Secretion of a lysophospholipase D activity by adipocytes: involvement in lysophosphatidic acid synthesis
2002; Elsevier BV; Volume: 43; Issue: 6 Linguagem: Inglês
10.1016/s0022-2275(20)30464-8
ISSN1539-7262
AutoresStéphane Gesta, Marie‐Françoise Simon, Astrid Rey, David Sibrac, Alexia Girard, Max Lafontan, Philippe Valet, Jean Sébastien Saulnier‐Blache,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoThe aim of the present work was to depict the metabolic pathways involved in extracellular production of lysophosphatidic acid (LPA) by adipocytes. LPA was followed by quantifying the accumulation of LPA in the incubation medium (conditioned medium, CM) of 3T3F442A adipocytes or human adipose tissue explants using a radioenzymatic assay. Surprisingly, after separation from the cells, the amount of LPA present in CM could be significantly increased by further incubation at 37°C. This suggested the presence of a LPA-synthesizing activity (LPA-SA) in CM. LPA-SA appeared as a soluble activity which was inhibited by divalent ion chelators EDTA and phenanthrolin. The effect of EDTA was preferentially reverted by CoCl2, as described for a lysophospholipase D (lyso-PLD) activity previously identified in rat plasma. LPA concentration could also be increased by treatment with a bacterial PLD, demonstrating the presence of PLD-sensitive LPA precursors (mainly lysophosphatidylcholine) in adipocyte CM. LPA-SA could be increased by the addition of exogenous lysophosphatidylcholine, lysophosphatidylglycerol, or lyso-platelet activating factor, demonstrating that LPA-SA resulted from the action of a lyso-PLD. LPA-SA was not inhibited, but rather activated, by primary alcohol (ethanol and 1-butanol), suggesting that adipocyte lyso-PLD was not a classical PLD. Finally, LPA-SA was found to be weaker in CM of undifferentiated adipocyte (preadipocytes) compared with CM of differentiated adipocytes.In conclusion, our results reveal the existence of a secreted lyso-PLD activity regulated during adipocyte-differentiation and involved in extra cellular production of synthesis of LPA by adipocytes. The aim of the present work was to depict the metabolic pathways involved in extracellular production of lysophosphatidic acid (LPA) by adipocytes. LPA was followed by quantifying the accumulation of LPA in the incubation medium (conditioned medium, CM) of 3T3F442A adipocytes or human adipose tissue explants using a radioenzymatic assay. Surprisingly, after separation from the cells, the amount of LPA present in CM could be significantly increased by further incubation at 37°C. This suggested the presence of a LPA-synthesizing activity (LPA-SA) in CM. LPA-SA appeared as a soluble activity which was inhibited by divalent ion chelators EDTA and phenanthrolin. The effect of EDTA was preferentially reverted by CoCl2, as described for a lysophospholipase D (lyso-PLD) activity previously identified in rat plasma. LPA concentration could also be increased by treatment with a bacterial PLD, demonstrating the presence of PLD-sensitive LPA precursors (mainly lysophosphatidylcholine) in adipocyte CM. LPA-SA could be increased by the addition of exogenous lysophosphatidylcholine, lysophosphatidylglycerol, or lyso-platelet activating factor, demonstrating that LPA-SA resulted from the action of a lyso-PLD. LPA-SA was not inhibited, but rather activated, by primary alcohol (ethanol and 1-butanol), suggesting that adipocyte lyso-PLD was not a classical PLD. Finally, LPA-SA was found to be weaker in CM of undifferentiated adipocyte (preadipocytes) compared with CM of differentiated adipocytes. In conclusion, our results reveal the existence of a secreted lyso-PLD activity regulated during adipocyte-differentiation and involved in extra cellular production of synthesis of LPA by adipocytes. Lysophosphatidic acid (LPA) is a bioactive phospholipid known to be involved in the control of numerous cell functions via its interaction with specific G-protein coupled receptors belonging to the endothelium differentiation gene family (EDG-2, EDG-4, and EDG-7) (1Goetzl E. An S. Diversity of cellular receptors and functions for the lysophospholipid growth factors lysophosphatidic acid and sphingosin 1-phosphate..FASEB J. 1998; 12: 1589-1598Crossref PubMed Scopus (490) Google Scholar, 2Chun J. Contos J.J. Munroe D. A growing family of receptor genes for lysophosphatidic acid (LPA) and other lysophospholipids (LPs)..Cell Biochem. Biophys. 1999; 30: 213-242Crossref PubMed Scopus (138) Google Scholar). Therefore, the availability of LPA at the extra-cellular face of plasma membrane conditions the intensity LPA responsiveness. Two major pathways of LPA synthesis have been described: i) phospholipase A2-dependent deacylation of phosphatidic acid (3McCrea J. Robinson P. Gerrard J. Mepacrine (quinacrine) inhibition of thrombin-induced platelet responses can be overcome by lysophosphatidic acid..Biochim. Biophys. Acta. 1985; 842: 189-194Crossref PubMed Scopus (31) Google Scholar, 4Gerrard J.M. Robinson P. Identification of the molecular species of lysophosphatidic acid produced when platelets are stimulated by thrombin..Biochim. Biophys. Acta. 1989; 1001: 282-285Crossref PubMed Scopus (179) Google Scholar, 5Fourcade O. Simon M.F. Viodé C. Rugani N. Leballe F. Ragab A. Fournié B. Sarda L. Chap H. Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells..Cell. 1995; 80: 919-927Abstract Full Text PDF PubMed Scopus (492) Google Scholar); and ii) lysophospholipase D (lyso-PLD) dependent hydrolysis of other lysophospholipids such as lysophosphatidylcholine (6Tokumura A. Harada K. Fukuzawa K. Tsukatani H. Involvement of lysophospholipase D in the production of lysophosphatidic acid in rat plasma..Biochim. Biophys. Acta. 1986; 875: 31-38Crossref PubMed Scopus (153) Google Scholar, 7van Dijk M. Postma F. Hilkmann H. Jalink K. van Blitterswijk W.J. Moolenaar W. Exogenous phospholipase D generates lysophosphatidic acid and activates Ras, Rho and Ca2+ signaling pathways..Curr. Biol. 1998; 8: 386-392Abstract Full Text Full Text PDF PubMed Google Scholar, 8Tokomura A. Miyake M. Nishioka Y. Yamano S. Aono T. Fukuzawa K. Production of lysophosphatidic acids by lysophospholipase D in human follicular fluids of in vitro fertilization patients..Biol. Reprod. 1999; 61: 195-199Crossref PubMed Scopus (119) Google Scholar). The precise contribution of these pathways in cellular production of LPA is still a matter of debate. LPA is found in abundance in serum (bound to albumin) (9Tigyi G. Miledi R. Lysophosphatidates bound to serum albumin activate membrane currents in Xenopus oocytes and neurite retraction in PC12 pheochomocytoma cells..J. Biol. Chem. 1992; 267: 21360-21367Abstract Full Text PDF PubMed Google Scholar, 10Eichholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. The bioactive phospholipid lysophosphatidic acid is released from activated platelets..Biochem. J. 1993; 291: 677-680Crossref PubMed Scopus (577) Google Scholar, 11Tokumura A. Iimori M. Nishioka Y. Kitahara M. Sakashita M. Tanaka S. Lysophosphatidic acids induce proliferation of cultured vascular smooth muscles cells from rat aorta..Am. J. Physiol. 1994; 267: C204-C210Crossref PubMed Google Scholar, 12Sasagawa T. Suzuki K. Shiota T. Kondo T. Okita M. The significance of plasma lysophospholipids in patients with renal failure on hemodialysis..J. Nutr. Sci. Vitaminol. 1998; 44: 809-818Crossref PubMed Scopus (75) Google Scholar) resulting from platelet aggregation (10Eichholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. The bioactive phospholipid lysophosphatidic acid is released from activated platelets..Biochem. J. 1993; 291: 677-680Crossref PubMed Scopus (577) Google Scholar, 13Gaits F. Fourcade O. Gueguen L.B.F.G. Gaigé B. Gassama-Diagne A. Fauvel J. Salles J-P. Mauco G. Simon M-F. Chap H. Lysophosphatidic acid as a phospholipid mediator: pathways of synthesis..FEBS Lett. 1997; 410: 54-58Crossref PubMed Scopus (147) Google Scholar). However, LPA is also found in other biological fluids such as plasma of patients with ovarian cancer (14Xu Y. Gaudette D. Boynton J. Frankel A. Fang X. Sharma A. Hurteau J. Casey G. Goodbody A. Mellors A. Holub B. Mills G. Characterization of an ovarian cancer activating factor in ascites from ovarian cancer patients..Clin. Cancer Res. 1995; 1: 1223-1232PubMed Google Scholar), ascites (15Westermann A. Havik E. Postma F. Beijnen J. Dalesio O. Moolenar M. Rodenhuis S. Malignant effusions contain lysophosphatidic acid (LPA)-like activity..Ann. Oncol. 1998; 9: 437-442Abstract Full Text PDF PubMed Scopus (156) Google Scholar), aqueous humor (16Liliom K. Guan Z. Tseng J-L. Desiderio D.M. Tigyi G. Watsky M.A. Growth factor-like phospholipids generated after corneal injury..Am. J. Physiol. 1998; 274: C1065-C1074Crossref PubMed Google Scholar), follicular fluids (8Tokomura A. Miyake M. Nishioka Y. Yamano S. Aono T. Fukuzawa K. Production of lysophosphatidic acids by lysophospholipase D in human follicular fluids of in vitro fertilization patients..Biol. Reprod. 1999; 61: 195-199Crossref PubMed Scopus (119) Google Scholar), and extracellular fluid of adipose tissue (17Valet P. Pages C. Jeanneton O. Daviaud D. Barbe P. Record M. Saulnier-Blache J.S. Lafontan M. Alpha2-adrenergic receptor-mediated release of lysophosphatidic acid by adipocytes. A paracrine signal for preadipocyte growth..J. Clin. Invest. 1998; 101: 1431-1438Crossref PubMed Scopus (126) Google Scholar). This suggests the involvement of other cell types in LPA production. LPA can indeed be produced by cancer cells (18Shen Z. Belinson J. Morton R. Yan X. Phorbol 12-myristate 13-acetate stimulates lysophosphatidic acid secretion from ovarian and cervical cancer cells but not from breast or leukemia cells..Gynecol. Oncol. 1998; 71: 364-368Abstract Full Text PDF PubMed Scopus (102) Google Scholar) and adipocytes (17Valet P. Pages C. Jeanneton O. Daviaud D. Barbe P. Record M. Saulnier-Blache J.S. Lafontan M. Alpha2-adrenergic receptor-mediated release of lysophosphatidic acid by adipocytes. A paracrine signal for preadipocyte growth..J. Clin. Invest. 1998; 101: 1431-1438Crossref PubMed Scopus (126) Google Scholar). The initial aim of the present study was to depict the metabolic pathways involved in extracellular production of LPA by adipocytes. We found that, in parallel to LPA, adipocytes secrete a soluble lyso-PLD catalyzing hydrolysis of lysophosphatidylcholine into LPA. 3T3F442A preadipocytes were seeded at a density of 1,200 cells/cm2 and grown in DMEM supplemented with 10% FBS. At confluence, differentiation of preadipocytes into adipocytes was achieved by growing cells in DMEM supplemented with 10% FBS plus 50 nM insuline as reported previously (17Valet P. Pages C. Jeanneton O. Daviaud D. Barbe P. Record M. Saulnier-Blache J.S. Lafontan M. Alpha2-adrenergic receptor-mediated release of lysophosphatidic acid by adipocytes. A paracrine signal for preadipocyte growth..J. Clin. Invest. 1998; 101: 1431-1438Crossref PubMed Scopus (126) Google Scholar, 19Bétuing S. Valet P. Lapalu S. Peyroulan D. Hickson G. Daviaud D. Lafontan M. Saulnier-Blache J.S. Functional consequences of constitutively active α2A-adrenergic receptor expression in 3T3F442A preadipocytes and adipocytes..Biochem. Biophys. Res. Commun. 1997; 235: 765-773Crossref PubMed Scopus (20) Google Scholar). Before utilization 3T3F442A adipocytes or preadipocytes were washed twice with sterile PBS containing 1% (w/v) fatty acid-free BSA in order to remove all the LPA of serum. Human adipose tissue was obtained from healthy, drug free women undergoing abdominal dermolipectomy for plastic surgery according to the regulation of the Ethical Committee of Faculty Hospital. Adipose tissue was carefully dissected out from blood vessels and cut in small pieces (average weight 20 mg) as previously described (20Viguerie-Bacands N. Saulnier-Blache J. Dandine M. Dauzats M. Daviaud D. Langin D. Increase in uncoupling protein-2 mRNA expression by BRL49653 and bromopalmitate in human adipocytes..Biochem. Biophys. Res. Commun. 1999; 256: 138-141Crossref PubMed Scopus (53) Google Scholar). Before utilization adipose tissue explants were washed twice in sterile PBS. In order to study extra-cellular production of LPA, 3T3F442A adipocytes (3 cm diameter plate) or human adipose tissue explants (0.7 g) were incubated in 1 ml of sterile HEPES-buffer (HB) (118 mM NaCl, 6 mM KCl, 1 mM CaCl2, 1.2 mM MgSO4, 12.4 mM HEPES, 6 mM glucose, pH 7.4) supplemented with 1% fatty acid-free BSA (HB-BSA). Incubations were performed at 37°C in an humidified atmosphere containing 7% CO2. At different time point (0 to 48 h), incubation medium was collected, centrifuged for 15 min at 800 g to eliminate detached cells and cell debris, and frozen at −20°C before LPA quantification. The collected incubation media were called conditioned media (CM). LPA quantification was performed either directly after defrosting (initial) or after further incubation of the defrosted sample at 37°C for various period of time in sterile condition. Quantification of LPA was performed using a radioenzymatic assay as previously described (21Saulnier-Blache J.S. Girard A. Simon M.F. Lafontan M. Valet P. A simple and highly sensitive radioenzymatic assay for lysophosphatidic acid quantification..J. Lipid Res. 2000; 41: 1947-1951Abstract Full Text Full Text PDF PubMed Google Scholar). Phospholipids were extracted from CM with 1 vol of 1-butanol followed by evaporation of the solvent under nitrogen. Dry phospholipids were resuspended in 200 μl of reaction medium (1 μl [14C]oleoyl CoA (RAS 55 mCi/mmole, NEN), 20 μl Tris (pH 7.5) 200 mM, 10 μl of semi-purified lysophosphatidic acid acyl transferase (LPAAT), 8 μl of sodium orthovanadate 500 μM, and 161 μl H2O (containing 1 mg/ml Tween 20), and incubated for 120 min at 20°C. The mixture was vortexed every 15 min. The reaction was stopped by addition of 400 μl of CHCl3/MeOH/HCl 12 N (1/1/0.26) followed by a vigorous shaking and by 10 min centrifugation at 3,000 g. The lower CHCl3 phase was evaporated under nitrogen, resuspended in 20 μl of CHCl3/MeOH (1/1), spotted on a silica gel 60 TLC glass plate (Merck), and separated using CHCl3/MeOH/NH4OH/ H2O (65/25/0.9/3) as a solvent. The plate was autoradiographed overnight to localize the [14C]phosphatidic acid spots, which were then scrapped and counted with 3 ml of scintillation cocktail. Palmitoyl-, stearoyl-, oleoyl-lysophosphatidylcholine palmitoyl-lyso-platelet activating factor, and dioleoyl-phosphatidic acid were from Sigma. Oleoyl-lysophosphatidylglycerol was from Avanti Polar Lipids. Linoleoyl-lysophosphatidylcholine was obtained by treatment of dilinoleoyl-phosphatidylcholine (Avanti Polar Lipids) with pancreatic phospholipase A2 (Sigma) followed by separation and extraction on TLC. Arachidonoyl-lysophosphatidylcholine was obtained by treatment of palmitoyl-arachidonoyl-phosphatidylcholine (Sigma) with type XI lipase from Rhizopus arrhizus (Sigma) followed by separation and extraction on TLC. All phospholipids were solubilized in methanol, evaporated under nitrogen, and resuspended in HB-BSA. The final concentration was verified by using phosphorus measurement (22Böttcher C. Gent C.V. Priest C. A rapid and sensitive sub microphosphorus determination..Anal. Chim. Acta. 1961; 24: 203-204Crossref Scopus (851) Google Scholar). As previously described (17Valet P. Pages C. Jeanneton O. Daviaud D. Barbe P. Record M. Saulnier-Blache J.S. Lafontan M. Alpha2-adrenergic receptor-mediated release of lysophosphatidic acid by adipocytes. A paracrine signal for preadipocyte growth..J. Clin. Invest. 1998; 101: 1431-1438Crossref PubMed Scopus (126) Google Scholar), adipocytes produce LPA in their extracellular medium. This was demonstrated using qualitative assays (bioassay and 32P-labeling). In the mean time we developed a high sensitive radioenzymatic assay for LPA quantification (21Saulnier-Blache J.S. Girard A. Simon M.F. Lafontan M. Valet P. A simple and highly sensitive radioenzymatic assay for lysophosphatidic acid quantification..J. Lipid Res. 2000; 41: 1947-1951Abstract Full Text Full Text PDF PubMed Google Scholar). This new method was used to determine the concentration of LPA present in CM obtained after incubation of 3T3F442A adipocytes, or human adipose tissue explants, in HB-BSA (see Materials and Methods) for various period of time. As shown in Fig. 1(initial curves) the amount of LPA present in CM increased with time of incubation with 3T3F442A adipocyte (Fig. 1) or human adipose tissue explants (Fig. 2). This corresponds to the basal extra-cellular production of LPA by adipocytes.Fig. 2.Presence of a LPA-synthesizing activity in human adipocyte conditioned medium. Human adipose tissue explants were incubated in HB-BSA. At different time points the conditioned medium was collected and frozen. LPA was quantified either immediatly after defrosting (initial) or 6 h after further incubation at 37°C of defrosted conditioned medium. A: Autoradiography of representative experiment. B: Mean ± SE of three separate experiments. ** P < 0.01 and * P < 0.05 when comparing with initial value (Student's t-test).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Surprisingly, when CM (which were centrifuged in order to eliminate detached cells and cell debris) was further incubated for 6 h at 37°C, a significant increase in their LPA concentration was observed (Fig. 1 and 2 after 6 h curves). This increase was observed with CM obtained from both 3T3F442A adipocytes (Fig. 1) and human adipose tissue explants (Fig. 2). Six hours incubation corresponded to the maximal increase (4-fold) (Fig. 3). Above observations suggested that adipocytes were able to secreted or release a LPA-synthesizing activity (LPA-SA) in their incubation medium. LPA-SA was completely inhibited by previous heating of CM at 90°C for 5 min (not shown). This suggested that LPA-SA likely resulted from a enzymatic rather than from a chemically catalyzed reaction. No LPA-SA was detected when HB-BSA (the buffer used to incubate adipocytes) alone was incubated in the same condition. This showed that LPA-SA was not brought by albumin. In addition, bacterial contamination had never been detected even after 48 h incubation of CM at 37°C (not shown). These observations showed that LPA-SA could only be produced by adipocytes. In order to determine whether LPA-SA was soluble or associated with cell particules (membranes, vesicules), CM was subjected to ultra-centrifugation (100,000 g, 60 min, 4°C). The totality of LPA-SA remained in a supernatant after ultracentrifugation (not shown), strongly suggesting that LPA-SA was a soluble enzyme-activity. Above results suggested that LPA-SA was resulting from a soluble enzyme-activity secreted or released by adipocytes. In order to better characterize LPA-SA, the influence of ion chelators and divalent ions was tested. EDTA and phenanthroline both inhibited LPA-SA present in 3T3F442A adipocyte incubation medium, with IC50 of 0.02 and 0.09 mM respectively (Fig. 4A, B). The inhibitory effect of 0.05 mM EDTA was completely reversed by 5 mM CoCl2, partially reverted by 5 mM CaCl2, and not affected by 5 mM MgCl2 (Fig. 5). These results showed that LPA-SA required divalent-ions, with a preference for Co++ ions. Similar metal ion preference was described for a lyso-PLD catalyzing hydrolysis of lysophospholipids into LPA in rat plasma (6Tokumura A. Harada K. Fukuzawa K. Tsukatani H. Involvement of lysophospholipase D in the production of lysophosphatidic acid in rat plasma..Biochim. Biophys. Acta. 1986; 875: 31-38Crossref PubMed Scopus (153) Google Scholar, 23Tokumura A. Miyabe M. Yoshimoto O. Shimizu M. Fukuzawa K. Metal-ion stimulation and inhibition of lysophospholipase D which generates bioactive lysophosphatidic acid in rat plasma..Lipids. 1998; 33: 1009-1015Crossref PubMed Scopus (52) Google Scholar). This resemblance suggested that LPA-SA could correspond to a lyso-PLD.Fig. 5.Reversion of EDTA effect on LPA-SA by CaCl2 and CoCl2. Conditioned medium was collected and frozen after 24 h incubation with 3T3F442A adipocytes. LPA was quantified either immediately after defrosting (initial) or 6 h after further incubation at 37°C in the absence (continuous, EDTA) or in the presence of 0.2 mM EDTA ± 5 mM of CaCl2, MgCl2, or CoCl2. Data represent the mean ± SE of three separate experiments. * P < 0.05 and ** P < 0.001 when comparing to continuous EDTA (Student's t-test).View Large Image Figure ViewerDownload Hi-res image Download (PPT) If LPA-SA was a lyso-PLD, the presence of PLD-sensitive LPA precursors (other lysophospholipids) in adipocyte CM was required. To examine that point, adipocyte CM was treated with a bacterial PLD (from Streptomyces chromofuscus, Sigma) for 45 min and LPA was quantified. The initial concentration of LPA (26 ± 3 nM, n = 3) was increased to 299 ± 25 nM (n = 3) by treatment with bacterial PLD. This indicated the presence of PLD-sensitive LPA precursors in adipocyte CM. Conversely, treatment with pancreatic phospholipase A2 (potentially able to lead to the formation of LPA by deacylation of phosphatidic acid), did not modify LPA concentration (26 ± 3 nM vs. 27 ± 3 nM). Among lysophospholipids, lysophosphatidylcholine is usually the most abundant. Phospholipids co-migrating with LPC were purified from 1 ml adipocyte CM by preparative TLC, solubilized in 1 ml HB-BSA, and treated with bacterial PLD before quantification of LPA. This led to a concentration of LPA of 323 ± 12 nM LPA (n = 3). This concentration was very close to what was obtained after treatment of whole CM with bacterial PLD: 299 ± 25 nM. Above results showed that, in addition to LPA, adipocyte CM also contained other lysophospholipids (mainly lysophosphatidylcholine) constituting potential substrates for a lyso-PLD. Results also showed that adipocyte CM did not contain detectable PLA2-sensitive LPA precursors, therefore excluding that LPA-SA could be a PLA2. If LPA-SA was a lyso-PLD, addition of exogenous lysophospholipids in adipocyte CM should increase LPA concentration in CM. As shown in Fig. 6, addition of exogenous (2 μM final concentration) oleoyl-LPC, oleoyl-LPG, or palmitoyl-LPAF significantly increased the amount of LPA generated by 6 h incubation at 37°C of adipocyte CM. Conversely, no increase was observed by addition of exogenous dioleoyl-PA (Fig. 6). In parallel, no LPA was detected when LPC, LPG, or LPAF were incubated for 6 h at 37°C in HB-BSA alone (not shown). Above results showed that adipocyte CM contained a lyso-PLD, and did not contain detectable PLA2 activity. As shown in Fig. 6, the order of potency of conversion of the different lysophospholipids into LPA was LPC > LPG > LPAF, showing that LPC was the best substrate for adipocyte lyso-PLD activity. In order to determine the substrate specificity of adipocyte lyso-PLD, different LPC species were added in adipocyte CM (2 μM final concentration) and the amount of LPA generated after 6 h incubation was determined. The rate of conversion of palmitoyl-LPC, stearoyl-LPC, oleoyl-LPC, linoleoyl-LPC, and arachidonoyl-LPC into LPA were 6.3 ± 0.7%, 2.9 ± 0.2%, 4.9 ± 0.5%, 5.2 ± 0.3%, and 4.0 ± 0.7% (n = 3), respectively. These data revealed that, except for stearoyl-LPC, which exhibited a slightly lower conversion, the other LPC substrates exhibited equivalent conversion into LPA, suggesting that adipocyte lyso-PLD did not exhibit substantial substrate specificity. Some phospholipase D such as the PLD1 and PLD2 are characterized by their ability to catalyze, in the presence of primary alcohol, a transphosphatidylation reaction producing phosphatidylacohol instead of phosphatidic acid (24Liskovitch M. Czarny M. Fiucci G. Tang X. Phospholipase D: molecular and cell biology of a novel gene family..Biochem. J. 2000; 345: 401-415Crossref PubMed Google Scholar). If adipocyte lyso-PLD activity was due to the action of PLD1 or PLD2, it should be blocked by primary alcohols. As shown in Fig. 7, ethanol (from 0.5% to 10%) did not inhibit, but rather increased (at 5% and 10%) adipocyte LPA-SA. Similar increase was observed with 0.5% and 1% 1-butanol (Fig. 7). Above results showed that PLD1 or PLD2 could not be responsible for adipocyte LPA-SA. Our laboratory has previously reported that extra-cellular production of LPA by adipocytes was increased after stimulation of α2-adrenergic receptors (17Valet P. Pages C. Jeanneton O. Daviaud D. Barbe P. Record M. Saulnier-Blache J.S. Lafontan M. Alpha2-adrenergic receptor-mediated release of lysophosphatidic acid by adipocytes. A paracrine signal for preadipocyte growth..J. Clin. Invest. 1998; 101: 1431-1438Crossref PubMed Scopus (126) Google Scholar). We found that α2-adrenergic receptor stimulation did not influence lyso-PLD secretion nor production of lyso-PC by adipocytes (not shown), showing that α2-adrenergic-dependent regulation of extra-cellular production of LPA is likely mediated by another pathway which remains to be clarified. In order to determine the existence of possible regulations of adipocyte lyso-PLD, its activity was measured during the course of adipocyte differentiation. For that purpose, confluent 3T3F442A undifferentiated preadipocytes were grown in a standardized differentiating medium (see Materials and Methods). Based upon analysis of specific markers (expression of the adipocyte-lipid binding protein mRNA, accumulation of triglycerides), adipocyte differentiation occurs between 2 and 5 days after confluence (25Pagès C. Daviaud D. An S. Krief S. Lafontan M. Valet P. Saulnier-Blache J. Endothelial differentiation gene-2 receptor is involved in lysophosphatidic acid-dependent control of 3T3F442A preadipocyte proliferation and spreading..J. Biol. Chem. 2001; 276: 11599-11605Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Confluent preadipocytes and adipocytes (day 7 after confluence) were incubated in HB-BSA for 7 h. LPA present in the conditioned medium was quantified either directly (this corresponded to the basal extra-cellular production of LPA by the cells), or after 6 h further incubation at 37°C (this corresponds to the secretion of lyso-PLD), or after 45 min further incubation at 37°C in the presence of bacterial PLD (this corresponded to the production of LPC). Preadipocytes produced significantly less LPA (3.6-fold) and less lyso-PLD activity (6-fold) than adipocytes (Table 1). In parallel, no major alteration in lyso-PC production was observed between preadipocytes and adipocytes (Table 1). These results suggested the existence of a differentiation-dependent regulation of lyso-PLD activity that could account for a differentiation-dependent regulation of extra-cellular production of LPA.TABLE 1.Influence of adipocyte differentiation of production of LPA, LPA-SA, and lyso-PC3T3F442A CellsLPA (pmoles/mg of cell protein)InitialAfter 6 h+ PLDPreadipocytes6 ± 221 ± 3814 ± 150Adipocytes22 ± 3aP < 0.01 when comparing preadipocytes and adipocytes (Student's t-test).126 ± 12aP < 0.01 when comparing preadipocytes and adipocytes (Student's t-test).1129 ± 122Confluent 3T3F442A preadipocytes were grown for 7 days in a standardized differentiating medium (see Materials and Methods) in order to obtain adipocytes. Conditioned media were collected and frozen after 7 h incubation of preadipocytes or adipocytes in HB-BSA. LPA was quantified either immediately after defrosting (Initial), or 6 h after further incubation at 37°C, or after 45 min additional incubation at 37°C in the presence of 1 U/ml bacterial PLD (+ PLD). Data represent the mean ± SE of three separate experiments.a P < 0.01 when comparing preadipocytes and adipocytes (Student's t-test). Open table in a new tab Confluent 3T3F442A preadipocytes were grown for 7 days in a standardized differentiating medium (see Materials and Methods) in order to obtain adipocytes. Conditioned media were collected and frozen after 7 h incubation of preadipocytes or adipocytes in HB-BSA. LPA was quantified either immediately after defrosting (Initial), or 6 h after further incubation at 37°C, or after 45 min additional incubation at 37°C in the presence of 1 U/ml bacterial PLD (+ PLD). Data represent the mean ± SE of three separate experiments. We previously demonstrated that adipocytes are able to produce LPA in their culture medium (17Valet P. Pages C. Jeanneton O. Daviaud D. Barbe P. Record M. Saulnier-Blache J.S. Lafontan M. Alpha2-adrenergic receptor-mediated release of lysophosphatidic acid by adipocytes. A paracrine signal for preadipocyte growth..J. Clin. Invest. 1998; 101: 1431-1438Crossref PubMed Scopus (126) Google Scholar); however the metabolic origin of this extra-cellular production was not clarified. In the present study we demonstrate that, in parallel to LPA, adipocyte is also able to secrete a LPA-synthesizing activity (LPA-SA). Biochemical analysis strongly suggests that LPA-SA corresponds to a soluble lyso-PLD catalyzing transformation of LPC into LPA. The hypothesis that LPA-SA could indeed correspond to a lyso-PLD is supported by several observations. First, LPA-SA exhibits similar sensitivity to the the ion-chelators (EDTA and phenanthrolin) and similar sensitivity to cobalt ion, than a lyso-PLD activity previously described in rat plasma (23Tokumura A. Miyabe M. Yoshimoto O. Shimizu M. Fukuzawa K. Metal-ion stimulation and inhibition of lysophospholipase D which generates bioactive lysophosphatidic acid in rat plasma..Lipids. 1998; 33: 1009-1015Crossref PubMed Scopus (52) Google Scholar). Second, adipocyte CM is able to transform exogenously added lysophospholipids into LPA, a reaction that can only be achieved by a lyso-PLD. Based on our results it appeared that the main substrate of adipocyte lyso-PLD is LPC since it is the main PLD-transformable LPA-precursor (mainly PLC) present in adipocyte CM. By using various exogenous lysophospholipids, we also found that LPC appears as the best substrate of adipocyte lyso-PLD as compared with LPG and LPAF. In addition, by using various LPC species we found that adipocyte lyso-PC does not exhibit substantial substrate specificity regarding the nature of the fatty acid composing its substrate. In that respect, adipocyte lyso-PLD appears to be different from the lyso-PLD previously described in rat plasma and which was demonstrated to hydrolyze polyunsaturated-LPC preferentially to the saturated-LPCs (6Tokumura A. Harada K. Fukuzawa K. Tsukatani H. Involvement of lysophospholipase D in the production of lysophosphatidic acid in rat plasma..Biochim. Biophys. Acta. 1986; 875: 31-38Crossref PubMed Scopus (153) Google Scholar, 26Tokumura A. Fujimoto H. Yoshimoto O. Nishioka Y. Miyake M. Fukuzawa K. Production of lysophosphatidic acid by lysophospholipase D in incubated plasma of spontaneously hypertensive rats and Wistar Kyoto rats..Life Sci. 1999; 65: 245-253Crossref PubMed Scopus (43) Google Scholar). In platelets and ovarian cancer cells, LPA synthesis has been proposed to result from deacylation of phosphatidic acid by a phospholipase A2-activity (3McCrea J. Robinson P. Gerrard J. Mepacrine (quinacrine) inhibition of thrombin-induced platelet responses can be overcome by lysophosphatidic acid..Biochim. Biophys. Acta. 1985; 842: 189-194Crossref PubMed Scopus (31) Google Scholar, 5Fourcade O. Simon M.F. Viodé C. Rugani N. Leballe F. Ragab A. Fournié B. Sarda L. Chap H. Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells..Cell. 1995; 80: 919-927Abstract Full Text PDF PubMed Scopus (492) Google Scholar, 18Shen Z. Belinson J. Morton R. Yan X. Phorbol 12-myristate 13-acetate stimulates lysophosphatidic acid secretion from ovarian and cervical cancer cells but not from breast or leukemia cells..Gynecol. Oncol. 1998; 71: 364-368Abstract Full Text PDF PubMed Scopus (102) Google Scholar). In adipocyte CM, we could not detect any transformation of phosphatidic acid into LPA, showing that phospholipase A2-activity could not account for LPA-SA. In addition, treatment of adipocyte CM with pancreatic phospholipase A2 did not increase LPA concentration, suggesting that PA was undetectable in adipocyte CM. Therefore, the direct involvement of phospholipase A2 in LPA-SA could reasonably be excluded. Nevertheless, phospholipase A2 very likely plays an indirect role by providing the substrate of the lyso-PLD, lysophosphatidylcholine, resulting from hydrolysis of phosphatidylcholine. Because of its relatively high polarity, it is unlikely that LPA could easily diffuse through phospholipid membranes. Therefore, when present extra-cellularly, LPA would result from secreted or ectopic enzyme(s), rather than from intracellular enzyme(s). This hypothesis is in agreement with previous report showing that exogenous bacterial PLD exhibiting lyso-PLD activity can produce LPA in the outer membrane leaflet of intact cells (7van Dijk M. Postma F. Hilkmann H. Jalink K. van Blitterswijk W.J. Moolenaar W. Exogenous phospholipase D generates lysophosphatidic acid and activates Ras, Rho and Ca2+ signaling pathways..Curr. Biol. 1998; 8: 386-392Abstract Full Text Full Text PDF PubMed Google Scholar). The fact that adipocyte lyso-PLD was found in CM after separation from the cells suggests that this enzyme could be secreted or released from adipocytes. This hypothesis is reinforced by the fact that adipocyte lyso-PLD activity was found to be soluble and not associated with a particulate fraction. Although the precise mechanisms of its secretion/release remain to be established, adipocyte lyso-PLD is, to our knowledge, the first extracellular lyso-PLD of known cellular origin in mammals. Tokomura et al. have reported the existence of a lyso-PLD activity in rat plasma (6Tokumura A. Harada K. Fukuzawa K. Tsukatani H. Involvement of lysophospholipase D in the production of lysophosphatidic acid in rat plasma..Biochim. Biophys. Acta. 1986; 875: 31-38Crossref PubMed Scopus (153) Google Scholar) as well as in human follicular fluids (8Tokomura A. Miyake M. Nishioka Y. Yamano S. Aono T. Fukuzawa K. Production of lysophosphatidic acids by lysophospholipase D in human follicular fluids of in vitro fertilization patients..Biol. Reprod. 1999; 61: 195-199Crossref PubMed Scopus (119) Google Scholar). Whereas the involvement of this lyso-PLD activity in LPA production has been demonstrated, the structure of the enzyme, as well as its cellular origin, remains completely unknown. Therefore, the discovery of an adipocyte lyso-PLD activity gives a unique opportunity to purify and clone the enzyme. Interestingly, our data show that lyso-PLD activity is up-regulated during adipocyte differentiation. It is therefore likely that the adipocyte lyso-PLD gene belongs to a set of genes positively regulated during adipocyte differentiation, and this could help for its identification. As we previously showed, LPA produced by adipocytes is able to activate the growth of the adipocyte precursors (preadipocytes) (17Valet P. Pages C. Jeanneton O. Daviaud D. Barbe P. Record M. Saulnier-Blache J.S. Lafontan M. Alpha2-adrenergic receptor-mediated release of lysophosphatidic acid by adipocytes. A paracrine signal for preadipocyte growth..J. Clin. Invest. 1998; 101: 1431-1438Crossref PubMed Scopus (126) Google Scholar), one of the key events of adipose tissue development (adipogenesis). Preadipocytes being located in the close vicinity of adipocytes in the adipose tissue, adipocyte production of LPA could play an important role in paracrine control of adipose tissue development. In order to control this paracrine function of LPA, it is required either to target LPA-receptors (EDG-2 receptors) present in preadipocytes (25Pagès C. Daviaud D. An S. Krief S. Lafontan M. Valet P. Saulnier-Blache J. Endothelial differentiation gene-2 receptor is involved in lysophosphatidic acid-dependent control of 3T3F442A preadipocyte proliferation and spreading..J. Biol. Chem. 2001; 276: 11599-11605Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), or to act on adipocyte-LPA synthesis. Consequently, adipocyte lyso-PLD could represent an interesting target to control adipose tissue development. This work was supported by grants from the Institut Nationale de la Santé et de la Recherche Médicale, the Institut de Recherche Servier, the Association pour la Recherche sur le Cancer (#5381) and the Laboratoires Clarins. chloroform conditioned medium lysophosphatidic acid lysophosphatidic acid acyltransferase lyso-platelet activated factor lysophosphatidic acid-synthesizing activity lysophosphatidylcholine lysophosphatidylglycerol methanol phosphatidic acid phosphatidylcholine phospholipase A2 phospholipase D
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