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• W2026202377 abstract "Our group has recently demonstrated (Gesta, S., Simon, M., Rey, A., Sibrac, D., Girard, A., Lafontan, M., Valet, P., and Saulnier-Blache, J. S. (2002) J. Lipid Res. 43, 904–910) the presence, in adipocyte conditioned-medium, of a soluble lysophospholipased-activity (LPLDact) involved in synthesis of the bioactive phospholipid lysophosphatidic acid (LPA). In the present report, LPLDact was purified from 3T3F442A adipocyte-conditioned medium and identified as the type II ecto-nucleotide pyrophosphatase phosphodiesterase, autotaxin (ATX). A unique ATX cDNA was cloned from 3T3F442A adipocytes, and its recombinant expression in COS-7 cells led to extracellular release of LPLDact. ATX mRNA expression was highly up-regulated during adipocyte differentiation of 3T3F442A-preadipocytes. This up-regulation was paralleled by the ability of newly differentiated adipocytes to release LPLDact and LPA. Differentiation-dependent up-regulation of ATX expression was also observed in a primary culture of mouse preadipocytes. Treatment of 3T3F442A-preadipocytes with concentrated conditioned medium from ATX-expressing COS-7 cells led to an increase in cell number as compared with concentrated conditioned medium from ATX non-expressing COS-7 cells. The specific effect of ATX on preadipocyte proliferation was completely suppressed by co-treatment with a LPA-hydrolyzing phospholipase, phospholipase B. Finally, ATX expression was found in mature adipocytes isolated from mouse adipose tissue and was substantially increased in genetically obese-diabeticdb/db mice when compared with their lean siblings. In conclusion, the present work shows that ATX is responsible for the LPLDact released by adipocytes and exerts a paracrine control on preadipocyte growth via an LPA-dependent mechanism. Up-regulations of ATX expression with adipocyte differentiation and genetic obesity suggest a possible involvement of this released protein in the development of adipose tissue and obesity-associated pathologies. Our group has recently demonstrated (Gesta, S., Simon, M., Rey, A., Sibrac, D., Girard, A., Lafontan, M., Valet, P., and Saulnier-Blache, J. S. (2002) J. Lipid Res. 43, 904–910) the presence, in adipocyte conditioned-medium, of a soluble lysophospholipased-activity (LPLDact) involved in synthesis of the bioactive phospholipid lysophosphatidic acid (LPA). In the present report, LPLDact was purified from 3T3F442A adipocyte-conditioned medium and identified as the type II ecto-nucleotide pyrophosphatase phosphodiesterase, autotaxin (ATX). A unique ATX cDNA was cloned from 3T3F442A adipocytes, and its recombinant expression in COS-7 cells led to extracellular release of LPLDact. ATX mRNA expression was highly up-regulated during adipocyte differentiation of 3T3F442A-preadipocytes. This up-regulation was paralleled by the ability of newly differentiated adipocytes to release LPLDact and LPA. Differentiation-dependent up-regulation of ATX expression was also observed in a primary culture of mouse preadipocytes. Treatment of 3T3F442A-preadipocytes with concentrated conditioned medium from ATX-expressing COS-7 cells led to an increase in cell number as compared with concentrated conditioned medium from ATX non-expressing COS-7 cells. The specific effect of ATX on preadipocyte proliferation was completely suppressed by co-treatment with a LPA-hydrolyzing phospholipase, phospholipase B. Finally, ATX expression was found in mature adipocytes isolated from mouse adipose tissue and was substantially increased in genetically obese-diabeticdb/db mice when compared with their lean siblings. In conclusion, the present work shows that ATX is responsible for the LPLDact released by adipocytes and exerts a paracrine control on preadipocyte growth via an LPA-dependent mechanism. Up-regulations of ATX expression with adipocyte differentiation and genetic obesity suggest a possible involvement of this released protein in the development of adipose tissue and obesity-associated pathologies. lysophosphatidic acid lysophospholipase D LPLD activity autotaxine Dulbecco's modified Eagle's medium bovine serum albumin lysophosphatidylcholine conditioned medium concentrated conditioned medium thin layer chromatography polyethylene glycol purification buffer mass spectrometry reverse transcription adipocyte fatty acid-binding protein hormone-sensitive lipase Because of its ability to store extra energy as triacylglycerol (lipogenesis) and to release fatty acids and glycerol (lipolysis), adipose tissue plays a crucial role in energy balance. In obesity, excessive accumulation of triacylglycerol in adipocytes (hypertrophy) results from an alteration in the balance between lipogenic and/or lipolytic activities of the adipocytes. It is now recognized that, beside their involvement in lipid homeostasis, adipocytes also produce and secrete numerous factors. Among them are endocrine peptides (leptin, adiponectin, angiotensinogen, etc.) which may play an important role in the development of morbid complications of obesity such as cardiovascular diseases, hypertension, diabetes, and cancer. Other adipocyte-secreted factors (tumor necrosis factor, fatty acids, eicosanoids, lysophosphatidic acid, etc.) are produced locally and may influence adipose tissue development and/or metabolism by exerting autocrine/paracrine effects on the different cells composing adipose tissue (adipocytes, preadipocytes, and endothelial cells) (1Spiegelman B.M. Flier J.S. Cell. 1996; 87: 377-389Abstract Full Text Full Text PDF PubMed Scopus (1166) Google Scholar, 2Mohamed-Ali V. Pinkney J. Coppack S. Int. J. Obes. 1998; 22: 1145-1158Crossref PubMed Scopus (812) Google Scholar, 3Hausman D.B. DiGirolamo M. Bartness T.J. Hausman G.J. Martin R.J. Obes. Rev. 2001; 2: 239-254Crossref PubMed Scopus (527) Google Scholar). Of particular interest is the ability of some adipocyte-secreted factors to exert a paracrine control on preadipocyte proliferation and differentiation, cellular processes leading to the recruitment of new fat cells in adipose tissue (adipogenesis) and a further increase in adipose tissue mass (3Hausman D.B. DiGirolamo M. Bartness T.J. Hausman G.J. Martin R.J. Obes. Rev. 2001; 2: 239-254Crossref PubMed Scopus (527) Google Scholar, 4MacDougald O.A. Mandrup S. Trends Endocrinol. Metab. 2002; 13: 5-11Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Our group has demonstrated that lysophosphatidic acid (LPA)1 is released from adipocytes in vitro and is present in vivo in the extracellular fluid of adipose tissue collected by microdialysis (5Valet P. Pages C. Jeanneton O. Daviaud D. Barbe P. Record M. Saulnier-Blache J.S. Lafontan M. J. Clin. Invest. 1998; 101: 1431-1438Crossref PubMed Scopus (127) Google Scholar). In parallel, LPA is able to activate preadipocyte motility and proliferation by interacting preferentially with the LPA1 receptor (LPA1-R) (6Pagès C. Daviaud D. An S. Krief S. Lafontan M. Valet P. Saulnier-Blache J. J. Biol. Chem. 2001; 276: 11599-11605Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Therefore LPA may participate to the paracrine control of adipogenesis. LPA is a bioactive phospholipid regulating a wide range of cellular responses (proliferation, survival, motility, ion flux, and secretion) through the activation of the G-protein-coupled receptors LPA1-, LPA2-, and LPA3-R (7Tigyi G. Prostaglandins Other Lipid Mediat. 2001; 64: 47-62Crossref PubMed Scopus (93) Google Scholar, 8Chun J. Goetzl E.J. Hla T. Igarashi Y. Lynch K.R. Moolenaar W. Pyne S. Tigyi G. Pharmacol. Rev. 2002; 54: 265-269Crossref PubMed Scopus (450) Google Scholar). Bioactive LPA was initially found in culture serum (9Tigyi G. Miledi R. J. Biol. Chem. 1992; 267: 21360-21367Abstract Full Text PDF PubMed Google Scholar) and was further detected in other biological fluids such as plasma (10Xu Y. Gaudette D. Boynton J. Frankel A. Fang X. Sharma A. Hurteau J. Casey G. Goodbody A. Mellors A. Holub B. Mills G. Clin. Cancer Res. 1995; 1: 1223-1232PubMed Google Scholar, 11Saulnier-Blache J.S. Girard A. Simon M.F. Lafontan M. Valet P. J. Lipid Res. 2000; 41: 1947-1951Abstract Full Text Full Text PDF PubMed Google Scholar), ascitic fluid (12Westermann A. Havik E. Postma F. Beijnen J. Dalesio O. Moolenar M. Rodenhuis S. Ann. Oncol. 1998; 9: 437-442Abstract Full Text PDF PubMed Scopus (157) Google Scholar), follicular fluid (13Tokumura A. Miyake M. Nishioka Y. Yamano S. Aono T. Fukuzawa K. Biol. Reprod. 1999; 61: 195-199Crossref PubMed Scopus (120) Google Scholar), aqueous humor (14Liliom K. Guan Z. Tseng J.-L. Desiderio D.M. Tigyi G. Watsky M.A. Am. J. Physiol. 1998; 274: C1065-C1074Crossref PubMed Google Scholar), and the extracellular fluid of adipose tissue (5Valet P. Pages C. Jeanneton O. Daviaud D. Barbe P. Record M. Saulnier-Blache J.S. Lafontan M. J. Clin. Invest. 1998; 101: 1431-1438Crossref PubMed Scopus (127) Google Scholar). Whereas serum LPA mainly originates from aggregating platelets (15Gaits F. Fourcade O. Le Balle F. Gueguen G. Gaige B. Gassama-Diagne A. Fauvel J. Salles J.P. Mauco G. Simon M.F. Chap H. FEBS Lett. 1997; 410: 54-58Crossref PubMed Scopus (148) Google Scholar, 16Aoki J. Taira A. Takanezawa Y. Kishi Y. Hama K. Kishimoto T. Mizuno K. Saku K. Taguchi R. Arai H. J. Biol. Chem. 2002; 277: 48737-48744Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar), the precise cellular origin of LPA in other biological fluids still remains unclear. We recently showed that, in parallel to LPA, adipocytes also release a LPA-synthesizing activity that was characterized as a lysophospholipase D activity (LPLDact), catalyzing transformation of lysophosphatidylcholine into LPA (17Gesta S. Simon M. Rey A. Sibrac D. Girard A. Lafontan M. Valet P. Saulnier-Blache J.S. J. Lipid Res. 2002; 43: 904-910Abstract Full Text Full Text PDF PubMed Google Scholar). Adipocyte LPL Dact is soluble and sensitive to cobalt ions (17Gesta S. Simon M. Rey A. Sibrac D. Girard A. Lafontan M. Valet P. Saulnier-Blache J.S. J. Lipid Res. 2002; 43: 904-910Abstract Full Text Full Text PDF PubMed Google Scholar). In addition, adipocyte lysophospholipase D activity is insensitive to primary alcohol, suggesting that it could be catalyzed by a non-conventional phospholipase D that remains to be identified. In the present study, adipocyte LPLD activity was purified and identified as the type II ecto-nucleotide pyrophosphate phosphodiesterase autotaxin (ATX). ATX was found to be expressed by and released from adipocytes, activates preadipocyte proliferation, and its expression was strongly up-regulated during adipocyte differentiation and in a model of genetic obesity. Because of its LPLDact, adipocyte-ATX leads to LPA synthesis and could be involved in the control of adipose tissue development as well as in obesity-associated pathologies. The mouse preadipose cell line 3T3F442A (18Green H. Kehinde O. Cell. 1976; 7: 105-113Abstract Full Text PDF PubMed Scopus (618) Google Scholar) was grown and differentiated as described previously (19Bétuing S. Valet P. Lapalu S. Peyroulan D. Hickson G. Daviaud D. Lafontan M. Saulnier-Blache J.S. Biochem. Biophys. Res. Com. 1997; 235: 765-773Crossref PubMed Scopus (20) Google Scholar). Briefly, cells were grown to confluence (day 0 of differentiation) in DMEM supplemented with 10% donor calf serum and then shifted in a differentiating medium consisting in DMEM supplemented with 10% fetal calf serum plus 50 nm insulin. In these culture conditions, quiescent preadipocytes differentiate into functional adipocytes. COS-7 monkey cells (American Type Culture Collection) were grown in DMEM supplemented with 10% fetal calf serum and transfected using DEAE-dextran as reported previously (20Vagner S. Gensac M.C. Maret A. Bayard F. Amalric F. Prats H. Prats A.C. Mol. Cell. Biol. 1995; 15: 35-44Crossref PubMed Scopus (289) Google Scholar). 3T3F442A cells or COS-7 cells were washed twice with phosphate-buffered saline to remove serum and incubated (5 ml for a 10-cm diameter plate; 1 ml for a 3-cm diameter plate) in serum-free DMEM supplemented (for LPA release) or not (for LPLDact release) with 1% free fatty acid BSA at 37 °C in a humidified atmosphere containing 7% CO2. Conditioned media were also prepared from adipose tissue by incubating 300–500 mg of finely cut out subcutaneous adipose tissue from db/+ ordb/db mice in 3 ml of serum-free DMEM to measure released LPLDact. After various time of incubation (0–5 h for measurement of LPLD activity; 7 h for quantification of LPA and lysophosphatidylcholine (LPC)), conditioned medium (CM) was separated from the cells or tissue, centrifuged to eliminate cell debris, and stored at −20 °C before measurement of LPLDact or quantification of LPA. In some experiments, conditioned media were concentrated (about 50-fold) using an Amicon Ultra 10,000 (Millipore). After adjustment of the protein concentration, concentrated conditioned media (CCM) were aliquoted and stored at −20 °C before use. LPLDact was measured by conversion of radiolabeled LPC into radiolabeled LPA as described previously (21Tokumura A. Miyabe M. Yoshimoto O. Shimizu M. Fukuzawa K. Lipids. 1998; 33: 1009-1015Crossref PubMed Scopus (52) Google Scholar) with minor modifications. A solution of [14C]palmitoyl-lysophosphatidylcholine (PerkinElmer Life Sciences; 55.8 mCi/mmol) at 0.0025 μCi/μl in DMEM supplemented with 1% free fatty acid BSA was first prepared, and 20 μl of this solution was incubated with 500 μl of thawed CM plus 1 μl of sodium orthovanadate (0.5 mm) for 90 min at 37 °C. At the end of the incubation period, phospholipids were extracted with 500 μl of 1-butanol, evaporated, spotted on a silica gel 60 TLC glass plate (Merck), and separated using CHCl3/MeOH/NH4OH (60:35:8) as the migration solvent. The plate was autoradiographed overnight at −80 °C using a Biomax-MS film (Kodak) to localize radiolabeled LPA spots, which were scraped and counted with 3 ml of scintillation mixture. All the procedures were performed at 0–4 °C. One hundred twenty milliliters of 3T3F442A adipocyte-conditioned medium containing LPLDact was centrifuged for 1 h at 100,000 × g to remove cell debris and concentrated 8-fold on polyethylene glycol 20,000 (PEG 20,000). After overnight dialysis against 10 liters of purification buffer (PB) using Spectra-Por 1.7 ml/cm tubing (Pierce Chemicals, Interchim) to remove salts and small proteins, 16 ml of the concentrated medium was applied (60 ml/h) onto a heparin-Sepharose column (CL+6B, Amersham Biosciences; 15-ml packed volume) equilibrated with 100 ml of PB. The column was washed with 150 ml of PB and eluted with 125 ml of PB containing 0.5 m NaCl. The elution fraction containing LPLDact was dialyzed overnight against 10 liters of PB using Spectra-Por 1.7 ml/cm tubing (Pierce Chemicals, Interchim) to remove NaCl and concentrated on PEG 20,000 to obtain a volume of 40 ml. This concentrate was applied (60 ml/h) on a phosphocellulose column (P11, Whatman, 15 ml packed volume) equilibrated with 100 ml PB. The column was washed with 150 ml of PB and eluted with 125 ml of PB containing 0.5 m NaCl. The elution fraction containing LPLDact was dialyzed overnight against 10 liters of purification buffer using Spectra-Por 1.7 ml/cm tubing (Pierce Chemicals, Interchim) to remove NaCl, and concentrated on PEG 20,000 to obtain a final volume of 5 ml. This concentrate was separated (30 ml/h) over a gel filtration column (HiLoad 16/60, Superdex 200 PrepGrade, Amersham Biosciences) on an Amersham Biosciences FPLC system previously equilibrated with PB. The column was eluted with the same buffer. Aliquots of the collected fractions were assayed for LPLDact. All the fractions containing LPLDact were pooled and concentrated 10 times on dialysis tubing (0.5 ml/cm) in PEG 20,000. The protein concentration was determined by the Bradford assay (Protassay, Bio-Rad) with bovine serum albumin as standard. SDS-PAGE (4–20%) was performed according to Laemmli (22Laemmli U. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207856) Google Scholar) followed by SYPRO Ruby or colloidal blue staining. After the addition of sample buffer (Novex, Invitrogen), concentrated fractions from gel filtration were boiled at 100 °C for 5 min. Electrophoretic separation of proteins was carried out on a 1-mm thick 8 × 6-cm gel 10% acrylamide. A 40-μg portion of total protein in sample buffer was loaded into a 4-mm well of the gel and separated at 40 mA. A total of 30 μg of standards (Mark12, Invitrogen) migrated in a neighboring lane. After coloration with colloidal Coomassie Blue (Biosafe, Bio-Rad), the 110-kDa protein was cut, reduced, and alkylated using dithiothreitol and iodoacetamide, respectively, and subjected to digestion with trypsin overnight following the protocol of Shevchenko et al. (23Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7853) Google Scholar). Gel pieces were successively washed with ammonium bicarbonate and dehydrated with acetonitrile. After drying in a Speedvac (Heto), the pieces were incubated at 56 °C with 10 mm dithiothreitol and then at room temperature with 55 mm iodoacetamide. After successive washing steps with ammonium bicarbonate and acetonitrile, the gel pieces were completely dried in the Speedvac. Trypsin solution (Promega) was added, and digestion was performed overnight at 37 °C. Peptide digests were extracted successively with acetonitrile and 5% formic acid. Before analysis, the peptides were dried in the Speedvac, diluted in 5% formic acid, and desalted on Poros R-2 resin. Peptides were eluted with 2 μl of 60% methanol/1% formic acid directly into nanospray capillary needles (Micromass, Manchester, UK). Peptides were analyzed on a Q-TOF 2 mass spectrometer (Micromass). MS acquisitions were performed within the mass range of 550–1300 m/zand MS/MS within 50–2000 m/z. Amino acid sequences were used to search Swiss-Prot and TREMBL with BLAST interface (United States National Center for Biotechnology Information). Partial amino acid sequences (sequence TAG) were used to search the data bases with PeptideSearch interface (European Molecular Biology Laboratory, Heidelberg, Germany). Hits were confirmed by performing a theoretical trypsin digestion with GPMAW (Lighthouse data, Odense, Denmark) and comparing the resulting peptide mass and sequence to those obtained experimentally. Total RNAs were isolated using the RNeasy mini kit (Qiagen). Total RNAs (1 μg) were reverse transcribed for 60 min at 37 °C using Superscript II reverse transcriptase (Invitrogen) in the presence of a random hexamer. A minus RT reaction was performed in parallel to ensure the absence of genomic DNA contamination. Real time RT-PCR was performed starting with 25 ng cDNA and 900 nm (human ATX and mouse ATX) or 900 nm (mouse ap2) concentration of both sense and antisense primers in a final volume of 25 μl using the SYBR green TaqMan Universal PCR Master Mix (Applied Biosystems). Fluorescence was monitored and analyzed in a GeneAmp 5700 detection system instrument (Applied Biosystems). Analysis of the 18 S ribosomal RNA was performed in parallel using the ribosomal RNA control Taqman Assay Kit (Applied Biosystem) to normalize gene expression. Results are expressed as 2(Ct18S − Ctgene), where Ct corresponds to the number of cycles needed to generate a fluorescent signal above a predefined threshold. Oligonucleotide primers were designed using the Primer Express software (PerkinElmer Life Sciences). Oligonucleotides used were as follows: mouse aP2, sense 5′-TTCGATGAAATCACCGCAGA-3′ and antisense 5′-GGTCGACTTTCCATCCCACTT-3′; mouse ATX, sense 5′-GACCCTAAAGCCATTATTGCTAA-3′ and antisense 5′-GGGAAGGTGCTGTTTCATGT-3′; mouse HSL, sense 5′-GGCTTACTGGGCACAGATACCT-3′ and antisense 5′-CTGAAGGCTCTGAGTTGCTCAA-3′. Total RNA from 15 days post-confluent 3T3F442A adipocytes was reverse transcribed using random hexamers and Superscript II reverse transcriptase (Invitrogen). First strand cDNA (corresponding to 1 μg of total RNA) was amplified using a program consisting of 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 3 min with a pre- and post-incubation of 95 °C for 2 min and 72 for 10 min, respectively. PCR amplification utilized oligonucleotide primers based on the GenBankTM entry for mouse autotaxin (forward 59–64, reverse 2630–2651, accession number BC003264). The PCR fragment was isolated and ligated into pcDNA3 (Invitrogen) downstream of the FLAG peptide sequence (Sigma). The recombinant plasmid, designated pcDNA-mATX-FLAG, was sequenced on both strands by automated sequencing. LPA was butanol-extracted from conditioned medium and quantified using a radioenzymatic assay as described previously (11Saulnier-Blache J.S. Girard A. Simon M.F. Lafontan M. Valet P. J. Lipid Res. 2000; 41: 1947-1951Abstract Full Text Full Text PDF PubMed Google Scholar). The amount of lysophosphatidylcholine present in conditioned media was determined as described previously (17Gesta S. Simon M. Rey A. Sibrac D. Girard A. Lafontan M. Valet P. Saulnier-Blache J.S. J. Lipid Res. 2002; 43: 904-910Abstract Full Text Full Text PDF PubMed Google Scholar) after 30 min of treatment with bacterial phospholipase D followed by LPA quantification. Male C57BL/KsJdb/db and db/+ mice (Jackson Laboratory) were housed in an animal room maintained at 22 °C on a 12:12 h light/dark cycle with ad libitum access to food and water. Animals were handled in accordance with the principles and guidelines established by the National Institute of Medical Research (INSERM). On the day of sacrifice, mouse blood was collected on heparin and glucose was immediately measured with a glucose meter (Glucometer 4; Bayer Diagnosis, Puteaux, France). Perigonadic and inguinal white adipose tissues were removed, and adipocytes were isolated as described above and immediately processed for RNA extraction using an RNeasy mini kit (Qiagen). The other tissues (interscapular brown adipose tissue, brain, kidney, and liver) were removed and snap frozen in liquid nitrogen before RNA isolation using RNA STAT kit (AMS Biotechnology Ltd., Oxon, UK). Adipose tissue was dissected from mice and digested in 5 ml of DMEM supplemented with 1 mg/ml collagenase, 1% BSA, and 2 μg/ml gentamycin for 45–60 min at 37 °C under shaking. Digestion was followed by filtration through a 100-μm screen and centrifugation at 800 × g for 10 min at room temperature. This phase allowed the separation of floating adipocytes from the pellet containing the stromal-vascular fraction. Adipocytes were washed twice in DMEM and further processed for RNA extraction using the RNeasy mini kit (Qiagen). The stromal-vascular pellet was washed twice in DMEM and resuspended in 1 ml of lysis buffer (7 mm tris, 0.83% NH4Cl, pH 7.6) for 3 min to get rid of erythrocytes and then seeded in 12-well plates (150,000 cells per plate) in DMEM/Ham's F-12 medium supplemented with 10% fetal calf serum. After overnight culture, the medium was replaced by a serum-free differentiating medium consisting of DMEM/Ham's F12 supplemented with transferrin (10 μg/ml), biotin (33 μm), insulin (66 nm), T3 (1 nm), and pantothenate (17 μm). That time point is considered as day 0. In these conditions, adipocyte differentiation occurs a few days later (24Deslex S. Negrel R. Ailhaud G. Exp. Cell Res. 1987; 168: 15-30Crossref PubMed Scopus (155) Google Scholar). Total RNAs were prepared at different time points after induction of the differentiation using the RNeasy mini kit (Qiagen). To identify the enzyme responsible for LPLDact present in adipocyte CM (17Gesta S. Simon M. Rey A. Sibrac D. Girard A. Lafontan M. Valet P. Saulnier-Blache J.S. J. Lipid Res. 2002; 43: 904-910Abstract Full Text Full Text PDF PubMed Google Scholar), its purification from 3T3F442A adipocyte CM was undertaken. LPLDact hydrolyzes the phosphocholine bound in LPC to generate LPA and free choline. In the present work, LPLDact was measured using [14C]lysopalmitoyl-phosphatidylcholine as substrate, followed by TLC separation of synthesized [14C]LPA (see “Experimental Procedures”). The initial release of LPLDact in adipocyte CM was observed in an Hepes buffer incubation medium (17Gesta S. Simon M. Rey A. Sibrac D. Girard A. Lafontan M. Valet P. Saulnier-Blache J.S. J. Lipid Res. 2002; 43: 904-910Abstract Full Text Full Text PDF PubMed Google Scholar). As shown in Fig.1, a stronger (3.3-fold after 5 h of incubation) LPLDact was measured when using DMEM as the incubation medium. In both incubation media, LPLDact was detectable after a 30-min incubation and reached a maximum after 5 h. Based upon these results, purification of LPLDact was undertaken from 3T3F442A adipocyte CM prepared after 5 h of incubation in DMEM. After 10-fold concentration, CM was first applied onto a heparin affinity chromatography column (Fig. 2,section 1), washed and eluted with 0.5m NaCl at once (lane e), and concentrated again. This allowed the recovery of LPLDact, but no specific band was identified by SDS-PAGE (lane f). As a second purification step, concentrated fraction eluted from the heparin column was applied onto a phosphocellulose column (Fig. 2), eluted at 0.2 m NaCl (lane i), concentrated 25 times (lane j), applied on a gel filtration column (Fig. 2, section 3), and eluted (Fig. 2, section 3,fractions 52 to 82). LPLDact was detected in fractions 68 to 76. SDS-PAGE analysis revealed the presence of a band (molecular mass between 116 and 97 kDa) in the fractions exhibiting maximal LPLD activity (fractions 70, 72, and 74). Fraction 70 was concentrated 100 times, separated by 4–20% SDS-polyacrylamide gel electrophoresis, stained with Coomassie Blue, and cut out from the gel. Tryptic cleavage followed by tandem mass spectrometry of the specific band of fraction 70 led to the generation of 27 peptides exhibiting 100% homology (TableI) with mouse ecto-nucleotide pyrophosphatase phosphodiesterase (ENPP2), also called autotaxin (25Stracke M.L. Krutzsch H.C. Unsworth E.J. Arestad A. Cioce V. Schiffmann E. Liotta L.A. J. Biol. Chem. 1992; 267: 2524-2529Abstract Full Text PDF PubMed Google Scholar,26Murata J. Lee H.Y. Clair T. Krutzsch H.C. Arestad A.A. Sobel M.E. Liotta L.A. Stracke M.L. J. Biol. Chem. 1994; 269: 30479-30484Abstract Full Text PDF PubMed Google Scholar). These results showed that adipocyte-LPLDact was co-purified with ATX.Table IPeptides and mouse autotoxin sequenceList of the peptides positively recognised by mass spectrometrySYSSCCHDFDELCLKTSYDILYHTDFESGYSEIFLMPLWTSYTISKQAEVPECPAGFVRPPLIIFSVDGFRVSSIPEHLTNCVRPDVRSCGTHAPYMRPVYPTKVSPGFSQNCLAYKWWGGKPLWITATKQMSYGFLFPPYLSSSPEAKWWGGKPLWITATKQGVRAYDAFLVTNMVPMYPAFKRGTFFWSVSIPHERNGVNVISGPIFDYNYDGLRILTILQWLSLPDNERPYASERNGVNVISGPIFDYNYDGLRSVYAFYSEQPDFSGHKYGPFVSPGFSQNCLAYKNDKGPEMTNPLRQYVEGSSIPVPTHYYSIITSCLDFTQPADKWWGGKPLWITATKQGVRVRDIEHLTGLDFYRTEFLSNYLTNVDDITLVPGTLGRDIEHLTGLDFYRKPDQHFKPYMKQHLPKDIEHLTGLDFYRKVNSMQTVFVGYGPTFKSYSEILTLKTYLHTYESEITYLHTYESEIMouse autotaxin sequenceMARQGCFGSYQVISLFTFAIGVNLCLGFTASRIKRAEWDEGPPTVLS↓DSPWTNTSGSCKGRCFELQEVGPPDCRCDNLCKSYSSCCHDFDELCLKTARGWECTKDRCGEVRNEENACHCSEDCLSRGDCCTNYQVVCKGESHWVDDDCEEIRVPECPAGFVRPPLIIFSVDGFRASYMKKGSKVMPNIEKLRSCGTHAPYMRPVYPTKTFPNLYTLATGLYPESHGIVGNSMYDPVFDATFHLRGREKFNHRWWGGKPLWITATKQGVRAGTFFWSVSIPHERRILTILQWLSLPDNERPSVYAFYSEQPDFSGHKYGPFGPEMTNPLREIDKTVGKLMDGLKQLKLHRCVNVIFVGDHGMEDVTCDRTEFLSNYLTNVDDITLVPGTLGRIRPKIPNNLKYDPKAIIANLTCKKPDQHFKPYMKQHLPKRLHYANNRRIEDLHLLVERRWHVARKPLDVYKKPSGKCFFQGDHGFDNKVNSMQTVFVGYGPTFKYRTKVPPFENIELYNVMCDLLGLKPAPNNGTHGSLNHLLRTNTFRPTLPEEVSRPNYPGIMYLQSDFDLGCTCDDKVEPKNKLEELNKRLHTKGSTEERHLLYGRPAVLYRTSYDILYHTDFESGYSEIFLMPLWTSYTISKQAEVSSIPEHLTNCVRPDVRVSPGFSQNCLAYKNDKQMSYGFLFPPYLSSSPEAKYDAFLVTNMVPMYPAFKRVWTYFQRVLVKKYASERNGVNVISGPIFDYNYDGLRDIEDEIKQYVEGSSIPVPTHYYSIITSCLDFTQPADKCDGPLSVSSFILPHRPDNDESCNSSEDESKWVEELMKMHTARVRDIEHLTGLDFYRKTSRSYSEILTLKTYLHTYESEISequence of the peptides derived from the 97-116 kDa protein co-purified with adipocyte LPLDact. The specific band of 97-116 kDa in fraction 70 of Fig. 2 was submitted to tryptic cleavage and analyzed by tandem mass spectrometry, allowing the identification of 27 peptides exhibiting 100 % homology with mouse ATX. Underlined and bold characters indicate the peptides identified by mass spectrometry. ↓ indicates potential proteolytic cleave site. Open table in a new tab Sequence of the peptides derived from the 97-116 kDa protein co-purified with adipocyte LPLDact. The specific band of 97-116 kDa in fraction 70 of Fig. 2 was submitted to tryptic cleavage and analyzed by tandem mass spectrometry, allowing the identification of 27 peptides exhibiting 100 % homology with mouse ATX. Underlined and bold characters indicate the peptides identified by mass spectrometry. ↓ indicates potential proteolytic cleave site. A unique cDNA encoding ATX was cloned from 3T3F442A adipocytes and subcloned in a pcDNA-FLAG vector. This cDNA exhibited 100% identity with previously identified mouse-ATX cDNA present in the GenBankTM data base (BC003264, NM_015744, AF123542) (TableI). The corresponding protein is composed of 863 amino acids (molecular mass of ∼100 kDa) which, by analogy with human, corresponds to the previously described β-form (27Kawagoe H. Soma O. Goji J. Nishimura N. Narita M. Inazawa J. Nakamura H. Sano K. Genomics. 1995; 30: 380-384Crossref PubMed Scopus (65) Google Scholar, 28Lee H.Y. Murata J. Clair T. Polymeropoulos M.H. Torres R. Manrow R.E. Liotta L.A. Stracke M.L. Biochem. Biophys. Res. Commun. 1996; 218: 714-719Crossref PubMed Scopus (96) Google Scholar, 29Piao J.H. Matsuda Y. Nakamura H. Sano K. Cytogenet. Cell Genet. 1999; 87: 172-174Crossref PubMed Google Scholar). In human ATX the site of cleavage has been located between Ser-48 and asp-49 in the amino acid sequence EGPPTVLS↓DSPWTN, which is entirely conserved in mouse (BC003264, NM_015744, AF123542, and herein the 3T3F442A-cDNA sequence). Based upon mouse ATX sequence, the exp" @default.
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• W2026202377 title "Autotaxin Is Released from Adipocytes, Catalyzes Lysophosphatidic Acid Synthesis, and Activates Preadipocyte Proliferation" @default.
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