Arachidonic acid-dependent inhibition of adipocyte differentiation requires PKA activity and is associated with sustained expression of cyclooxygenases
2003; Elsevier BV; Volume: 44; Issue: 12 Linguagem: Inglês
10.1194/jlr.m300192-jlr200
ISSN1539-7262
AutoresRasmus K. Petersen, Claus J⊘rgensen, Arild C. Rustan, Livar Fr⊘yland, Karin Müller‐Decker, Gerhard Fürstenberger, Rolf K. Berge, Karsten Kristiansen, Lise Madsen,
Tópico(s)Eicosanoids and Hypertension Pharmacology
ResumoArachidonic acid inhibits adipocyte differentiation of 3T3-L1 cells via a prostaglandin synthesis-dependent pathway. Here we show that this inhibition requires the presence of a cAMP-elevating agent during the first two days of treatment. Suppression of protein kinase A activity by H-89 restored differentiation in the presence of arachidonic acid. Arachidonic acid treatment led to a prolonged activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 activity by the addition of U0126 rescued differentiation. Upon induction of differentiation, expression of cyclooxygenase-2 (COX-2) was transiently induced and then declined, whereas COX-1 expression declined gradually as differentiation progressed. Treatment with arachidonic acid led to sustained expression of COX-1 and COX-2. Omission of a cAMP-elevating agent or addition of H-89 or U0126 prevented sustained expression of COX-2. Unexpectedly, we observed that selective COX-1 or COX-2 inhibitors rescued adipocyte differentiation in the presence of arachidonic acid as effectively as did the nonselective COX-inhibitor indomethacin.De novo fatty acid synthesis, diacylglycerol acyltransferase (DGAT) activity, and triacylglycerol accumulation were repressed in cells treated with arachidonic acid. Indomethacin restored DGAT activity and triacylglycerol accumulation without restoring de novo fatty acid synthesis, resulting in an enhanced incorporation of arachidonic acid into cellular triacylglycerols. Arachidonic acid inhibits adipocyte differentiation of 3T3-L1 cells via a prostaglandin synthesis-dependent pathway. Here we show that this inhibition requires the presence of a cAMP-elevating agent during the first two days of treatment. Suppression of protein kinase A activity by H-89 restored differentiation in the presence of arachidonic acid. Arachidonic acid treatment led to a prolonged activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 activity by the addition of U0126 rescued differentiation. Upon induction of differentiation, expression of cyclooxygenase-2 (COX-2) was transiently induced and then declined, whereas COX-1 expression declined gradually as differentiation progressed. Treatment with arachidonic acid led to sustained expression of COX-1 and COX-2. Omission of a cAMP-elevating agent or addition of H-89 or U0126 prevented sustained expression of COX-2. Unexpectedly, we observed that selective COX-1 or COX-2 inhibitors rescued adipocyte differentiation in the presence of arachidonic acid as effectively as did the nonselective COX-inhibitor indomethacin. De novo fatty acid synthesis, diacylglycerol acyltransferase (DGAT) activity, and triacylglycerol accumulation were repressed in cells treated with arachidonic acid. Indomethacin restored DGAT activity and triacylglycerol accumulation without restoring de novo fatty acid synthesis, resulting in an enhanced incorporation of arachidonic acid into cellular triacylglycerols. Fatty acids derived from dietary fat act directly on preadipocytes to positively or negatively influence proliferation and differentiation. However, the underlying mechanisms dictating the opposing effects of distinct classes of dietary fatty acids are far from being fully understood. Whereas high-fat diets have been demonstrated to induce both hypertrophy and hyperplasia of adipose tissue in rats (1Klyde B.J. Hirsch J. Increased cellular proliferation in adipose tissue of adult rats fed a high-fat diet.J. Lipid Res. 1979; 20: 705-715Abstract Full Text PDF PubMed Google Scholar, 2Belzung F. Raclot T. Groscolas R. Fish oil n-3 fatty acids selectively limit the hypertrophy of abdominal fat depots in growing rats fed high-fat diets.Am. J. 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Requirement and role of arachidonic acid in the differentiation of pre-adipose cells.Biochem. J. 1989; 15: 389-397Crossref Scopus (162) Google Scholar). The proadipogenic effect of arachidonic acid in this cell system is cyclooxygenase (COX)-dependent and mediated by prostacyclin (16Gaillard D. Negrel R. Lagarde M. Ailhaud G. Requirement and role of arachidonic acid in the differentiation of pre-adipose cells.Biochem. J. 1989; 15: 389-397Crossref Scopus (162) Google Scholar, 17Negrèl R. Gaillard D. Ailhaud G. Prostacyclin as a potent effector of adipose-cell differentiation.Biochem. J. 1989; 257: 399-405Crossref PubMed Scopus (130) Google Scholar, 18Catalioto R.M. Gaillard D. Maclouf J. Ailhaud G. Negrel R. Autocrine control of adipose cell differentiation by prostacyclin and PGF2 alpha.Biochim. Biophys. Acta. 1991; 1091: 364-369Crossref PubMed Scopus (62) Google Scholar). In sharp contrast, another arachidonic acid-derived metabolite, prostaglandin F2α (PGF2α), inhibits differentiation of primary preadipocytes (19Serrero G. Lepak N.M. Goodrich S.P. Prostaglandin F2 alpha inhibits the differentiation of adipocyte precursors in primary culture.Biochem. Biophys. Res. Commun. 1992; 183: 438-442Crossref PubMed Scopus (42) Google Scholar, 20Serrero G. Lepak N.M. Prostaglandin F2alpha receptor (FP receptor) agonists are potent adipose differentiation inhibitors for primary culture of adipocyte precursors in defined medium.Biochem. Biophys. Res. Commun. 1997; 233: 200-202Crossref PubMed Scopus (61) Google Scholar), 1246 cells (21Serrero G. Lepak N.M. Goodrich S.P. Paracrine regulation of adipose differentiation by arachidonate metabolites: prostaglandin F2 alpha inhibits early and late markers of differentiation in the adipogenic cell line 1246.Endocrinology. 1992; 131: 2545-2551Crossref PubMed Google Scholar), and 3T3-L1 cells (22Miller C.W. Casimir D.A. Ntambi J.M. The mechanism of inhibition of 3T3-L1 preadipocyte differentiation by prostaglandin F2alpha.Endocrinology. 1996; 137: 5641-5650Crossref PubMed Scopus (99) Google Scholar, 23Casimir D.A. Wilson Miller C. Ntambi J.M. Preadipocyte differentiation blocked by prostaglandin stimulation of prostanoid FP2 receptor in murine 3T3-L1 cells.Differentiation. 1996; 60: 203-210Crossref PubMed Google Scholar, 24Kamon J. Naitoh N. Kitahara M. Tsuruzoe N. Prostaglandin F2alpha enhances glucose consumption through neither adipocyte differentiation nor GLUT1 expression in 3T3-L1 cells.Cell. Signal. 2001; 13: 105-109Crossref PubMed Scopus (10) Google Scholar). PGF2α has been suggested to inhibit differentiation of 3T3-L1 preadipocytes through a PGF2α receptor-mediated increase in intracellular calcium and increased DNA synthesis (22Miller C.W. Casimir D.A. Ntambi J.M. The mechanism of inhibition of 3T3-L1 preadipocyte differentiation by prostaglandin F2alpha.Endocrinology. 1996; 137: 5641-5650Crossref PubMed Scopus (99) Google Scholar) and through activation of mitogen-activated protein kinase (MAPK), resulting in an inhibitory phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ) (25Reginato M.J. Krakow S.L. Bailey S.T. Lazar M.A. Prostaglandins promote and block adipogenesis through opposing effects on peroxisome proliferator-activated receptor gamma.J. Biol. Chem. 1998; 273: 1855-1858Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar).The rate-limiting step in prostaglandin synthesis is catalyzed by COXs comprising the constitutive isoform, COX-1, and the inducible isoform, COX-2. COX-2 has been implicated in the regulation of body fat accumulation, as haplo-insufficient mice are prone to develop obesity (26Fain J.N. Ballou L.R. Bahouth S.W. Obesity is induced in mice heterozygous for cyclooxygenase-2.Prostaglandins Other Lipid Mediat. 2001; 65: 199-209Crossref PubMed Scopus (62) Google Scholar). Recently, 3T3-L1 preadipocytes were shown to express both COX isoforms, and both isoforms were suggested to negatively influence adipocyte differentiation (27Yan H. Kermouni A. Abdel-Hafez M. Lau D.C. Role of cyclooxygenases COX-1 and COX-2 in modulating adipogenesis in 3T3-L1 cells.J. Lipid Res. 2003; 44: 424-429Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar).The present study was undertaken to elucidate the molecular mechanisms by which arachidonic acid prevents adipocyte differentiation of 3T3-L1 preadipocytes and to investigate the role(s) of COX-1 and COX-2 in mediating the inhibitory effect of arachidonic acid. We show that arachidonic acid-mediated inhibition of differentiation requires both COX-1 and COX-2 activity. COX-2 expression was transiently induced upon induction of differentiation, and arachidonic acid treatment led to a sustained expression of COX-2. Addition of arachidonic acid led to a pronounced repression of de novo fatty acid synthesis, diacylglycerol acyltransferase (DGAT) activity, and triacylglycerol accumulation. Interestingly, indomethacin restored DGAT activity and triacylglycerol accumulation without fully restoring de novo fatty acid synthesis, suggesting a prostaglandin-independent repression of de novo fatty acid synthesis. Under these conditions, enhanced incorporation of arachidonic acid into cellular triacylglycerol was observed. We demonstrate that arachidonic acid-mediated inhibition of adipocyte differentiation and sustained expression of COX-2 require the inclusion of a cAMP-elevating agent in the adipogenic cocktail and are dependent on protein kinase A (PKA) and MAPK activity.MATERIALS AND METHODSCell culture and differentiation3T3-L1 cells were cultured to confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) (28Madsen L. Petersen R.K. S⊘rensen M.B. J⊘rgensen C. Hallenborg P. Pridal L. Fleckner J. Amri E-Z. Krieg P. Furstenberger G. Berge R.K. Kristiansen K. Adipocyte differentiation of 3T3-L1 preadipocytes is dependent on lipoxygenase activity during the initial stages of the differentiation process.Biochem. J. 2003; 375: 539-549Crossref PubMed Scopus (114) Google Scholar). Two days postconfluent (designated day 0), cells were induced to differentiate with DMEM supplemented with 10% fetal bovine serum (FBS), 1 μM dexamethasone (DEX) (Sigma), 0.5 mM methylisobutylxanthine (MIX) (Sigma), and 1 μg/ml insulin (Novo Nordisk A/S). Fatty acids (Sigma) and inhibitors were dissolved in DMSO and added when differentiation was induced. Cells not treated with fatty acids or inhibitors received similar volumes of DMSO. After 48 h, the media were replaced with DMEM supplemented with 10% FBS and 1 μg/ml insulin. The cells were subsequently refed every 48 h with DMEM supplemented with 10% FBS.Oil Red O stainingStaining of lipids by Oil Red O was performed as described previously (29Hansen J.B. Petersen R.K. Larsen B. Bartkova J. Alsner J. Kristiansen K. Activation of peroxisome proliferator-activated receptor gamma bypasses the function of the retinoblastoma protein in adipocyte differentiation.J. Biol. Chem. 1999; 274: 2386-2393Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar).Whole-cell extracts and Western blot analysisWhole-cell extracts, electrophoresis, blotting, visualization, and stripping of membranes were performed as described previously (29Hansen J.B. Petersen R.K. Larsen B. Bartkova J. Alsner J. Kristiansen K. Activation of peroxisome proliferator-activated receptor gamma bypasses the function of the retinoblastoma protein in adipocyte differentiation.J. Biol. Chem. 1999; 274: 2386-2393Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The primary antibodies used were rabbit anti-p44/p42 MAPK and mouse anti-phospho-p44/p42 MAPK (Thr-202/Tyr-204) obtained from Cell Signaling Technology; rabbit anti-TFIIB, rabbit anti-PPARγ, rabbit anti-C/EBPβ, goat anti-COX-1, and goat anti-COX-2 antibodies obtained from Santa Cruz Biotechnology; and rabbit anti-ALBP/aP2 kindly provided by D. A. Bernlohr. Secondary antibodies were horseradish peroxidase-conjugated anti-mouse, anti-goat, or anti-rabbit antibodies obtained from DAKO.De novo fatty acid synthesisCells were incubated with [1(2)-14C] acetic acid sodium salt (0.2 μCi/ml medium) (Amersham Pharmacia Biotech) for 4 h, harvested in water, and frozen. Fatty acids were extracted according to the procedure of Folch, Lees, and Sloane-Stanley (30Folch J. Lees M. Sloane-Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) with minor modifications. Cells sonicated in 0.5 ml water were added drop-wise to 2.5 ml methanol with constant shaking, and 1.25 ml chloroform was then added. After shaking (300 rpm, 5 min) and centrifugation (2,000 g, 5 min), the pellet was dissolved in 2.5 ml methanol and 3.75 ml chloroform. After a second shaking (300 rpm, 5 min) and centrifugation (2,000 g, 5 min), the supernatants were collected, and 4 ml water was added. Separation was allowed to proceed overnight at −20°C. After centrifugation (2,000 g, 5 min) at room temperature, the upper phase was removed and the lower phase dried under N2, at 35°C. The lipids were hydrolyzed in 2 ml 0.5 M KOH in ethanol:water (9:1; v/v) for 60 min at 80°C, followed by acidification by 0.35 ml 4 M HCl. The fatty acids were extracted twice with 3 ml hexane, dried under N2, at 35°C, dissolved in 8 ml scintillation liquid, and quantified by scintillation counting.Cellular levels of triacylglycerolsCells grown in 6-well plates were harvested in 1 ml water and frozen. The cells were sonicated, and the cellular levels of triacylglycerols were measured on an AXON Byer spectrophotometer using the TRINDER reaction kit from bioMèrieux.DGAT activityCells grown in 15 cm plates were washed and harvested in 1 ml homogenzation buffer [0.25 M sucrose, 10 mM HEPES (pH 7.4) and 2 mM EDTA] followed by homogenization by repeated forcing of the cell suspensions through a ball bearing homogenizer, and a postnuclear fraction was prepared as described previously (31Tronstad K.J. Berge K. Dyr⊘y E. Madsen L. Berge R.K. Growth reduction in glioma cells after treatment with tetradecylthioacetic acid: changes in fatty acid metabolism and oxidative status.Biochem. Pharmacol. 2001; 61: 639-649Crossref PubMed Scopus (19) Google Scholar). DGAT activity was measured in the postnuclear fractions as originally described by Coleman and Bell (32Coleman R. Bell R.M. Triacylglycerol synthesis in isolated fat cells. Studies on the microsomal diacylglycerol acyltransferase activity using ethanol-dispersed diacylglycerols.J. Biol. Chem. 1976; 251: 4537-4543Abstract Full Text PDF PubMed Google Scholar) with modifications as described by Rustan et al. (33Rustan A.C. Nossen J.Ø. Christiansen E.N. Drevon C.A. Eicosapentaenoic acid reduces hepatic synthesis and secretion of triacylglycerol by decreasing the activity of acyl-coenzyme A:1,2-diacylglycerol acyltransferase.J. Lipid Res. 1988; 29: 1417-1426Abstract Full Text PDF PubMed Google Scholar) using oleoyl-CoA and 1,2-di[1-14C]oleoylglycerol as substrates.Arachidonic acid incorporation into glycerolipidsCells grown in 6-well plates were washed twice with PBS. The cells were subsequently incubated with 2 ml serum-free medium containing 10 μM U-[14C]arachidonic acid for 4 h. The incubations were terminated by cooling on ice, and the medium was subsequently centrifuged at 600 g for 5 min. 14CO2 was trapped from cell-free medium in sealed tubes as described previously (31Tronstad K.J. Berge K. Dyr⊘y E. Madsen L. Berge R.K. Growth reduction in glioma cells after treatment with tetradecylthioacetic acid: changes in fatty acid metabolism and oxidative status.Biochem. Pharmacol. 2001; 61: 639-649Crossref PubMed Scopus (19) Google Scholar). The cells were washed twice with PBS and harvested in 2 ml PBS. The cell suspensions were centrifuged and resuspended in 0.5 ml distilled water and frozen. The lipids were extracted from the cell suspensions (30Folch J. Lees M. Sloane-Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar), dissolved in n-hexane, and separated by TLC on silica gel plates using hexane-diethyl ether-acetic acid (65:35:1; v/v/v). The bands were visualized by iodine vapor, cut into pieces, and assayed for radioactivity by scintillation counting.Transient transfectionThe UASx4-TK-luc reporter, containing four Gal4-responsive elements, was kindly provided by Ronald M. Evans. The pcDNA1-GAL4-mPPARγ(LBD) encoding the Gal4-DBD-mPPARγ-LBD fusion has been described previously(28). The CMV-β-galactosidase plasmid used for normalization was from Clontech. 3T3-L1 cells were plated in 12-well plates 12 h before transfection. One hour prior to transfection, the medium was changed, and each well was transfected with 400 ng UASGALx4-TK-luc reporter, 200 ng Gal4-DBD-mPPARγ-LBD, and 50 ng CMV-β-galactosidase using the calcium-phosphate protocol. Five hours later, fresh medium containing resin-charcoal-stripped 10% FCS and indomethacin dissolved in DMSO, or DMSO alone, was added. After 24 h, the cells were harvested. Luciferase and β-galactosidase activities were measured in a Berthold MicroLumat LB96P luminometer using a commercial kit (Galacto-Light, Applied Biosystems, Foster City, CA). Luciferase values were normalized to β-galactosidase values.Statistical analysis and presentation of dataAll experiments were performed in duplicate or triplicate and were repeated two to five times. The data presented in bars represent means ± standard deviation of triplicates in a representative experiment. Where relevant, a two-tailed Student's t-test was applied for equality of means at the significance level of P < 0.05.RESULTSArachidonic acid-dependent inhibition of adipocyte differentiation requires both COX-1 and COX-2 activityArachidonic acid-mediated inhibition of differentiation of 3T3-L1 cells is reversed by addition of COX inhibitors, suggesting that the inhibitory effect of arachidonic acid is mediated by prostaglandins (23Casimir D.A. Wilson Miller C. Ntambi J.M. Preadipocyte differentiation blocked by prostaglandin stimulation of prostanoid FP2 receptor in murine 3T3-L1 cells.Differentiation. 1996; 60: 203-210Crossref PubMed Google Scholar). Both the constitutively expressed COX-1 and the inducible COX-2 are expressed in undifferentiated 3T3-L1 preadipocytes (27Yan H. Kermouni A. Abdel-Hafez M. Lau D.C. Role of cyclooxygenases COX-1 and COX-2 in modulating adipogenesis in 3T3-L1 cells.J. Lipid Res. 2003; 44: 424-429Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), but the roles of the individual COX isoforms in adipogenesis have not been elucidated.Here we induced 3T3-L1 cells to differentiate by ny linje methylisobutylxanthine, dexamethasone, and insulin (MDI) in the presence of arachidonic acid and the selective COX-1 inhibitors piroxicam and SC-560, the selective COX-2 inhibitor NS-398, as well as the nonselective COX inhibitor indomethacin. Confirming previous results (23Casimir D.A. Wilson Miller C. Ntambi J.M. Preadipocyte differentiation blocked by prostaglandin stimulation of prostanoid FP2 receptor in murine 3T3-L1 cells.Differentiation. 1996; 60: 203-210Crossref PubMed Google Scholar), administration of the nonselective COX inhibitor indomethacin fully rescued adipocyte differentiation of 3T3-L1 cells in the presence of arachidonic acid (Fig. 1A). Surprisingly, however, administration of the selective COX-1 inhibitors piroxicam and SC-560, as well as the selective COX-2 inhibitor NS-398, also rescued differentiation in the presence of arachidonic acid (Fig. 1A). Inhibition of COX activity has been reported to stimulate differentiation of 3T3-L1 cells (3Shillabeer G. Lau D.C.V. Regulation of new fat cell formation in rats: the role of dietary fats.J. Lipid Res. 1994; 35: 592-600Abstract Full Text PDF PubMed Google Scholar, 27Yan H. Kermouni A. Abdel-Hafez M. Lau D.C. Role of cyclooxygenases COX-1 and COX-2 in modulating adipogenesis in 3T3-L1 cells.J. Lipid Res. 2003; 44: 424-429Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 34Williams I.H. Polakis S.E. Differentiation of 3T3-L1 fibroblasts to adipocytes. The effect of indomethacin, prostaglandin E1 and cyclic AMP on the process of differentiation.Biochem. Biophys. Res. Commun. 1977; 77: 175-186Crossref PubMed Scopus (84) Google Scholar, 35Hopkins N.K. Gorman R.R. Regulation of 3T3-L1 fibroblast differentiation by prostacyclin (prostaglandin I2).Biochim. Biophys. Acta. 1981; 663: 457-466Crossref PubMed Scopus (30) Google Scholar), but the biological significance of these observations is obscured by the fact that several COX inhibitors, including indomethacin, are also PPARγ agonists, with reported EC50 values in the micromolar range (36Lehmann J.M. Lenhard J.M. Oliver B.B. Ringold G.M. Kliewer S.A. Peroxisome proliferator-activated receptors alpha and gamma are activated by indomethacin and other non-steroidal anti-inflammatory drugs.J. Biol. Chem. 1997; 272: 3406-3410Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar, 37Jaradat M.S. Wongsud B. Phornchirasilp S. Rangwala S.M. Shams G. Sutton M. Romstedt K.J. Noonan D.J. Feller D.R. Activation of peroxisome proliferator-activated receptor isoforms and inhibition of prostaglandin H(2) synthases by ibuprofen, naproxen, and indomethacin.Biochem. Pharmacol. 2001; 62: 1587-1595Crossref PubMed Scopus (113) Google Scholar). In our cell system, indomethacin at 1 μM did not enhance PPARγ-dependent transactivation (Fig. 1B). Furthermore, the selective inhibitors of COX-1 and -2, piroxicam, SC-560, and NS-398, did not enhance PPARγ-dependent transactivation at the concentrations used in this study (results not shown) and did not affect MDI-induced differentiation (Fig. 1A).The finding that piroxicam, SC-560, and NS-398 rescued adipocyte differentiation in the presence of arachidonic acid suggests that arachidonic acid-dependent inhibition requires both COX-1 and COX-2 activity. To determine whether constitutive COX activity was necessary for expression of one or the other isoform, the expression of COX-1 and COX-2 was determined by Western blotting of 3T3-L1 cells induced to differentiate by MDI in the presence of arachidonic acid and the nonselective COX inhibitor indomethacin. As reported (27Yan H. Kermouni A. Abdel-Hafez M. Lau D.C. Role of cyclooxygenases COX-1 and COX-2 in modulating adipogenesis in 3T3-L1 cells.J. Lipid Res. 2003; 44: 424-429Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), we found that both COX-1 and COX-2 were expressed in undifferentiated 3T3-L1 cells at day 0. In the differentiated state, COX-1 expression was reduced but still clearly detectable, whereas expression of COX-2 was barely discernible in day 8 adipocytes (Fig. 1C). Both COX-1 and COX-2 expression remained high in cells treated with arachidonic acid (Fig. 1C). The sustained expression of COX-1 in arachidonic acid-treated cells is not solely a result of impaired differentiation, inasmuch as COX-1 expression remained high even in the presence of indomethacin (Fig. 1C). Thus, prostaglandin synthesis is not a prerequisite for the sustained COX-1 expression in cells treated with arachidonic acid. In contrast, indomethacin very strongly reduced COX-2 expression in cells treated with arachidonic acid, indicating either that prostaglandins are necessary for maintaining COX-2 expression or that COX-2 expression is downregulated in response to adipocyte differentiation (Fig. 1C).To follow the immediate changes in COX expression upon induction of differentiation, 3T3-L1 cells were induced to differentiate by MDI and cells were harvested at several time points after induction during the first 24 h. Figure 1D shows that COX-2 expression is increased 1.0 h after induction in both the presence and the absence of arachidonic acid. After 12 h, COX-2 expression declined in DMSO-treated cells, but expression was maintained in cells treated with arachidonic acid (Fig. 1D).The finding that both selective COX-1 and selective COX-2 inhibitors rescued arachidonic acid-dependent inhibition of adipocyte differentiation indicates that products from both COX isoforms are necessary for mediating the effect of arachidonic acid. To investigate this possibility, 3T3-L1 cells were induced to differentiate by MDI in the presence of arachidonic acid and the selective COX-1 inhibitors piroxicam and SC-560, the selective COX-2 inhibitor NS-398, as well as the nonselective COX inhibitor indomethacin. The cells were harvested after 3 and 6 h, and the levels of prostaglandins were measured in the supernatants. Interestingly, we found that production of the inhibitory PGE2 in arachidonic acid-treated cells was reduced by both the COX-1 and COX-2 selective inhibitors (results not shown).Arachidonic acid regulates triacylglycerol accumulation at the level of de novo fatty acid synthesis and DGAT activityDuring adipocyte differentiation, triacylglycerol accumulation is dependent on the overall supply of fatty acid from uptake or de novo synthesis and the rate of triacylglycerol synthesis, in which the last and rate-determining step is catalyzed by DGAT1. To determine how arachidonic acid affects these processes quantitatively, we first measured triacylglycerol accumulation in vehicle-treated cells (DMSO) and cells treated with arachidonic acid in the absence or presence of different inhibitors affecting prostaglandin synthesis. Figure 2Ashows that the nonselective COX inhibitor indomethacin, as well as the selective COX-1 and COX-2 inhibitors piroxicam, SC-560, and NS-398, respectively, completely rescued triacylglycerol accumulation of arachidonic acid-treated cells. However, the rate of de novo fatty acid synthesis was only partially restored, reaching ∼50% of the level in cells differentiated in the absence of arachidonic acid (Fig. 2B). This suggests that arachidonic acid reduces the rate of de novo fatty acid synthesis, at least partially, in a prostaglandin-independent manner. We hypothesized that the inhibitory action of arachidonic acid on fatty acid synthesis was related to its polyunsaturated nature, and therefore, we compared the effects of arachidonic acid with those of eicosapentaenoic aci
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