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• W2085475071 abstract "Alterations in lipid secondary messenger generation and lipid metabolic flux are essential in promoting the differentiation of adipocytes. To determine whether specific subtypes of intracellular phospholipases A2 (PLA2s) facilitate hormone-induced differentiation of 3T3-L1 cells into adipocytes, we examined alterations in the mRNA level, protein mass, and activity of three previously characterized mammalian intracellular PLA2s. Hormone-induced differentiation of 3T3-L1 cells resulted in 7.3 ± 0.5- and 7.4 ± 1.4-fold increases of mRNA encoding the calcium-independent phospholipases, iPLA2β and iPLA2γ, respectively. In contrast, the temporally coordinated loss of at least 90% of cPLA2α mRNA was manifest. Western analysis demonstrated the near absence of both iPLA2β and iPLA2γ protein mass in resting 3T3-L1 cells that increased dramatically during differentiation. In vitro measurement of PLA2 activities demonstrated an increase in both iPLA2β and iPLA2γ activities that were discriminated using the chiral mechanism based inhibitors (S)- and (R)-BEL, respectively. Remarkably, treatment of 3T3-L1 cells with small interfering RNA directed against either iPLA2β or iPLA2γ prevented hormone-induced differentiation. Moreover, analysis of the temporally programmed expression of transcription factors demonstrated that the small interfering RNA knockdown of iPLA2β or iPLA2γ resulted in down-regulation of the expression of peroxisome proliferator-activated receptor γ and the CCAAT enhancer-binding protein α (C/EBPα). No alterations in the expression of the early stage transcription factors C/EBPβ and C/EBPδ were observed. Collectively, these results demonstrate prominent alterations in intracellular PLA2s during 3T3-L1 cell differentiation into adipocytes and identify the requirement of iPLA2β and iPLA2γ for the adipogenic program that drives resting 3T3-L1 cells into adipocytes after hormone stimulation. Alterations in lipid secondary messenger generation and lipid metabolic flux are essential in promoting the differentiation of adipocytes. To determine whether specific subtypes of intracellular phospholipases A2 (PLA2s) facilitate hormone-induced differentiation of 3T3-L1 cells into adipocytes, we examined alterations in the mRNA level, protein mass, and activity of three previously characterized mammalian intracellular PLA2s. Hormone-induced differentiation of 3T3-L1 cells resulted in 7.3 ± 0.5- and 7.4 ± 1.4-fold increases of mRNA encoding the calcium-independent phospholipases, iPLA2β and iPLA2γ, respectively. In contrast, the temporally coordinated loss of at least 90% of cPLA2α mRNA was manifest. Western analysis demonstrated the near absence of both iPLA2β and iPLA2γ protein mass in resting 3T3-L1 cells that increased dramatically during differentiation. In vitro measurement of PLA2 activities demonstrated an increase in both iPLA2β and iPLA2γ activities that were discriminated using the chiral mechanism based inhibitors (S)- and (R)-BEL, respectively. Remarkably, treatment of 3T3-L1 cells with small interfering RNA directed against either iPLA2β or iPLA2γ prevented hormone-induced differentiation. Moreover, analysis of the temporally programmed expression of transcription factors demonstrated that the small interfering RNA knockdown of iPLA2β or iPLA2γ resulted in down-regulation of the expression of peroxisome proliferator-activated receptor γ and the CCAAT enhancer-binding protein α (C/EBPα). No alterations in the expression of the early stage transcription factors C/EBPβ and C/EBPδ were observed. Collectively, these results demonstrate prominent alterations in intracellular PLA2s during 3T3-L1 cell differentiation into adipocytes and identify the requirement of iPLA2β and iPLA2γ for the adipogenic program that drives resting 3T3-L1 cells into adipocytes after hormone stimulation. Recently, there has been a dramatic increase in the incidence of obesity in industrialized and newly developed countries (1Friedman J.M. Nature. 2000; 404: 632-634Crossref PubMed Scopus (629) Google Scholar). Obesity results from abnormal increases in white adipose tissue (WAT) 1The abbreviations used are: WAT, white adipose tissue; PLA2, phospholipases A2; iPLA2, calcium-independent phospholipase A2; cPLA2, cytosolic phospholipase A2; sPLA2, secretory phospholipase A2; TAG, LPA, lysophosphatidic acid; LPC, lysophosphatidylcho-triacylglycerol; line; FFA, free fatty acid; PG, prostaglandin; siRNA, small interfering RNA; ESI/MS, electrospray ionization mass spectrometry; PPARγ, peroxisome proliferator-activated receptor γ; C/EBP, CCAAT enhancer binding protein; BEL, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; CAPS, 3-(cyclohexylamino)propanesulfonic acid. mass leading to alterations in whole organism energy storage and utilization (2Spiegelman B.M. Flier J.S. Cell. 2001; 104: 531-543Abstract Full Text Full Text PDF PubMed Scopus (1942) Google Scholar, 3Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Crossref PubMed Scopus (1862) Google Scholar, 4Friedman J.M. Halaas J.L. Nature. 1998; 395: 763-770Crossref PubMed Scopus (4523) Google Scholar). Increased adipose tissue mass can result from either an increase in individual adipocyte cell size (hypertrophy) or from an increase in total adipocyte number (hyperplasia). Alterations in whole organism lipid homeostasis leading to increased adipocyte tissue mass are highly correlated with the metabolic syndrome that is accompanied by its lethal sequelae of diabetes, hypertension, and atherosclerosis (2Spiegelman B.M. Flier J.S. Cell. 2001; 104: 531-543Abstract Full Text Full Text PDF PubMed Scopus (1942) Google Scholar, 3Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Crossref PubMed Scopus (1862) Google Scholar, 4Friedman J.M. Halaas J.L. Nature. 1998; 395: 763-770Crossref PubMed Scopus (4523) Google Scholar, 5Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. Nature. 2001; 409: 307-312Crossref PubMed Scopus (3974) Google Scholar). During the last decade, substantial progress has been made in understanding the biochemical events leading to adipocyte differentiation utilizing the hormone-induced 3T3-L1 cell model of adipocyte differentiation (6Moustaid N. Sul H.S. J. Biol. Chem. 1991; 266: 18550-18554Abstract Full Text PDF PubMed Google Scholar, 7MacDougald O.A. Lane M.D. Annu. Rev. Biochem. 1995; 64: 345-373Crossref PubMed Scopus (942) Google Scholar, 8Xue J.-C. Schwarz E.J. Chawla A. Lazar M.A. Mol. Cell. Biol. 1996; 16: 1567-1575Crossref PubMed Google Scholar, 9Prusty D. Park B.-H. Davis K.E. Farmer S.R. J. Biol. Chem. 2002; 277: 46226-46232Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). Central to this understanding has been the detailed characterization of temporally coordinated changes in the expression of specific genes that collectively define the adipocyte phenotype. Differentiation of adipocytes is accomplished by the programmed activation of transcriptional regulatory proteins that modulate the expression of mRNA and proteins that effectively reprogram 3T3-L1 cell lipid metabolism to that of a mature adipocyte. Such alterations include increased de novo fatty acid synthesis, accumulation of perilipin-coated triglyceride droplets, and the generation of lipid secondary messengers including eicosanoids and lysophosphatic acid that serve as potent and specific regulators of coordinated differentiation programs (3Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Crossref PubMed Scopus (1862) Google Scholar, 7MacDougald O.A. Lane M.D. Annu. Rev. Biochem. 1995; 64: 345-373Crossref PubMed Scopus (942) Google Scholar, 10Mandrup S. Lane M.D. J. Biol. Chem. 1997; 272: 5367-5370Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 11MacDougald O.A. Mandrup S. Trends Endocrinol. Metab. 2002; 13: 5-11Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 12Cornelius P. MacDougald O.A. Lane M.D. Annu. Rev. Nutri. 1994; 14: 99-129Crossref PubMed Scopus (573) Google Scholar, 13Rosen E.D. Spiegelman B.M. J. Biol. Chem. 2001; 276: 37731-37734Abstract Full Text Full Text PDF PubMed Scopus (1080) Google Scholar, 14Gao G. Serrero G. J. Biol. Chem. 1990; 265: 2431-2434Abstract Full Text PDF PubMed Google Scholar). Phospholipases A2 (PLA2s) catalyze the hydrolysis of the sn-2 fatty acid substituents from glycerophospholipid substrates to yield free fatty acid (e.g. arachidonic acid) and lysophospholipid (15Gross R.W. Trends Cardiovasc. Med. 1992; 2: 115-121Crossref PubMed Scopus (49) Google Scholar, 16Gross R.W. J. Lipid Mediat. Cell Signal. 1995; 12: 131-137Crossref PubMed Scopus (17) Google Scholar, 17Miyake R. Gross R.W. Biochim. Biophys. Acta. 1992; 1165: 167-176Crossref PubMed Scopus (36) Google Scholar). Mammalian phospholipases A2 have been categorized into several classes based on their requirement for calcium ion in in vitro activity assays (i.e. millimolar, nanomolar, or no calcium requirement) leading to their broad classification into three classes of enzymes: calcium-independent phospholipase A2 (iPLA2), cytosolic phospholipase A2 (cPLA2), and secretory phospholipase A2 (sPLA2) (18Kudo I. Murakami M. Prostaglandins Other Lipid Mediat. 2002; 68/69: 3-58Crossref Scopus (659) Google Scholar). Prior studies have demonstrated that eicosanoids are potent modulators of adipocyte differentiation underscoring the roles of PGE2 and PGI2 in inducing transformation of progenitor cells into mature adipocytes (19Vassaux G. Gaillard D. Darimont C. Ailhaud G. Negrel R. Endocrinology. 1992; 131: 2393-2398Crossref PubMed Google Scholar, 20Vassaux G. Gaillard D. Ailhaud G. Negrel R. J. Biol. Chem. 1992; 267: 11092-11097Abstract Full Text PDF PubMed Google Scholar). In contrast, PGF2α inhibits hormone-induced differentiation of 3T3-L1 cells into mature adipocytes (21Reginato 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). In most mammalian cells, the rate-determining step in the production of biologically active eicosanoids is the release of arachidonic acid from the sn-2 position of glycerophospholipids. Despite the known importance of eicosanoids in modulating adipocyte differentiation, there is a paucity of information on the molecular identity of the specific types of intracellular phospholipases A2 present in differentiating adipocytes, the alterations in protein mass and activity levels of the different intracellular phospholipase A2 classes, and the importance of each specific type of phospholipase A2 in adipocyte differentiation (14Gao G. Serrero G. J. Biol. Chem. 1990; 265: 2431-2434Abstract Full Text PDF PubMed Google Scholar). Recent studies have demonstrated that lysophosphatidic acid (LPA) serves a dual function in adipocyte differentiation acting both as an extracellular ligand for EDG receptors (22Ferry G. Tellier E. Try A. Gres S. Naime I. Simon M.F. Rodriguez M. Boucher J. Tack I. Gesta S. Chomarat P. Dieu M. Raes M. Galizzi J.P. Valet P. Boutin J.A. Saulnier-Blache J.S. J. Biol. Chem. 2003; 278: 18162-18169Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 23Hooks S.B. Santos W.L. Im D.-S. Heise C.E. Macdonald T.L. Lynch K.R. J. Biol. Chem. 2001; 276: 4611-4621Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) and as the endogeneous intracellular ligand for the adipocyte transcriptional regulator peroxisome proliferator-activated receptor (PPAR) γ (24McIntyre T.M. Pontsler A.V. Silva A.R. St. Hilaire A. Xu Y. Hinshaw J.C. Zimmerman G.A. Hama K. Aoki J. Arai H. Prestwich G.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 131-136Crossref PubMed Scopus (460) Google Scholar). According to current dogma, LPA produced during adipocyte differentiation results from the sequential hydrolysis of phosphatidylcholine to lysophosphatidylcholine (LPC) by endogenous phospholipases A2 and the subsequent extracellular hydrolysis of LPC to LPA catalyzed by a secreted lysophospholipase D, autotaxin (22Ferry G. Tellier E. Try A. Gres S. Naime I. Simon M.F. Rodriguez M. Boucher J. Tack I. Gesta S. Chomarat P. Dieu M. Raes M. Galizzi J.P. Valet P. Boutin J.A. Saulnier-Blache J.S. J. Biol. Chem. 2003; 278: 18162-18169Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). However, there is no information presently available on the types of phospholipases A2 present in the adipocyte that contribute to eicosanoid and lysolipid production in the adipocyte. Recent analyses of the transcriptional programs utilized for adipocyte differentiation have identified the critical roles of the CCAAT/enhancer-binding protein (C/EBP) family and PPARγ in mediating the transcriptional alterations required for adipocyte differentiation (3Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Crossref PubMed Scopus (1862) Google Scholar, 10Mandrup S. Lane M.D. J. Biol. Chem. 1997; 272: 5367-5370Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). Hormone-induced growth-arrested 3T3-L1 cells treated with insulin, methylisobutylxanthine, and dexamethasone express the early transcription factors C/EBPβ and C/EBPδ, which lead to their re-entry into the cell cycle (25Tang Q.Q. Otto T.C. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 850-855Crossref PubMed Scopus (406) Google Scholar, 26Tang Q.Q. Otto T.C. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 44-49Crossref PubMed Scopus (644) Google Scholar). C/EBPβ and C/EBPδ then activate the transcription of C/EBPα and PPARγ, which are believed to both be antimitotic and act synergistically to activate the expression of adipocyte-specific genes leading to the differentiated adipocyte phenotype (27Altiok S. Xu M. Spiegelman B.M. Genes Dev. 1997; 11: 1987-1998Crossref PubMed Scopus (337) Google Scholar, 28Wang H. Iakova P. Wilde M. Welm A. Goode T. Roesler W.J. Timchenko N.A. Mol. Cell. 2001; 8: 817-828Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). In this study, we demonstrate the dramatic up-regulation of both iPLA2β and iPLA2γ mRNA levels, protein content, and enzymatic activities during hormone-induced differentiation of 3T3-L1 cells temporally coordinated with the down-regulation of cPLA2α to near background levels. Moreover, the essential roles of iPLA2β and iPLA2γ in adipocyte differentiation and their interplay with C/EBP and PPAR transcription factors have been identified by specific siRNA knockdown of either iPLA2β or iPLA2γ activity. The results demonstrate that downregulation of iPLA2β or iPLA2γ inhibits adipocyte differentiation via preventing PPARγ and C/EBPα expression without affecting the expression of C/EBPβ and C/EBPδ. Collectively, these results are the first to demonstrate the central roles of both iPLA2β and iPLA2γ in the differentiation of a mammalian preadipocyte cell line into adipocytes. Materials—3T3-L1 cells were obtained from ATCC (Manassas, VA). Fetal calf serum and Dulbecco's modified Eagle's medium (DMEM) were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum was obtained from BioWhittaker, Inc. (Walkersville, MD). Reagents for reverse transcription and quantitative PCR were supplied from Applied Biosystems (Foster City, CA). Oligonucleotide primer pairs and probes used in quantitative PCR were ordered from Applied Biosystems (Foster City, CA). SiRNA construction and transfection kits were purchased from Ambion (Austin, TA). All radiolabeled lipids were obtained from American Radiolabeled Chemicals Inc. (St. Louis, MO). Most other chemicals were obtained from Sigma. Anti-PPARγ, anti-C/EBPα, anti-C/EBPβ, anti-C/EBPδ, anti-SCD I, and anti-cPLA2α antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antiperilipin and anti-GLUT4 antibodies were kindly provided by Dr. Michael M. Mueckler (Washington University, St. Louis, MO). Anti-PMP70 antibody was obtained from Affinity Bioreagents (Golden, CO). Rabbit anti-iPLA2β or anti-iPLA2γ polyclonal antibodies were produced utilizing the synthetic peptides CEFLKREFGEHTKMTDVKKP (iPLA2β) or CENIPLDESRNEKLDQ (iPLA2γ) and immunoaffinity purified as previously described (29Mancuso D.J. Jenkins C.M. Gross R.W. J. Biol. Chem. 2000; 275: 9937-9945Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Cell Culture of 3T3-L1 Cells and Differentiation into the Adipoctye Phenotype—3T3-L1 cells were cultured to confluence in DMEM containing 10% calf serum by changing the medium every 2 days as previously described (30Frost S.C. Lane M.D. J. Biol. Chem. 1985; 260: 2646-2652Abstract Full Text PDF PubMed Google Scholar). Two days after cell confluence, differentiation was initiated by adding differentiation medium 1 (0.5 mm methylisobutylxanthine, 0.25 μm dexamethasone, 1 μg/ml insulin in DMEM containing 10% fetal bovine serum). Two days later, methylisobutylxanthine and dexamethasone were removed and insulin (1 μg/ml) was maintained for 2 more days. Thereafter, cells were grown in DMEM containing 10% fetal bovine serum in the absence of differentiating reagents by replacing the media every 2 days. Reverse Transcription and Quantitative PCR—Total RNA was purified from 3T3-L1 cell pellets utilizing a RNeasy® Mini Kit from Qiagen (Valencia, CA) according to the manufacturer's instructions. For cDNA preparation, 250 pmol of random hexamers were hybridized by incubation for 10 min at 25 °C and extended by incubation for 30 min at 48 °C in the presence of 125 units of reverse transcriptase in 100 μl of PCR buffer (5.5 mm MgCl2, 0.5 mm of each dNTP, and 40 units of RNase inhibitor). Reverse transcriptase was inactivated by incubation at 95 °C for 5 min. Amplification of each target cDNA was performed with TaqMan® PCR reagent kits and quantified by the ABI PRISM 7700 detection system according to the protocol provided by the manufacturer (Applied Biosystems, Foster City, CA). A traditionally utilized standard gene, glyceraldehyde-3-phosphate dehydrogenase, was measured and used as internal standard. Oligonucleotide primer pairs and probes specific for cPLA2α (5′-CCTTTGAGTTCATTTTGGATCCTAA/5′-TGTAGCTGTGCCTAGGGTTTCAT/5′-AGGAAAATGTTTTGGAGATCACACTGATGGATG), iPLA2β (5′-CCTTCCATTACGCTGTGCAA/5′-GAGTCAGCCCTTGGTTGTT/5′-CCAGGTGCTACAGCTCCTAGGAAAGAATGC), and iPLA2γ (5′-GAGGAGAAAAAGCGTGTGCTACTTC/5′-GGTTGTTCTTCTTAAGGCCTGAA/5′-TCTGTTATCAATACTCACTCTTGCAATA) were employed. Protein Extraction and Western Blot—Proteins from 3T3-L1 cells were extracted as described previously (31Gomez F.E. Miyazaki M. Kim Y.C. Marwah P. Lardy H.A. Ntambi J.M. Fox B.G. Biochemistry. 2002; 41: 5473-5482Crossref PubMed Scopus (34) Google Scholar). Briefly, the cell monolayer was washed with ice-cold PBS and subsequently scraped into 1 ml of ice-cold lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mm phenylmethylmethanesulfonyl fluoride, 2 μg/ml aprotinin, and 1 μg/ml leupeptin). The solution was incubated on ice for 10 min after vortexing for 10 s. The cell homogenate was spun at 10,000 × g at 4 °C in a tabletop centrifuge for 10 min and the supernatant was transferred to a new tube and stored at –70 °C until used for Western blot analysis. Nuclear extracts were prepared with NE-PER® Nuclear and Cytoplasmic Extraction Reagents from Pierce according to manufacturer's protocol. Proteins were separated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore) in 10 mm CAPS buffer (pH 11) containing 10% methanol. Powdered milk (5% (w/v)) was used to block nonspecific binding sites prior to incubation with primary antibody directed against each specific protein as indicated. After incubation with secondary antibody (IgG-HRP conjugate diluted 1:5000 in blocking buffer), proteins were visualized by enhanced chemiluminescence according to the instructions of the manufacturer (Amersham Biosciences). Phospholipase A2 Assays—On the day of the experiment, 3T3-L1 cells at different stages of differentiation were washed briefly with PBS and detached by incubation in trypsin-EDTA (0.25%, w/v) at 37 °C for 5 min. The cells were washed again with 5 volumes of CMRL-1066, transferred to a 50-ml Falcon centrifuge tube, and centrifuged for 5 min at 1700 rpm at 4 °C. The resulting cell pellets were resuspended in CMRL-1066 medium and centrifuged as above two more times. The cell pellets from 4 plates (10 mm diameter) were resuspended in 3 ml of lysis buffer (0.25 m sucrose, 25 mm imidazole, pH 7.2) and were sonicated six times for 1 s each. The tubes were placed on ice for 3 min and then re-sonicated. PLA2 assays were performed as described previously (32Jenkins C.M. Han X. Mancuso D.J. Gross R.W. J. Biol. Chem. 2002; 277: 32807-32814Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Briefly, PLA2 activity were assessed by incubating 3T3-L1 cell protein (100–200 μg) with radiolabeled phosphatidylcholine, l-α-[oleoyl-1-14C]palmitoyl-2-oleoyl (POPC, 50 mCi/mmol, 5 μm final concentration, introduced by ethanol injection (2 μl)) in assay buffer (final conditions: 100 mm Tris-HCl, 4 mm EGTA, pH 7.2) at 37 °C for 30 min in a final volume of 200 μl. Reactions were quenched by addition of butanol (100 μl). 30 μlofthe organic phase of each sample were spotted on a Whatman silica plate that was developed with a nonpolar acidic mobile phase (100 ml of 70/30/1, petroleum ether/ethyl ether/acetic acid). Spots corresponding to fatty acids were scrapped into scintillation vials and radioactivity was quantified by scintillation spectrometry as described previously (32Jenkins C.M. Han X. Mancuso D.J. Gross R.W. J. Biol. Chem. 2002; 277: 32807-32814Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). BEL enantiomers were resolved by chiral high performance liquid chromatography as described previously (32Jenkins C.M. Han X. Mancuso D.J. Gross R.W. J. Biol. Chem. 2002; 277: 32807-32814Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). For the inhibition assays of iPLA2 by BEL, proteins were incubated with 10 μm (R)-BEL, (S)-BEL, racemic BEL, or ethanol vehicle for 3 min at 22 °C prior to the addition of radiolabeled substrate. SiRNA Construction and Transfection—The siRNAs directed against iPLA2β and iPLA2γ were constructed employing the Silencer™ siRNA construction kit (Ambion) according to the protocol provided by manufacturer. Upon confluence, the 3T3-L1 cell media were changed to growth media without antibiotics. One to 2 days later, cells were transfected with siRNAs (20 nm) using the siPORT™ lipid transfection reagent (Ambion) according to the manufacturer's instructions. Five volumes of 1.2× differentiation medium 1 without antibiotics were added 4 h after transfection and the cells were maintained at normal growing conditions and induced to differentiate as described above. Among four siRNAs for each targeting gene, the sequences specific for iPLA2β (5′-AACAGCACAGAGAAUGAGGAG-3′) and iPLA2γ (5′-AAGAUAAACAGCUUCAGGACA-3′) were selected based upon their potency to inhibit target gene expression. A scrambled siRNA was used as a negative control. Triacylglycerol Extraction and Electrospray Ionization Mass Spectrometry—After siRNA transfection, 3T3-L1 cells were grown to day 8 as described above. The cell monolayer was washed with ice-cold PBS and scraped into 1 ml of 50 mm LiCl. The lipids were extracted by the method of Bligh-Dyer (33Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42878) Google Scholar) in the presence of an internal standard (Tri17:0TAG, 200 nmol/mg of protein). Mass spectral analysis of TAG was performed by electrospray ionization utilizing a Finnigan TSQ Quantum spectrometer (Finnigan MAT, San Jose, CA) as previously described (34Han X. Gross R.W. Anal. Biochem. 2001; 295: 88-100Crossref PubMed Scopus (296) Google Scholar). Protein Extraction from White Adipose Tissue of Zucker Rats—Female obese Zucker (fa/fa) rats and lean congenic controls (5–6 weeks old) were housed and maintained with a 12-h light/12-h dark photoperiod. Water and food were given ad libitum. Animals were sacrificed (asphyxiated by CO2) and inguinal fat pads (WAT) were removed, rapidly frozen in liquid nitrogen, and ground with a motor and pestle. To the tissue powder was added lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mm phenylmethylmethanesulfonyl fluoride, 2 μg/ml aprotinin, and 1 μg/ml leupeptin) and the resulting mixtures were homogenized with a Potter-Elvehjem apparatus. The homogenates were spun at 10,000 × g at 4 °C in a tabletop centrifuge for 10 min and the supernatant was transferred to a new tube and stored at –70 °C until used for Western blot analysis. Miscellaneous—Protein concentration was determined utilizing a BCA protein assay kit (Pierce) with bovine serum albumin as a standard. All data were normalized to protein content and are presented as the mean ± S.E. Statistically significant differences between mean values were determined using unpaired Student's t tests. Alterations in the mRNA Levels of Intracellular Phospholipases A2 during Differentiation of 3T3-L1 Preadipocytes— Prior work has underscored the essential roles of eicosanoid metabolites and LPC-derived LPA in adipocyte differentiation (19Vassaux G. Gaillard D. Darimont C. Ailhaud G. Negrel R. Endocrinology. 1992; 131: 2393-2398Crossref PubMed Google Scholar, 20Vassaux G. Gaillard D. Ailhaud G. Negrel R. J. Biol. Chem. 1992; 267: 11092-11097Abstract Full Text PDF PubMed Google Scholar, 21Reginato 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, 22Ferry G. Tellier E. Try A. Gres S. Naime I. Simon M.F. Rodriguez M. Boucher J. Tack I. Gesta S. Chomarat P. Dieu M. Raes M. Galizzi J.P. Valet P. Boutin J.A. Saulnier-Blache J.S. J. Biol. Chem. 2003; 278: 18162-18169Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Because these metabolites are all downstream products of PLA2 catalyzed reactions, we sought to determine the specific types and amounts of PLA2 mRNA, protein, and activity corresponding to each of the previously characterized mammalian intracellular PLA2 as a function of time after hormone-induced differentiation of 3T3-L1 preadipocytes. In resting cells, cPLA2α mRNA was prominent, with only minimal amounts of mRNA encoding iPLA2 detectable. However, after hormone-induced differentiation, the levels of cPLA2α mRNA decreased dramatically to near background levels (Fig. 1A). Remarkably, the levels of iPLA2β and iPLA2γ mRNA increased 7.3 ± 0.5- and 7.4 ± 1.4-fold respectively (Fig. 1, B and C). Collectively, these results demonstrate the dramatic and temporally coordinated changes in the mRNA levels of each of the previously characterized mammalian intracellular PLA2 during adipocyte differentiation. Alterations of Intracellular Phospholipase A2 Protein Mass and Activity during Differentiation of 3T3-L1 Preadipocytes—To further substantiate the functional importance of the observed alterations in mRNA levels, Western blot analysis was performed. Western analyses demonstrated a decrease in cPLA2α protein mass to near background levels (as predicted by the decreased mass content of cPLA2α mRNA in the differentiating adipocyte) and the dramatic increases of both iPLA2β and iPLA2γ protein products (as predicted by increased mRNA levels encoding iPLA2β and iPLA2γ from quantitative PCR) (Fig. 2). The temporal course of the increased amounts of iPLA2β and iPLA2γ protein and the decreased amount of cPLA2α protein were inversely regulated. Thus, the protein mass of each intracellular PLA2 closely paralleled the intrinsic mRNA levels of each of three mammalian intracellular PLA2 (i.e. cPLA2α, iPLA2β, and iPLA2γ). Collectively, these results demonstrate the importance of transcriptional regulation in modulating reciprocal alterations in specific classes of intracellular PLA2 during adipocyte differentiation. To further investigate if alterations in the protein content of iPLA2β and iPLA2γ present during differentiation of 3T3-L1 cells were paralleled by changes in their activities, phospholipase A2 activity assays were performed. During adipocyte differentiation iPLA2 activity increased ≈4-fold (Fig. 3A). As anticipated, the measured increase in iPLA2 activity was inhibited by the mechanism-based inhibitor, racemic BEL (Fig. 3B). Previously, we demonstrated that (S)-BEL was approximately 1 order of magnitude more selective for iPLA2β in comparison to iPLA2γ, whereas (R)-BEL was approximately an order of magnitude more selective for iPLA2γ (32Jenkins C.M. Han X. Mancuso D.J. Gross R.W. J. Biol. Chem. 2002; 277: 32807-32814Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The measured iPLA2 activity in 3T3-L1 adipocyte homogenate was inhibited to similar levels by either (S)-BEL or (R)-BEL (Fig. 3B) demonstrating that both iPLA2β and iPLA2γ contribute similarly to the total amounts of measured iPLA2 activity in differentiated adipocytes. Concomitant with the increase in iPLA2 activity, calcium-dependent PLA2 activity in the homogenate decreased by 90% in day 8 3T3-L1 cells (data not shown). Collectively, these results showed an increase of iPLA2 activity and a concomitant decrease of calcium-dependent phospholipase A2 activity. Pretreatment of siRNAs Targeting iPLA2β or iPLA2γ Inhibits Hormone-induced Differentiation of 3T3-L1 Preadipocytes— These results, in the context of prior work on the importance of eicosanoids and lysolipids in adipocyte differentiation, suggested that iPLA2 activity may be required to promote adipocyte differentiation. To determine whether iPLA2β or iPLA2γ were r" @default.
• W2085475071 created "2016-06-24" @default.
• W2085475071 creator A5016963123 @default.
• W2085475071 creator A5021354628 @default.
• W2085475071 creator A5041289693 @default.
• W2085475071 creator A5045592928 @default.
• W2085475071 creator A5081268367 @default.
• W2085475071 date "2004-05-01" @default.
• W2085475071 modified "2023-10-13" @default.
• W2085475071 title "Small Interfering RNA Knockdown of Calcium-independent Phospholipases A2 β or γ Inhibits the Hormone-induced Differentiation of 3T3-L1 Preadipocytes" @default.
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• W2085475071 cites W1526978170 @default.
• W2085475071 cites W1538168419 @default.
• W2085475071 cites W1539862553 @default.
• W2085475071 cites W1569457616 @default.
• W2085475071 cites W1577992062 @default.
• W2085475071 cites W1587876647 @default.
• W2085475071 cites W1631449371 @default.
• W2085475071 cites W1673496785 @default.
• W2085475071 cites W1675830194 @default.
• W2085475071 cites W1907219612 @default.
• W2085475071 cites W1908257788 @default.
• W2085475071 cites W1971517790 @default.
• W2085475071 cites W1977595115 @default.
• W2085475071 cites W1978589050 @default.
• W2085475071 cites W1982460743 @default.
• W2085475071 cites W1984044242 @default.
• W2085475071 cites W1984900499 @default.
• W2085475071 cites W2000867519 @default.
• W2085475071 cites W2001807027 @default.
• W2085475071 cites W2002519246 @default.
• W2085475071 cites W2004571567 @default.
• W2085475071 cites W2004967964 @default.
• W2085475071 cites W2005674716 @default.
• W2085475071 cites W2026202377 @default.
• W2085475071 cites W2027321140 @default.
• W2085475071 cites W2027330535 @default.
• W2085475071 cites W2034810986 @default.
• W2085475071 cites W2041895698 @default.
• W2085475071 cites W2046833545 @default.
• W2085475071 cites W2047842452 @default.
• W2085475071 cites W2049629344 @default.
• W2085475071 cites W2051265887 @default.
• W2085475071 cites W2057475017 @default.
• W2085475071 cites W2071304121 @default.
• W2085475071 cites W2073079404 @default.
• W2085475071 cites W2086353275 @default.
• W2085475071 cites W2088926355 @default.
• W2085475071 cites W2091466360 @default.
• W2085475071 cites W2091673549 @default.
• W2085475071 cites W2101981691 @default.
• W2085475071 cites W2103633745 @default.
• W2085475071 cites W2115459204 @default.
• W2085475071 cites W2120738384 @default.
• W2085475071 cites W2131270588 @default.
• W2085475071 cites W2133067519 @default.
• W2085475071 cites W2168880873 @default.
• W2085475071 cites W2176176727 @default.
• W2085475071 cites W2300552860 @default.
• W2085475071 cites W2415214386 @default.
• W2085475071 cites W4247315158 @default.
• W2085475071 cites W4248955105 @default.
• W2085475071 cites W62702228 @default.
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