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• W2034883068 abstract "Diacylglycerol (DAG) is a versatile molecule that participates as substrate in the synthesis of structural and energetic lipids, and acts as the physiological signal that activates protein kinase C. Diacylglycerol acyltransferase (DGAT), the last committed enzyme in triacylglycerol synthesis, could potentially regulate the content and use of both signaling and glycerolipid substrate DAG by converting it into triacylglycerol. To test this hypothesis, we stably overexpressed the DGAT1 mouse gene in human lung SV40-transformed fibroblasts (DGAT cells), which contains high levels of DAG. DGAT cells exhibited a 3.9-fold higher DGAT activity and a 3.2-fold increase in triacylglycerol content, whereas DAG and phosphatidylcholine decreased by 70 and 20%, respectively, compared with empty vector-transfected SV40 cells (Control cells). Both acylation and de novo synthesis of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were reduced by 30–40% in DGAT cells compared with controls, suggesting that DGAT used substrates for triacylglycerol synthesis that had originally been destined to produce phospholipids. The incorporation of [14C]DAG and [14C]fatty acids released from plasma membrane by additions of either phospholipase C or phospholipase A2 into triacylglycerol was increased by 6.2- and 2.8-fold, respectively, in DGAT cells compared with control cells, indicating that DGAT can attenuate signaling lipids. Finally, DGAT overexpression reversed the neoplastic phenotype because it dramatically reduced the cell growth rate and suppressed the anchorage-independent growth of the SV40 cells. These results strongly support the view that DGAT participates in the regulation of membrane lipid synthesis and lipid signaling, thereby playing an important role in modulating cell growth properties. Diacylglycerol (DAG) is a versatile molecule that participates as substrate in the synthesis of structural and energetic lipids, and acts as the physiological signal that activates protein kinase C. Diacylglycerol acyltransferase (DGAT), the last committed enzyme in triacylglycerol synthesis, could potentially regulate the content and use of both signaling and glycerolipid substrate DAG by converting it into triacylglycerol. To test this hypothesis, we stably overexpressed the DGAT1 mouse gene in human lung SV40-transformed fibroblasts (DGAT cells), which contains high levels of DAG. DGAT cells exhibited a 3.9-fold higher DGAT activity and a 3.2-fold increase in triacylglycerol content, whereas DAG and phosphatidylcholine decreased by 70 and 20%, respectively, compared with empty vector-transfected SV40 cells (Control cells). Both acylation and de novo synthesis of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were reduced by 30–40% in DGAT cells compared with controls, suggesting that DGAT used substrates for triacylglycerol synthesis that had originally been destined to produce phospholipids. The incorporation of [14C]DAG and [14C]fatty acids released from plasma membrane by additions of either phospholipase C or phospholipase A2 into triacylglycerol was increased by 6.2- and 2.8-fold, respectively, in DGAT cells compared with control cells, indicating that DGAT can attenuate signaling lipids. Finally, DGAT overexpression reversed the neoplastic phenotype because it dramatically reduced the cell growth rate and suppressed the anchorage-independent growth of the SV40 cells. These results strongly support the view that DGAT participates in the regulation of membrane lipid synthesis and lipid signaling, thereby playing an important role in modulating cell growth properties. Amphipatic lipids, such as diacylglycerols (DAG) 1The abbreviations used are: DAGdiacylglycerolBSAbovine serum albuminDGATdiacylglycerol acyltransferaseFBSfetal bovine serumMEMminimal essential mediumPCphosphatidylcholinePEphosphatidylethanolaminePKCprotein kinase CPLA2phospholipase A2PLCphospholipase CPBSphosphate-buffered salineSMsphingomyelin. and acyl-CoAs exhibit dual actions, as substrates for membrane and energy storage glycerolipids, as well as second messengers for signaling transduction events. As a substrate for de novo glycerolipid synthesis, DAG is a common intermediate for both triacylglycerol and phospholipid synthesis. Studies performed with permeabilized cells indicate that the utilization of de novo synthesized DAG for either neutral or polar lipid synthesis is controlled by DGAT and CDP-choline (ethanolamine) phosphotransferase activities, suggesting the presence of a common DAG pool that is shared for both lipid synthetic routes (1Stals H.K. Top W. Declercq P.E. FEBS Lett. 1994; 343: 99-102Crossref PubMed Scopus (36) Google Scholar). Nevertheless, at least one other DAG pool is available for glycerolipid synthesis, because DAG that is released from triacylglycerol stores in human fibroblasts can be converted to phospholipids (2Igal R.A. Coleman R.A. J. Biol. Chem. 1996; 271: 16644-16651Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Moreover, hepatic cells might contain different DAG pools that are used to synthesize triacylglycerol for storage or for lipoproteins (3Wiggins D. Gibbons G.F. Biochem. J. 1992; 284: 457-462Crossref PubMed Scopus (201) Google Scholar, 4Owen M.R. Corstorphine C.C. Zammit V.A. Biochem. J. 1997; 323: 17-21Crossref PubMed Scopus (110) Google Scholar). Although no definitive evidence exists for specific DAG pools for polar and neutral glycerolipid synthetic pathways, segregation of DAG toward different metabolic routes seems to occur according to the need of the cell. For instance, when phospholipid synthesis is inhibited, DAG originally destined to form phospholipids is re-directed toward triacylglycerol (5Jackowski S. Wang J. Baburina I. Biochim. Biophys. Acta. 2000; 1483: 301-315Crossref PubMed Scopus (91) Google Scholar). diacylglycerol bovine serum albumin diacylglycerol acyltransferase fetal bovine serum minimal essential medium phosphatidylcholine phosphatidylethanolamine protein kinase C phospholipase A2 phospholipase C phosphate-buffered saline sphingomyelin. Growth factors and hormones activate phospholipases C and D to promote a bi-phasic accumulation of DAG that triggers signaling events (6Wakelam M.J. Biochim. Biophys. Acta. 1998; 1436: 117-126Crossref PubMed Scopus (160) Google Scholar). As a signaling effector, DAG regulates cell growth and differentiation by activating several isoforms of protein kinase C (PKC). A short-term release of DAG is caused by the hydrolysis of phosphatidylinositol by several isoforms of phosphoinositide-specific PLC (7Katan M. Biochim. Biophys. Acta. 1998; 1436: 5-17Crossref PubMed Scopus (192) Google Scholar). A second wave of DAG produced by cytokine-activated PC hydrolysis is needed to fully develop mitogenesis. In this regard, quiescent fibroblasts treated with platelet-derived growth factor or bacterial PCPLC increase DAG levels and a concomitant strong mitogenic response (8Larrodera P. Cornet M.E. Diaz-Meco M.T. Lopez-Barahona M. Diaz-Laviada I. Guddal P.H. Johansen T. Moscat J. Cell. 1990; 61: 1113-1120Abstract Full Text PDF PubMed Scopus (119) Google Scholar). Persistent accumulation of intracellular DAG has also been linked to oncogenic transformation. Thus, sustained high levels of DAG produced by overactivation of PC-PLC induce a transformed phenotype in NIH 3T3 cells (9Johansen T. Bjørkøy G. Øvervatn A. Díaz-Meco M.T. Traavik T. Moscat J. Mol. Cell. Biol. 1994; 14: 646-654Crossref PubMed Scopus (124) Google Scholar). Moreover, neoplastic transformation by simian virus 40 (sv40), and by ras-, src-, and fps oncogenes is accompanied by an excess content of intracellular DAG (10Díaz-Laviada I. Larrodera P. Díaz-Meco M.T. Cornet M.E. Guddal P.H. Johansen T. Moscat J. EMBO J. 1990; 9: 3907-3912Crossref PubMed Scopus (73) Google Scholar, 11Martin A. Duffy P.A. Liossis C. Gómez-Muñoz A. O'Brien L. Stone J.C. Brindley D.N. Oncogene. 1997; 14: 1571-1580Crossref PubMed Scopus (50) Google Scholar, 12Luberto C. Hannun Y.A. J. Biol. Chem. 1998; 273: 14550-14559Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Not only are appropriate intracellular levels of signaling DAG necessary for mitogenesis, but a proper supply of DAG substrate for glycerolipid synthesis is also required by proliferating cells. To prepare for mitosis, cells must synthesize new membrane phospholipids; hence precursors for phospholipid synthesis must be available. Thus, mitogenic signals stimulate the formation of new PC by activating the expression of CTP: phosphocholine cytidylyltransferase (13Jackowski S. J. Biol. Chem. 1994; 269: 3858-3867Abstract Full Text PDF PubMed Google Scholar) and by diverting DAG toward PC synthesis, rather than triacylglycerol formation (5Jackowski S. Wang J. Baburina I. Biochim. Biophys. Acta. 2000; 1483: 301-315Crossref PubMed Scopus (91) Google Scholar). Moreover, in neurite outgrowth induced by nerve growth factor, PC synthesis is enhanced by an increase in DAG levels together with an activation of the CDP-choline DAG phosphotransferase (14Araki W. Wurtman R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11946-11950Crossref PubMed Scopus (75) Google Scholar). Membrane lipid synthesis is also up-regulated in neoplastic cells, hence a constant supply of lipid precursors for new membranes is required to sustain the unrestricted proliferation of tumor cells. In this regard, increased synthesis and turnover of phospholipids has been observed in neoplastic cells (10Díaz-Laviada I. Larrodera P. Díaz-Meco M.T. Cornet M.E. Guddal P.H. Johansen T. Moscat J. EMBO J. 1990; 9: 3907-3912Crossref PubMed Scopus (73) Google Scholar, 11Martin A. Duffy P.A. Liossis C. Gómez-Muñoz A. O'Brien L. Stone J.C. Brindley D.N. Oncogene. 1997; 14: 1571-1580Crossref PubMed Scopus (50) Google Scholar, 15Teegarden D. Taparowsky E.J. Kent C. J. Biol. Chem. 1990; 265: 6042-6047Abstract Full Text PDF PubMed Google Scholar). Cells can regulate the content and destiny of DAG by lipolysis (16Chuang M. Severson D.L. Biochim. Biophys. Acta. 1998; 1390: 149-159Crossref PubMed Scopus (7) Google Scholar), phosphorylation by DAG kinases (17van Blitterswijk W.J. Hilkmann H. de Widt J. van der Bend R.L. J. Biol. Chem. 1991; 266: 10337-10343Abstract Full Text PDF PubMed Google Scholar, 18van der Bent R.L. de Widt J. Hilkmann H. van Blitterswijk W.J. J. Biol. Chem. 1994; 269: 4098-4102Abstract Full Text PDF PubMed Google Scholar, 19Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (253) Google Scholar, 20Topham M.K. Prescott S.M. J. Cell Biol. 2001; 152: 1135-1143Crossref PubMed Scopus (101) Google Scholar), and synthesis of PC (21Florin-Christensen J. Florin-Christensen M. Delfino J.M. Stegmann T. Rasmussen H. J. Biol. Chem. 1992; 267: 14783-14789Abstract Full Text PDF PubMed Google Scholar). DAG released from plasma membrane may also be directly incorporated into triacylglycerol, suggesting the presence of a novel mechanism for terminating DAG signals based on the synthesis of a storage lipid (22Igal R.A. Caviglia J.M. de Gómez Dumm I.N. Coleman R.A. J. Lipid Res. 2001; 42: 88-95Abstract Full Text Full Text PDF PubMed Google Scholar). A key enzyme involved in DAG and triacylglycerol metabolism is acyl-CoA-diacylglycerol acyltransferase 1 (DGAT1) (23Cases S. Smith S.J. Zheng Y-W. Myers H.M. Lear S.R. Sande E. Novak S. Collins C. Welch C.B. Lusis A.L. Erickson S.K. Farese Jr., R.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13018-13023Crossref PubMed Scopus (879) Google Scholar). DGAT1, and the recently discovered DGAT2 (24Cases S. Stone S.J. Zhou P. Yen E. Tow B. Lardizabal K.D. Voelker T. Farese Jr., R.V. J. Biol. Chem. 2001; 276: 38870-38876Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar), catalyze the last committed step in mammalian triacylglycerol synthesis by esterifying DAG with a fatty acid. The DAG used as a DGAT substrate is primarily derived from the de novo glycerol 3-phosphate pathway. Other sources of DAG for triacylglycerol synthesis include that produced from monoacylglycerol in intestinal cells (25Cao J. Lockwood J. Burn P. Shi Y. J. Biol. Chem. 2003; 278: 13860-13866Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), from the hydrolysis of triacylglycerol (2Igal R.A. Coleman R.A. J. Biol. Chem. 1996; 271: 16644-16651Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 3Wiggins D. Gibbons G.F. Biochem. J. 1992; 284: 457-462Crossref PubMed Scopus (201) Google Scholar), and from DAG released from plasma membrane phospholipids (22Igal R.A. Caviglia J.M. de Gómez Dumm I.N. Coleman R.A. J. Lipid Res. 2001; 42: 88-95Abstract Full Text Full Text PDF PubMed Google Scholar). Because both DGAT genes are widely expressed in mouse and human tissues (23Cases S. Smith S.J. Zheng Y-W. Myers H.M. Lear S.R. Sande E. Novak S. Collins C. Welch C.B. Lusis A.L. Erickson S.K. Farese Jr., R.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13018-13023Crossref PubMed Scopus (879) Google Scholar, 24Cases S. Stone S.J. Zhou P. Yen E. Tow B. Lardizabal K.D. Voelker T. Farese Jr., R.V. J. Biol. Chem. 2001; 276: 38870-38876Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar), the presence of two DGAT enzymes in non-adipose tissues suggests that the synthesis of triacylglycerol might be linked to cell functions other than energy storage. For instance, adenoviral expression of DGAT1 in pancreatic islets increased the formation of triacylglycerol by 100% but impaired the secretion of insulin after glucose stimulation (26Kelpe C.L. Johnson S.L. Poitout V. Endocrinology. 2002; 43: 3326-3332Crossref Scopus (51) Google Scholar). To understand the role of DGAT in terminating DAG signals, we tested the hypothesis that DGAT overexpression channels DAG and/or fatty acids toward triacylglycerol, thereby sequestering either proliferative lipid signals and/or lipid substrates for membrane biogenesis with a consequent decrease in phospholipid synthesis, normalization of cell proliferation, and a reversion in the abnormal cytological phenotype. Materials—Normal human lung fibroblasts (WI38) and the derived SV40-transformed strain were obtained from the American Type Culture Collection (Manassas, VA). LipofectAMINE™, cell culture media, G418 (Geneticin™) antibiotic, and other culture reagents were from Invitrogen. Ultrafiltered fetal bovine serum (FBS) was from Gensa (Buenos Aires, Argentina). Cell culture supplies were from Greiner BioOne (Frickenhausen, Germany). Restriction enzymes and other molecular biology reagents were purchased from Promega (Madison, WI). [14C]Oleic acid and [γ-32P]ATP were from Amersham Biosciences, [methyl-14C]choline and [3H]glycerol were purchased from PerkinElmer Life Sciences. Fatty acid-free bovine serum albumin (BSA), phospholipase A2 (PLA2, from Naja naja), PC-specific phospholipase C (PCPLC, from Bacillus cereus), and anti-FLAG M2 monoclonal antibody were from Sigma. Pure lipid standards were from Doosan Serdary (Yongin, Korea). 1,6-Diphenyl-1,3,5-hexatriene was purchased from Aldrich. Silica Gel 60 chromatography plates were from Merck (Darmstadt, Germany). Analytical-grade solvents were from Carlo Erba (Milano, Italy). DAG kinase membrane suspension was from Calbiochem. Cell Culture—Cells were routinely cultured in 100-mm Petri dishes in minimum essential medium with Earle's salts (MEM) with 10% heat-inactivated FBS, 1% penicillin (100 units/ml), streptomycin (10 μg/ml), 1% nonessential amino acids, and 1% MEM vitamins (growing medium), at 37 °C, 5% CO2, and 100% humidity. Normal human lung fibroblasts were used for the experiments before reaching the 20th passage. Generation of Stable DGAT1 Overexpressing SV40-transformed Cells—Mouse DGAT1 cDNA with an N-terminal FLAG epitope (kind gift of Dr. Robert V. Farese Jr., Gladstone Institute of Cardiovascular Disease, University of California) was cloned into the restriction sites EcoRV and XbaI of a pCDNA3 mammalian expression vector (Invitrogen). SV40-transformed cells, grown in 100-mm dishes up to 50% confluence, were washed twice with serum-free Opti-MEM and then transfected with 12 μg of either DGAT1-pCDNA3 plasmid or empty vector plus 35 μl of LipofectAMINE™ (2 mg/ml) in 0.5 ml of Opti-MEM plus 20% FBS. After 5 h incubation, medium was replaced by 10% FBS MEM and transfected cells were grown for an additional 24 h. Then, positive transfectants were selected by culturing the cells in 10% FBS, MEM containing geneticin (600 μg/ml) for 15 days. Ten DGAT1-transfected clones were isolated by using cloning cylinders and were grown in 100-mm Petri dishes with 10% FBS, MEM plus G418 antibiotic (300 μg/ml) for 15 days. Triacylglycerol synthetic rate and content were analyzed in each DGAT-transfected clone, as well as in empty vector-transfected SV40 cells (Control cells), and two DGAT clones (DGAT-A and DGAT-V cells) that showed the highest synthesis of triacylglycerol (measured by the incorporation of [14C]oleate into lipids during a 24-h period) were selected for the experiments. Metabolic Labeling—Preconfluent control and DGAT cells, cultured in 60-mm dishes, were incubated for up to 48 h either with [14C]oleic acid (0.25 μCi/dish), [3H]glycerol (5 μCi/dish) or [methyl-14C]choline (0.5μCi/dish), in the presence or absence of 100 μm oleate, in 10% FBS, MEM supplemented with 0.5% BSA. At the end of each labeling period, the radioactive medium was discarded and cell monolayers were washed twice with 0.1% BSA in ice-cold phosphate-buffered saline (PBS), to eliminate residual label on the cell surface. Next, cells were scraped from the dishes with two additions of 1 ml of ice-cold methanol and 0.5 ml of H2O. Total cellular lipids were obtained according to the procedure of Bligh and Dyer (27Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42875) Google Scholar) and aqueous and lipid fractions were concentrated in a Savant SpeedVac concentrator. Analysis and Quantitation of Lipids and Choline-derived Metabolites—For mass lipid determinations, cells were grown until near confluence in 100-mm dishes and total lipid extracts were obtained as described above. Neutral and polar lipids species were separated on Silica Gel 60 thin-layer chromatography (TLC) plates using one-dimensional single development procedures. Routinely, neutral lipid separation was carried out with the following solvent system:hexane:ethyl ether:acetic acid, 80:20:2 (by volume). Polar lipid species were resolved in a solvent system consisting of chloroform:methanol:ammonium hydroxide:water, 50:37.5:3.5:2 (by volume). Known amounts of pure lipid standards were seeded and run on the TLC plates in parallel to samples. To both solvent systems 100 μm 1,6-diphenyl-1,3,5-hexatriene was added to visualize the lipid spots on the plate under UV light (22Igal R.A. Caviglia J.M. de Gómez Dumm I.N. Coleman R.A. J. Lipid Res. 2001; 42: 88-95Abstract Full Text Full Text PDF PubMed Google Scholar). Fluorescent lipid spots from samples and standards were photographed and lipid mass was quantified using a DS120 Kodak Image system. For the [14C] and [3H] labeling experiments, individual fluorescent lipid spots were scraped into plastic vials and radioactivity levels were determined in a liquid scintillation counter. To separate water-soluble choline metabolites, aqueous phases were dried and dissolved in H2O, spotted on silica gel chromatoplates, and resolved with 0.6% sodium chloride, methanol, 30% ammonium hydroxide; 50:50:5 (by volume). Pure standards were used as carriers and were added to the samples before chromatography. Radioactive spots were detected in a Berthold II radiometric scanner, scraped into vials, and counted. Total 1,2-DAG was quantitated by the DAG kinase assay according to the procedure of Preiss et al. (28Preiss J. Loomis C.R. Bishop W.R. Stein R. Niedel J.E. Bell R.M. J. Biol. Chem. 1986; 261: 8597-8600Abstract Full Text PDF PubMed Google Scholar), using [γ-32P]ATP as substrate. The results of lipid determinations were expressed in nanomoles of lipid per mg of protein. Cell Proliferation—To evaluate cell growth, 1.5 × 104 cells were seeded in duplicate 60-mm dishes. Twenty-four hours later, medium was removed and replaced with fresh growing medium. At this time and up to 144 h cells were then trypsinized, and counted in an hemocytometer. Cell viability was determined by trypan blue exclusion. [3H]Thymidine Incorporation into Total DNA—To measure the rate of DNA synthesis, cells grown in triplicate 60-mm dishes were pulsed with [3H]thymidine (1 μCi/dish) in 2 ml of growing media for 3 h at 37 °C. Media was removed and cell monolayers were washed twice with ice-cold PBS, and then precipitated with 5% trichloroacetic acid for 10 min at 4 °C. The acid-insoluble material was solubilized with 0.2% SDS in 0.1 n NaOH and an aliquot was counted in a liquid scintillation counter. Cloning in Soft Agar—Control and DGAT cells were plated in quadruplicates at 1 × 104 cells per 60-mm dishes in MEM containing 10% FBS and 0.3% (w/v) agar onto a bottom layer of 0.6 (w/v) agar in MEM. After 3 and 5 weeks, the presence or absence of multicellular colonies (more than eight cells) in both cell groups was verified under the microscope. For macroscopic analysis, cell colonies were stained with ethidium bromide in PBS for 30 min, visualized, and photographed under UV light. Cell Radiolabeling and Phospholipase Treatments—Near confluent control and DGAT cells grown in 60-mm dishes were incubated with trace amounts of [14C]oleic acid (0.25 μCi/dish) in growing medium supplemented with 0.5% BSA for 48 h. After labeling, medium was removed and monolayers were washed twice with 0.1% BSA in warm PBS. Next, cells were treated with 10 units/dish of either PC-PLC or PLA2, or 100 μm oleic acid, for 6 h. After these treatments, the medium was discarded, and cell monolayers were washed with ice-cold PBS and scraped with two additions of ice-cold methanol. Lipid extraction and separation, as well as the quantitation of radioactivity levels of lipid species, were performed as stated above. DGAT Enzyme Assay—Cells were grown in 100-mm Petri dishes until 80–90% confluence. Cells were then trypsinized, washed twice with ice-cold PBS, and resuspended in 10 mm Tris-HCl buffer, pH 7.4, with protease inhibitor mixture (Sigma). Cell homogenates were obtained by sonication on an ice-water bath for 10 s at 50% output and stored at -70 °C until use. DGAT activity was determined using 100 and 200 μg of cell homogenate protein, 200 μm diacylglycerol in acetone, and 30 μm [3H]palmitoyl-CoA following the procedure of Coleman (29Coleman R.A. Methods Enzymol. 1992; 209: 134-146Crossref PubMed Scopus (23) Google Scholar). Western Blot Analysis of FLAG-DGAT—Preconfluent control and DGAT cells were homogenized in 25 mm Tris-HCl, pH 7.4, 1 mm EDTA, 0.1% SDS plus 1% protease inhibitor mixture by sonication in an ice-water bath. Proteins from total cell homogenates (150 μg) were separated by SDS-polyacrylamide gel electrophoresis on a 12.5% polyacrylamide gel and then transferred onto nitrocellulose membranes. After blocking, membranes were incubated with mouse anti-FLAG as primary antibody and donkey anti-mouse IgG-horseradish peroxidase conjugate as secondary antibody. Visualization of FLAG-DGAT protein was performed using a SuperSignal West Pico detection kit (Pierce). Other Methods—Protein content was measured as described by Lowry et al. (30Lowry O.R. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). [3H]Palmitoyl-CoA was synthesized enzymatically (31Merrill Jr., A.H. Gidwitz S. Bell R.M. J. Lipid Res. 1982; 23: 1368-1373Abstract Full Text PDF PubMed Google Scholar). Stable DGAT Overexpression Results in Massive Accumulation of Triacylglycerol and a Decrease in the Levels of DAG and PC—From a pool of SV40-transformed cells transfected with DGAT1 cDNA and selected by resistance to G418 antibiotic resistance, several clones of DGAT overexpressing cells were isolated. Two clones, DGAT-A and DGAT-V, which had the highest rate of TAG synthesis and TAG content were selected for the experiments. DGAT-A cells exhibited a high level of FLAG-DGAT protein expression (Fig. 1A), which correlated with a 3.9-fold increase in the in vitro DGAT activity (Fig. 1B), when compared with control cells. In addition, the DGAT-V cells exhibited a 10-fold increase in DGAT activity over the controls. However, because this DGAT-overexpressing clone showed an extremely low cell proliferation rate (see below), because of feasibility, most of the experiments were performed using the DGAT-A clone. To determine whether FLAG-DGAT protein was functional in the whole cells, the content of triacylglycerol mass in control and DGAT-A cells was analyzed by TLC and densitometric scanning. Accordingly, DGAT-A cells showed a 3.2-fold increase in triacylglycerol levels, with respect to control cells (Fig. 1, C and D). These cells also exhibited a higher level of cholesteryl esters, but this finding was not further investigated. We also observed that triacylglycerol accumulated as numerous small cytosolic droplets (Oil Red O sensitive) surrounding the nucleus (data not shown). This pattern of triacylglycerol depot was similar to that observed in other non-adipose cells (32Wolins N.E. Rubin B. Brasaemle D.L. J. Biol. Chem. 2001; 276: 5101-5108Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar), indicating that no aberrant localization of triacylglycerol droplets occurred in the DGAT-overexpressing cells. Taken as a whole, this group of observations indicate that the DGAT-transfected clones contained high amounts of functional DGAT enzyme that promoted the accumulation of its product triacylglycerol. Additionally, the mass of the main phospholipids, PC and PE, as well as 1,2-DAG was determined in control cells and DGAT overexpressors (Table I). PC content decreased ∼20% in DGAT-A cells compared with empty vector-transfected SV40 cells, whereas the levels of PE did not show significant changes between the cell groups. Interestingly, the content of total 1,2-DAG in DGAT cells was reduced by 70% with respect to control values, strongly suggesting that the overexpressed DGAT activity is depleting the DAG sources that can be used for phospholipid production by preferentially channeling this lipid intermediate into triacylglycerol stores.Table ILevels of PC, PE, and 1,2-DAG in control and DGAT-A cells SV40-transformed cells stably transfected with either empty vector (Control) or DGAT1 cDNA (DGAT-A) were grown until near confluence in MEM, 10% FBS in the presence of 300 μg/ml geneticin. Cells were harvested and the content of lipid species was determined as described under “Experimental Procedures.” Values represent mean ± S.D. of three separate determinations (PC and PE) or the average of duplicate determinations for two independent experiments, with a difference between determinations of less than 10% (1,2-DAG).Nanomole of lipid/mg proteinControlDGAT-APC156 ± 14121 ± 16ap < 0.05, Student's t testPE58 ± 1267 ± 111,2-DAG0.4090.116a p < 0.05, Student's t test Open table in a new tab Synthesis of Neutral Lipids Versus Polar Lipids in DGAT Overexpressing Cells: Regulation of Lipid Partitioning by DGAT—Because it has been hypothesized that PC and triacylglycerol biosynthetic pathways share the same pool of lipid substrates, we analyzed both the synthesis of neutral and polar lipids in control and DGAT-overexpressing cells incubated with traces of [14C]oleic acid. Labeling of total lipids was slightly decreased (∼20%) in DGAT-A cells compared with controls. When the synthesis of the [14C]lipid species was analyzed, it was observed that the formation of triacylglycerol was 2.3- and 5.5-fold higher in DGAT cells than in control cells at 24 and 48 h, respectively (Fig. 2A). On the other hand, synthesis of total polar lipids was decreased by 35% in DGAT overexpressors, both at 24 and 48 h (Fig. 2B). Decreases in both PC and PE, the only polar lipid species labeled with [14C]oleate in both cell groups, accounted for the lower phospholipid labeling (data not shown). Additionally, the levels of cellular [14C]1,2-DAG, measured after a 24-h incubation with trace amounts of [14C]oleate, was ∼30% decreased in DGAT cells compared with empty vector-transfected cells (6694 ± 836 versus 4602 ± 608 dpm/mg of protein, respectively). This observation further confirms that DGAT overexpression preferentially redirects DAG molecules toward the synthesis of triacylglycerol, decreasing the DAG pool available for the formation of phospholipids. Oleic acid can act as a substrate for both acylation of preexisting phospholipids as well as for the de novo synthesis of polar lipids, whereas labeled glycerol is a substrate exclusively for the de novo synthetic pathway, therefore labeling of lipids with [3H]glycerol would help to determine whether this route is specifically affected by DGAT overexpression. Thus, preconfluent control and DGAT-A cells were labeled with [3H]glycerol for 24 h in the presence of 100 μm oleate to enhance lipid synthesis, and labeled neutral and total phospholipids were analyzed. The incorporation of [3H]glycerol into total cell lipids was significantly depressed in DGAT cells compared with the mock-transfected group (Fig. 3), suggesting a specific down-regulation of the de novo pathway for glycerolipid formation in the DGAT overexpressors. However, despite a decreased de novo lipid formation, most of the [3H]glycerol was incorporated into triacylglycerol in DGAT cells at the expense of both PC and PE labeling (Fig. 3, inset), indicating that de novo synthesized lipid substrates were re-directed toward the synthesis of triacylglycero" @default.
• W2034883068 created "2016-06-24" @default.
• W2034883068 creator A5054812638 @default.
• W2034883068 creator A5084487481 @default.
• W2034883068 date "2003-12-01" @default.
• W2034883068 modified "2023-10-05" @default.
• W2034883068 title "Overexpression of Diacylglycerol Acyltransferase-1 Reduces Phospholipid Synthesis, Proliferation, and Invasiveness in Simian Virus 40-transformed Human Lung Fibroblasts" @default.
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