasmids encoding either HSL or CFP-HSL were transiently transfected into 293 cells, and the cholesteryl esterase activity in extracts was determined as described.View Large Image Figure ViewerDownload (PPT)Transient transfection of C8PA cells with CFP-HSL revealed that the fusion protein was broadly distributed within the cytoplasm but excluded from organelles and nuclei (Fig. 4A). When C8PA cells were stimulated with forskolin, some but not all CFP-HSL was reorganized and coalesced into a structure coincident with the droplet surface Fig. 4B). To demonstrate that the structure CFP-HSL translocated to is the droplet surface, the fluorescence of BODIPY-oleate staining of the droplets was compared with that of CFP-HSL. As shown in Fig. 4, E and F, CFP-HSL translocation resulted in association with BODIPY-FA-labeled droplets. As such, the C8PA lipocyte system reproduces that exhibited by true adipocytes. It should be noted that these studies do not address the molecular organization" @default.
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