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• W2057723954 abstract "Phospholipases A2 (PLA2s) catalyze hydrolysis of fatty acids from the sn-2 position of phospholipids. Here we report the identification and characterization of a membrane-associated intracellular calcium-dependent, adipose-specific PLA2 that we named AdPLA (adipose-specific phospholipase A2). We found that AdPLA was highly expressed specifically in white adipose tissue and was induced during preadipocyte differentiation into adipocytes. Clearance of AdPLA by immunoprecipitation significantly decreased PLA activity in white adipose tissue lysates but had no effect on liver lysates, where expression was hardly detectable. In characterizing AdPLA, we employed radiochemical assays with TLC analysis of the enzyme activity of lysates from COS-7 cells overexpressing AdPLA. For kinetic studies, we produced purified recombinant AdPLA for use in a lipoxidase-coupled spectrophotometric assay. AdPLA generated free fatty acid and lysophospholipid from phosphatidylcholine with a preference for hydrolysis at the sn-2 position. Although we found low but detectable lysophospholipase activity, AdPLA showed no significant activity against a variety of other lipid substrates. Calcium was found to activate AdPLA but was not essential for activity. Studies with known phospholipase inhibitors, including bromoenolactone, methyl arachidonyl fluorophosphate, AACOCF3, 7,7-dimethyl-5,8-eicosadienoic acid, and thioetheramide, supported that AdPLA is a phospholipase. Mutational studies showed that His-23 and Cys-113 are critical for activity of AdPLA and suggested that AdPLA is likely a His/Cys PLA2. Overall, although AdPLA is similar to other histidine phospholipases in pH and calcium dependence, AdPLA showed different characteristics in many regards, including predicted catalytic mechanism. AdPLA may therefore represent the first member of a new group of PLA2s, group XVI. Phospholipases A2 (PLA2s) catalyze hydrolysis of fatty acids from the sn-2 position of phospholipids. Here we report the identification and characterization of a membrane-associated intracellular calcium-dependent, adipose-specific PLA2 that we named AdPLA (adipose-specific phospholipase A2). We found that AdPLA was highly expressed specifically in white adipose tissue and was induced during preadipocyte differentiation into adipocytes. Clearance of AdPLA by immunoprecipitation significantly decreased PLA activity in white adipose tissue lysates but had no effect on liver lysates, where expression was hardly detectable. In characterizing AdPLA, we employed radiochemical assays with TLC analysis of the enzyme activity of lysates from COS-7 cells overexpressing AdPLA. For kinetic studies, we produced purified recombinant AdPLA for use in a lipoxidase-coupled spectrophotometric assay. AdPLA generated free fatty acid and lysophospholipid from phosphatidylcholine with a preference for hydrolysis at the sn-2 position. Although we found low but detectable lysophospholipase activity, AdPLA showed no significant activity against a variety of other lipid substrates. Calcium was found to activate AdPLA but was not essential for activity. Studies with known phospholipase inhibitors, including bromoenolactone, methyl arachidonyl fluorophosphate, AACOCF3, 7,7-dimethyl-5,8-eicosadienoic acid, and thioetheramide, supported that AdPLA is a phospholipase. Mutational studies showed that His-23 and Cys-113 are critical for activity of AdPLA and suggested that AdPLA is likely a His/Cys PLA2. Overall, although AdPLA is similar to other histidine phospholipases in pH and calcium dependence, AdPLA showed different characteristics in many regards, including predicted catalytic mechanism. AdPLA may therefore represent the first member of a new group of PLA2s, group XVI. Phospholipases A2 (PLA2) 4The abbreviations used are: PLA2phospholipase A2AdPLAadipose-specific phospholipase A2cPLA2cytosolic phospholipase A2DAGdiacylglycerolFFAfree fatty acidGFPgreen fluorescent proteiniPLA2calcium-independent phospholipase A2LRATlecithin:retinol acyltransferasePCphosphatidylcholinesPLA2secretory phospholipase A2TAGtriacylglycerolWATwhite adipose tissueHAhemagglutininBELbromoenolactoneAACOCF3arachidonyl trifluoromethyl ketoneDMEMDulbecco's modified Eagle's mediumBSAbovine serum albuminMAFPmethyl arachidonyl fluorophosphateMIXmethylisobutylxanthineESTexpressed sequence tag. 4The abbreviations used are: PLA2phospholipase A2AdPLAadipose-specific phospholipase A2cPLA2cytosolic phospholipase A2DAGdiacylglycerolFFAfree fatty acidGFPgreen fluorescent proteiniPLA2calcium-independent phospholipase A2LRATlecithin:retinol acyltransferasePCphosphatidylcholinesPLA2secretory phospholipase A2TAGtriacylglycerolWATwhite adipose tissueHAhemagglutininBELbromoenolactoneAACOCF3arachidonyl trifluoromethyl ketoneDMEMDulbecco's modified Eagle's mediumBSAbovine serum albuminMAFPmethyl arachidonyl fluorophosphateMIXmethylisobutylxanthineESTexpressed sequence tag. catalyze hydrolysis of the sn-2 ester bond of phospholipids (1Schaloske R.H. Dennis E.A. Biochim. Biophys. Acta. 2006; 1761: 1246-1259Crossref PubMed Scopus (726) Google Scholar). They have diverse functions ranging from digestion of dietary phospholipids to membrane remodeling by acylation/deacylation cycles, to cell signaling through the liberation of lysophospholipid and arachidonic acid that is utilized in eicosanoid biosynthesis (2Yuan C. Rieke C.J. Rimon G. Wingerd B.A. Smith W.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6142-6147Crossref PubMed Scopus (102) Google Scholar). PLA2s therefore play important biological roles in a variety of tissues. In adipose tissue, arachidonic acid and eicosanoids have been implicated in diverse processes, including regulation of differentiation (3Aubert J. Saint-Marc P. Belmonte N. Dani C. Negrel R. Ailhaud G. Mol. Cell. Endocrinol. 2000; 160: 149-156Crossref PubMed Scopus (54) Google Scholar, 4Fajas L. Miard S. Briggs M.R. Auwerx J. J. Lipid Res. 2003; 44: 1652-1659Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 5Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2728) Google Scholar, 6Reginato M.J. Krakow S.L. Bailey S.T. Lazar M.A. J. Biol. Chem. 1998; 273: 1855-1858Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 7Yan H. Kermouni A. Abdel-Hafez M. Lau D.C. J. Lipid Res. 2003; 44: 424-429Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), lipolysis (8Cohen-Luria R. Rimon G. Cell. Signal. 1992; 4: 331-335Crossref PubMed Scopus (20) Google Scholar, 9Fain J.N. Leffler C.W. Bahouth S.W. J. Lipid Res. 2000; 41: 1689-1694Abstract Full Text Full Text PDF PubMed Google Scholar, 10Kather H. Simon B. J. Clin. Investig. 1979; 64: 609-612Crossref PubMed Scopus (38) Google Scholar), and glucose transport (11Nugent C. Prins J.B. Whitehead J.P. Wentworth J.M. Chatterjee V.K. O'Rahilly S. J. Biol. Chem. 2001; 276: 9149-9157Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), highlighting the importance of PLA2 in this tissue. phospholipase A2 adipose-specific phospholipase A2 cytosolic phospholipase A2 diacylglycerol free fatty acid green fluorescent protein calcium-independent phospholipase A2 lecithin:retinol acyltransferase phosphatidylcholine secretory phospholipase A2 triacylglycerol white adipose tissue hemagglutinin bromoenolactone arachidonyl trifluoromethyl ketone Dulbecco's modified Eagle's medium bovine serum albumin methyl arachidonyl fluorophosphate methylisobutylxanthine expressed sequence tag. phospholipase A2 adipose-specific phospholipase A2 cytosolic phospholipase A2 diacylglycerol free fatty acid green fluorescent protein calcium-independent phospholipase A2 lecithin:retinol acyltransferase phosphatidylcholine secretory phospholipase A2 triacylglycerol white adipose tissue hemagglutinin bromoenolactone arachidonyl trifluoromethyl ketone Dulbecco's modified Eagle's medium bovine serum albumin methyl arachidonyl fluorophosphate methylisobutylxanthine expressed sequence tag. PLA2s are currently classified into 15 major groups that are subclassified into 5 distinct types of enzymes as follows: secretory PLA2s (sPLA2s), cytosolic PLA2s (cPLA2s), and calcium-independent PLA2s (iPLA2s), as well as platelet-activating factor acetylhydrolases and lysosomal PLA2s (1Schaloske R.H. Dennis E.A. Biochim. Biophys. Acta. 2006; 1761: 1246-1259Crossref PubMed Scopus (726) Google Scholar). The sPLA2s are characterized by relatively low molecular weights (typically <20 kDa), millimolar Ca2+ dependence for optimum catalytic activity, and extensive disulfide-stabilized tertiary structure (12Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1217) Google Scholar). Most sPLA2s contain a catalytic histidine residue within a conserved CCXHDXC motif (12Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1217) Google Scholar). The cytosolic PLA2s (cPLA2) are much larger, ranging in size to over 100 kDa but have few disulfide bonds and are localized intracellularly (13Ghosh M. Tucker D.E. Burchett S.A. Leslie C.C. Prog. Lipid Res. 2006; 45: 487-510Crossref PubMed Scopus (308) Google Scholar). They require Ca2+ for translocation from the cytosol to their site of action on membrane phospholipids, but they do not require calcium for catalytic activity per se (14Diaz B.L. Arm J.P. Prostaglandins Leukot. Essent. Fatty Acids. 2003; 69: 87-97Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). All contain a conserved Ser/Asp catalytic dyad and Arg that are required for catalytic activity. c-PLA2α is the only PLA2 that shows specificity for arachidonic acid in the sn-2 position (13Ghosh M. Tucker D.E. Burchett S.A. Leslie C.C. Prog. Lipid Res. 2006; 45: 487-510Crossref PubMed Scopus (308) Google Scholar, 14Diaz B.L. Arm J.P. Prostaglandins Leukot. Essent. Fatty Acids. 2003; 69: 87-97Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). iPLA2s are characterized by a GXSXG consensus lipase motif with a catalytic serine, and these are also localized intracellularly. iPLA2sdonot require Ca2+ for membrane association or for catalytic activity (1Schaloske R.H. Dennis E.A. Biochim. Biophys. Acta. 2006; 1761: 1246-1259Crossref PubMed Scopus (726) Google Scholar). In our efforts to identify novel proteins of significance in adipocyte metabolism, we found a transcript encoding an 18-kDa protein that is highly and differentially expressed only in adipose tissue, and we named it AdPLA for adipose-specific PLA2. Sequence analysis indicated that this protein has been identified previously as HRASLS3 (Ha-RAS like suppressor 3 or H-Rev-107-1), a class II tumor suppressor with no known molecular function (15Sers C. Emmenegger U. Husmann K. Bucher K. Andres A.C. Schafer R. J. Cell Biol. 1997; 136: 935-944Crossref PubMed Scopus (98) Google Scholar). This protein has been reported to be ubiquitously expressed at low levels in a variety of tissues, although prior to this study, expression in adipose tissue had not been examined. Thus far, no enzymatic activity has been attributed to this protein. Sequence alignment indicated shared homology with lecithin:retinol acyltransferase (LRAT) (16Jahng W.J. Xue L. Rando R.R. Biochemistry. 2003; 42: 12805-12812Crossref PubMed Scopus (37) Google Scholar). However, we found no acyltransferase activity of AdPLA, but rather phospholipase activity. We found that this protein catalyzes the efficient release of free fatty acids (FFA) and lysophospholipid from phosphatidylcholine (PC), indicating that it is a phospholipase A (PLA). Furthermore, we found a preference for hydrolysis at the sn-2 position of phospholipids that classifies AdPLA to the PLA2 superfamily of enzymes. Further characterization of this enzyme has led us to propose that AdPLA may be the first member of an entirely new group of calcium-dependent intracellular PLA2s, group XVI. Materials—[U-14C]Palmitic acid (specific activity, 850 mCi/mmol) was from PerkinElmer Life Sciences; 1,2-di[1-14C]palmitoyl-sn-glycerol-3-phosphocholine (specific activity, 110 mCi/mmol), 1-palmitoyl-2[1-14C]palmitoyl-sn-glycerol-3-phosphocholine (specific activity, 60 mCi/mmol), 1-[1-14C]palmitoyl-2-hydroxy-sn-glycerol-3-lysophosphocholine (specific activity, 53 mCi/mmol), and [9,10-3H]-triolein (specific activity, 53 Ci/mmol) were from GE Healthcare. 1,2-Dilineoyl-PC, 1-palmitoyl-2-linoleoyl-PC, 1-palmitoyl-2-arachidonyl-PC, 1-palmitoyl-2-linoleoyl-phosphatidylethanolamine, 1-palmitoyl-2-linoleoyl-phosphatidylserine, 1-palmitoyl-2-linoleoyl-phosphatidic acid, 1-palmitoyl-2-linoleoyl-phosphatidylinositol, and egg PC were from Avanti Polar Lipids (Alabaster, AL). Sodium deoxycholate, lipoxidase preparation from soybeans (type V, 701,000 units/mg protein), and pancreatic phospholipase A2 from bovine pancreas were from Sigma. Racemic BEL ((E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one), 7,7-dimethyl-5,8-eicosadienoic acid, AACOCF3, methyl arachidonyl fluorophosphate (MAFP), and thioetheramide were from Cayman Chemicals (Ann Arbor, MI). Lipofectamine 2000 was from Invitrogen, and other chemicals and high pressure liquid chromatography grade solvents were from Fisher. GeneFilter® Microarray Analysis—Identification of genes expressed exclusively in adipose tissue was achieved by comparing the gene expression patterns of different mouse tissues using rat GeneFilter® membranes (Research Genetics) as described previously (17Kim K.H. Lee K. Moon Y.S. Sul H.S. J. Biol. Chem. 2001; 276: 11252-11256Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar). Briefly, filters were hybridized with 33P-labeled cDNAs synthesized by reverse transcription using 5 μg of total RNA from WAT, brain, muscle, and liver. Only those spots found exclusively in filters hybridized with WAT cDNAs were further analyzed. Candidate EST clones were sequenced, and adipose tissue-specific expression was verified by Northern blot analysis using RNA from liver, brain, muscle, and WAT (data not shown). One of these clones showed exclusive expression in WAT. Using the sequence of the selected EST clone as a query, a BLASTn search of the mouse genome data base of NCBI identified a match with HRASLS3/H-Rev107-1. The full-length cDNA was prepared and sequenced (GenBank™ accession number NM_139269) (18Roder K. Latasa M.J. Sul H.S. Biochem. Biophys. Res. Commun. 2002; 293: 793-799Crossref PubMed Scopus (11) Google Scholar). Generation of AdPLA Mammalian and Bacterial Expression Vectors and Preparation of Purified Recombinant NUS-AdPLA—We amplified the coding region of AdPLA, including a C-terminal hemagglutinin (HA) tag using mouse cDNA reverse-transcribed from RNA prepared from WAT, and we subcloned this into the BamHI/XhoI sites of pCR3.1. Two AdPLA-GFP constructs bearing either the complete coding region of AdPLA or a truncated form missing the last 36 codons was prepared by subcloning enhanced green fluorescent protein (GFP) in-frame with the C terminus of AdPLA in pCR3.1. Lipofectamine 2000 was used for all transfections. For protein expression in Escherichia coli, HA-tagged AdPLA was subcloned into the NotI-XhoI site of the pET-43.1a prokaryotic expression vector (Novagen) containing the N-terminal His tag for purification and a hydrophilic NUS A tag. AdPLA mutants were prepared using a QuikChange site-directed mutagenesis kit (Stratagene Inc.) according to the vendor's instructions (for primers see supplemental Table S1). For AdPLA and AdPLA mutants, the plasmids were transformed into E. coli BL21STAR-(DE3), expression was induced by 0.5 mm isopropyl 1-thio-β-d-galactopyranoside, and cultures were grown at 27 °C for an additional 3.5 h. His-tagged AdPLA and mutants were affinity-purified using HisPur cobalt resin from Pierce. Protein was visualized by Coomassie staining relative to BSA standards for quantification. Cell Culture, Differentiation, and Production of Primary Preadipocytes—3T3-L1 and COS-7 cells (American Type Culture Collection) were cultured in DMEM containing 10% fetal bovine serum. To induce differentiation of 3T3-L1 cells into adipocytes, 2-day post-confluent preadipocytes (day 0) were treated with 1 μm dexamethasone, 0.5 mm methylisobutylxanthine (MIX), and 1 μg/ml insulin for 48 h. After the induction period, cells were switched to maintenance medium (DMEM supplemented with 10% fetal bovine serum) and maintained for 5–7 days, at which point 90% of the cells exhibited the typical adipocyte morphology. Subcutaneous inguinal fat depots from female Zucker rats were dissected, and the lymph nodes were removed. The stromal vascular cells were obtained by collagenase digestion (1 mg/ml) at 37 °C for 45 min in HEPES buffer (10 mm HEPES, pH 7.4, 135 mm NaCl, 2.2 mm CaCl2, 1.25 mm MgSO4, 0.45 mm KH2PO4, 2.17 mm Na2HPO4, 5 mm d-glucose, and 2% w/v BSA). The cell suspension was filtered through a 100-μm nylon filter and centrifuged at 400 × g for 10 min. The pellets were washed, filtered through a 25-μm nylon filter, and plated at a density of 2.5 × 104 cells/cm2 in DMEM with 10% FBS. At confluence, differentiation was initiated by the addition of 0.1 μm dexamethasone, 0.25 mm MIX, and 17 nm insulin. After 2 days, medium was replaced by DMEM with 10% FBS and insulin only. Cells were harvested for RNA isolation at the time points indicated. Subcellular Localization of AdPLA—COS-7 or 3T3-L1 cells were grown on coverslips. COS-7 cells transfected with AdPLA-GFP expression vector were fixed with 4% paraformaldehyde, incubated with polyclonal antibodies against COX-1 for 1 h (Santa Cruz Biotechnology), washed with phosphate-buffered saline, and incubated with Alexa Fluor 546-conjugated secondary antibody at 5 μg/ml for 3 h (Molecular Probes). For endoplasmic reticulum staining, 3T3-L1 cells transfected with AdPLA-GFP were differentiated into adipocytes and were stained with concanavalin A Alexa Fluor 633 conjugate (Molecular Probes). The samples were mounted on glass microscope slides using Antifade and Prolong mounting media (Molecular Probes), and images were captured using a Zeiss Axiophot LSM 510Meta confocal microscope. Polyclonal Anti-AdPLA Antiserum and Western Blot Analysis—To generate polyclonal antisera, rabbits were immunized with purified recombinant AdPLA-His expressed in E. coli. For Western blotting, proteins were subjected to 12% SDS-PAGE and transferred to nitrocellulose membrane for immunodetection using primary antibodies against HA (Covance, 1:1000), AdPLA (1:2000), or glyceraldehyde-3-phosphate dehydrogenase (1:1000) (Santa Cruz Biotechnology). RNA Extraction, Northern Blotting, and Real Time Reverse Transcription-PCR—Total RNA was isolated from tissues using TRIzol Reagent (Invitrogen). For Northern blot analysis, 5–15 μg of total RNA was subjected to electrophoresis on formaldehyde-containing 1.2% agarose gels and transferred onto Hybond N+ nylon membranes (Amersham Biosciences). Hybridization was carried out in ExpressHyb solution (Clontech) using 32P-labeled cDNA-specific probes for AdPLA, adipocyte fatty acid-binding protein, stearoyl-CoA desaturase 1, or peroxisome proliferator-activated receptor-γ. For reverse transcription-quantitative PCR, cDNA was synthesized from 2 μg of total RNA by oligo(dT) priming and Superscript II reverse transcriptase (Invitrogen). Tissue expression of AdPLA, iPLA2β, or cPLA2α was determined with an ABI PRISM 7700 sequence fast detection system (Applied Biosystems) using specific primers from Applied Biosystems. Gene expression level was quantified by measuring the threshold cycle normalized to glyceraldehyde-3-phosphate dehydrogenase and is expressed relative to levels in liver. In Vitro Phospholipase Activity Assay—The continuous spectrophotometric assay was carried out as described (19Jimenez M. Cabanes J. Gandia-Herrero F. Escribano J. Garcia-Carmona F. Perez-Gilabert M. Anal. Biochem. 2003; 319: 131-137Crossref PubMed Scopus (36) Google Scholar) with minor modifications. Briefly, aliquots of stock lipid substrate solutions in chloroform were dried under a stream of N2 and then sonicated into suspension up to a maximum concentration of 100 μm in 50 mm Tris buffer, pH 8, containing 2 mm deoxycholate. The resulting micellar solution was allowed to equilibrate for 10 min at 25 °C. Phospholipase A activity was determined by means of a coupled enzyme assay. Released polyunsaturated fatty acids (i.e. linoleic acid or arachidonic acid) were subsequently oxidized by lipoxygenase (0.36 mg/ml), giving rise to a hydroperoxide derivative that could be measured by spectrophotometric assessment of the increase in absorbance at 234 nm (ϵ234 = 25,000 m-1 cm-1). The reaction was started by adding purified NUS-AdPLA into the substrate/lipoxidase mixture, and the A234 was monitored continuously for 3 min. Controls without either AdPLA or lipoxygenase were carried out routinely. For studies with inhibitors, the compounds were incorporated at the indicated concentrations into micelles containing 100 μm 1-palmitoyl-2-linoleoyl-PC with 2 mm deoxycholate and 2 mm CaCl2. For assay of activity from cell lysates, samples were homogenized in Buffer A (0.1 m sucrose, 50 mm KCl, 40 mm KH2PO4, and 30 mm EDTA, pH 7.2) and centrifuged at 100,000 × g for 60 min to separate the supernatant (cytosol) from the membrane fraction. For assay from WAT or liver, homogenates were centrifuged at 20,000 × g and then cleared by incubation with preimmune serum or serum from rabbits immunized against AdPLA (1:100 dilution) followed by immunoprecipitation of antibody-protein complexes on protein A-agarose beads. PLA activity with radioactive substrate was determined essentially as described (20Lucas K.K. Dennis E.A. Prostaglandins Other Lipid Mediat. 2005; 77: 235-248Crossref PubMed Scopus (57) Google Scholar) with minor modifications. Micelles were created by sonification of radiolabeled lipid substrates in assay buffer (50 mm Tris, pH 8, 2 mm deoxycholate, 5 mm EDTA), and hydrolytic activity was monitored by the generation of [14C]palmitate. Reactions were started by the addition of purified enzyme or cell lysates and terminated by the addition of (2:1) methanol:chloroform. Lipids were extracted by the method of Bligh and Dyer (21Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42828) Google Scholar) and resolved by TLC on a hexane:diethyl ether:acetic acid solvent front (80:20:2). Bands corresponding to [14C]palmitate were identified by comparison with lipid standards and scraped and quantified by liquid scintillation counting. For resolution of phospholipid from lysophospholipid and FFA, lipid extracts were resolved by TLC on a more aqueous chloroform:methanol:acetic acid:water (60:30: 8.4:3.6) solvent front. For in vitro TAG hydrolase activity, lysates were prepared from COS-7 cells overexpressing AdPLA-HA or desnutrin-HA (positive control) by lysis in Buffer A followed by centrifugation at 16,000 × g. Supernatants (100 μg of protein in 0.1 ml) were incubated with 0.1 ml of substrate containing 100 μm [3H]triolein in mixed micelles with 25 μm egg yolk lecithin, 100 μm taurocholate, 2% BSA (w/v), and 50 mm Tris-HCl, pH 8.0. Reactions were terminated by the addition of 3.25 ml of methanol: chloroform:heptane (10:9:7), and fatty acids were extracted with1 ml of 0.1 m potassium carbonate, 0.1 m boric acid, pH 10.5. Radioactivity in 0.7 ml of upper phase obtained after centrifugation for 20 min at 800 × g was quantified by liquid scintillation counting. Statistical Analysis—The results are expressed as means ± S.E. Statistically significant differences between two groups were assessed by Student's t test. Differences between multiple groups were assessed by one-way analysis of variance with Bonferroni's post hoc test. PLA2 has been reported to function in adipocyte cell signaling (22Nazarenko I. Kristiansen G. Fonfara S. Guenther R. Gieseler C. Kemmner W. Schafer R. Petersen I. Sers C. Am. J. Pathol. 2006; 169: 1427-1439Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 23Nazarenko I. Schafer R. Sers C. J. Cell Sci. 2007; 120: 1393-1404Crossref PubMed Scopus (36) Google Scholar), differentiation (3Aubert J. Saint-Marc P. Belmonte N. Dani C. Negrel R. Ailhaud G. Mol. Cell. Endocrinol. 2000; 160: 149-156Crossref PubMed Scopus (54) Google Scholar, 4Fajas L. Miard S. Briggs M.R. Auwerx J. J. Lipid Res. 2003; 44: 1652-1659Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 5Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2728) Google Scholar, 6Reginato M.J. Krakow S.L. Bailey S.T. Lazar M.A. J. Biol. Chem. 1998; 273: 1855-1858Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 7Yan H. Kermouni A. Abdel-Hafez M. Lau D.C. J. Lipid Res. 2003; 44: 424-429Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and in the regulation of important adipocyte metabolic processes such as lipolysis and glucose transport (8Cohen-Luria R. Rimon G. Cell. Signal. 1992; 4: 331-335Crossref PubMed Scopus (20) Google Scholar, 9Fain J.N. Leffler C.W. Bahouth S.W. J. Lipid Res. 2000; 41: 1689-1694Abstract Full Text Full Text PDF PubMed Google Scholar, 10Kather H. Simon B. J. Clin. Investig. 1979; 64: 609-612Crossref PubMed Scopus (38) Google Scholar, 11Nugent C. Prins J.B. Whitehead J.P. Wentworth J.M. Chatterjee V.K. O'Rahilly S. J. Biol. Chem. 2001; 276: 9149-9157Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Because dysregulated adipocyte differentiation and metabolism are linked to obesity and associated pathologies, understanding phospholipid metabolism in adipose tissue is critical. Using EST cDNA microarray analysis we identified AdPLA as a differentially expressed gene that was found to be present at high levels specifically in adipocytes. We have used this approach to identify other important adipocyte-specific genes, including adipose-specific secretory factor (ADSF/resistin) (17Kim K.H. Lee K. Moon Y.S. Sul H.S. J. Biol. Chem. 2001; 276: 11252-11256Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar) and desnutrin (24Villena J.A. Roy S. Sarkadi-Nagy E. Kim K.H. Sul H.S. J. Biol. Chem. 2004; 279: 47066-47075Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar). Although we have previously found AdPLA to be expressed at low levels in a number of tissues and cultured cell lines (18Roder K. Latasa M.J. Sul H.S. Biochem. Biophys. Res. Commun. 2002; 293: 793-799Crossref PubMed Scopus (11) Google Scholar, 25Roder K. Kim K.H. Sul H.S. Biochem. Biophys. Res. Commun. 2002; 294: 63-70Crossref PubMed Scopus (15) Google Scholar, 26Roder K. Latasa M.J. Sul H.S. J. Biol. Chem. 2002; 277: 30543-30550Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), prior to this work, expression of AdPLA in adipocytes or in adipose tissue had not been examined. Although we observed that two other phospholipases, iPLA2β(Fig. 1A, right panel) and cPLA2α (Fig. 1A, middle panel), were present at varying levels in a host of tissues, AdPLA expression was clearly predominant in white adipose tissue (WAT) (40 to >150-fold more abundant than in liver) and, to a lesser extent, in brown adipose tissue (Fig. 1A, left panel). Separation of WAT into adipocytes and stromovascular fraction indicated expression of AdPLA exclusively in adipocytes (data not shown). In agreement, AdPLA was not detected in 3T3-L1 or primary rat preadipocytes but was induced upon differentiation (Fig. 1, B and C). Induction of AdPLA tended to occur late in differentiation as compared with the appearance of other marker genes of early to late stage adipocyte differentiation (i.e. peroxisome proliferator-activated receptor-γ, adipocyte fatty acid-binding protein, or stearoyl-CoA desaturase 1). Furthermore, induction of AdPLA most likely resulted from effects occurring during differentiation rather than from transcriptional modulation by either dexamethasone or MIX, because neither agent alone affected AdPLA expression in 3T3-L1 cells (Fig. 1D). We examined the localization of HA-tagged AdPLA in COS-7 cells and found it both in the 100,000 × g membrane fraction and also in the cytoplasmic fraction (Fig. 1E). Confocal imaging of full-length AdPLA-GFP showed a predominantly perinuclear localization as well as evidence of the presence within the cytoplasm of COS-7 cells (Fig. 1, F and G, left panel) and 3T3-L1 adipocytes (Fig. 1H, left panel). Notably, truncation of the C-terminal hydrophobic domain of AdPLA resulted in a more diffuse localization of the GFP fusion construct in cells (Fig. 1F, right panel). Because some PLA2s act on intracellular membrane phospholipids to provide arachidonic acid to cyclooxygenase 1 (COX-1) for eicosanoid biosynthesis, we examined the localization of AdPLA relative to both the endoplasmic reticulum and COX-1. We found AdPLA-GFP localized, at least in part, in proximity to COX-1 in COS-7 cells (Fig. 1G). Similarly, AdPLA-GFP partly co-localized with the endoplasmic reticulum in differentiated 3T3-L1 adipocytes (Fig. 1H). Taken together, these findings suggest that AdPLA may function in eicosanoid biosynthesis, although further studies will be required to determine the effects of AdPLA on adipose tissue metabolism. To study the activity of AdPLA, we produced cell lysates from COS-7 cells overexpressing HA-tagged AdPLA (Fig. 2A), as well as bacterially derived recombinant AdPLA for use in a continuous spectrophotometric based assay (Fig. 2B). Recombinant AdPLA tagged with HA and polyhistidine was also tagged with NUS A protein to enhance solubility and expression (27Marblestone J.G. Edavettal S.C. Lim Y. Lim P. Zuo X. Butt T.R. Protein Sci. 2006; 15: 182-189Crossref PubMed Scopus (335) Google Scholar), and was affinity-purified on Co3+-agarose beads to over 90% hom" @default.
• W2057723954 created "2016-06-24" @default.
• W2057723954 creator A5017939890 @default.
• W2057723954 creator A5023735639 @default.
• W2057723954 creator A5036039665 @default.
• W2057723954 creator A5038863786 @default.
• W2057723954 creator A5057486939 @default.
• W2057723954 date "2008-09-01" @default.
• W2057723954 modified "2023-10-16" @default.
• W2057723954 title "Identification and Functional Characterization of Adipose-specific Phospholipase A2 (AdPLA)" @default.
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