Arachidonic acid and prostacyclin signaling promote adipose tissue development: a human health concern?
2003; Elsevier BV; Volume: 44; Issue: 2 Linguagem: Inglês
10.1194/jlr.m200346-jlr200
ISSN1539-7262
AutoresFlorence Massiéra, Perla Saint-Marc, Josiane Seydoux, Tatsunori Murata, Takuya Kobayashi, Shuh Narumiya, Philippe Guesnet, Ez‐Zoubir Amri, Raymond Négrel, Gérard Ailhaud,
Tópico(s)Adipose Tissue and Metabolism
ResumoHigh fat intake is associated with fat mass gain through fatty acid activation of peroxisome proliferator-activated receptors δ and γ, which promote adipogenesis. We show herein that, compared to a combination of specific agonists to both receptors or to saturated, monounsaturated, and ω-3 polyunsaturated fatty acids, arachidonic acid (C20:4, ω-6) promoted substantially the differentiation of clonal preadipocytes. This effect was blocked by cyclooxygenase inhibitors and mimicked by carbacyclin, suggesting a role for the prostacyclin receptor and activation of the cyclic AMP-dependent pathways that regulate the expression of the CCAAT enhancer binding proteins β and δ implicated in adipogenesis. During the pregnancy-lactation period, mother mice were fed either a high-fat diet rich in linoleic acid, a precursor of arachidonic acid (LO diet), or the same isocaloric diet enriched in linoleic acid and α-linolenic acid (LO/LL diet). Body weight from weaning onwards, fat mass, epididymal fat pad weight, and adipocyte size at 8 weeks of age were higher with LO diet than with LO/LL diet. In contrast, prostacyclin receptor-deficient mice fed either diet were similar in this respect, indicating that the prostacyclin signaling contributes to adipose tissue development.These results raise the issue of the high content of linoleic acid of i) ingested lipids during pregnancy and lactation, and ii) formula milk and infant foods in relation to the epidemic of childhood obesity. High fat intake is associated with fat mass gain through fatty acid activation of peroxisome proliferator-activated receptors δ and γ, which promote adipogenesis. We show herein that, compared to a combination of specific agonists to both receptors or to saturated, monounsaturated, and ω-3 polyunsaturated fatty acids, arachidonic acid (C20:4, ω-6) promoted substantially the differentiation of clonal preadipocytes. This effect was blocked by cyclooxygenase inhibitors and mimicked by carbacyclin, suggesting a role for the prostacyclin receptor and activation of the cyclic AMP-dependent pathways that regulate the expression of the CCAAT enhancer binding proteins β and δ implicated in adipogenesis. During the pregnancy-lactation period, mother mice were fed either a high-fat diet rich in linoleic acid, a precursor of arachidonic acid (LO diet), or the same isocaloric diet enriched in linoleic acid and α-linolenic acid (LO/LL diet). Body weight from weaning onwards, fat mass, epididymal fat pad weight, and adipocyte size at 8 weeks of age were higher with LO diet than with LO/LL diet. In contrast, prostacyclin receptor-deficient mice fed either diet were similar in this respect, indicating that the prostacyclin signaling contributes to adipose tissue development. These results raise the issue of the high content of linoleic acid of i) ingested lipids during pregnancy and lactation, and ii) formula milk and infant foods in relation to the epidemic of childhood obesity. Obesity is associated with metabolic disorders such as dyslipidemia, diabetes, and hypertension, and fat mass excess in severe obesities is typically due to an increase in adipocyte size and number. The formation of adipocytes is a critical event, as mature adipocytes do not divide in vivo and do not undergo significant turnover under physiological conditions. The capacity for proliferation of precursor cells and their differentiation into adipocytes is highest at early age and decrease thereafter in humans and rodents. A limited number of hormones can affect the adipose tissue mass and possibly its distribution (1Ailhaud G. Hauner H. Development of white adipose tissue.in: Bray G. Bouchard C. James P.T. Handbook of Obesity. M. Dekker Inc., New York, NY1997: 359-378Google Scholar). High dietary fat intake is now recognized to be associated with a gain of fat mass in animals and humans at all ages (2Troiano R.P. Briefel R.R. Carrol D.M. Bialostosky K. Energy and fat intakes of children and adolescents in the United States: data from the National Health and Nutrition examination surveys.Am. J. Clin. Nutr. 2000; 72: 1343S-1353SGoogle Scholar, 3Oscai L.B. Brown N.M. Miller W.C. Effect of dietary fat on food intake, growth and body composition in rats.Growth. 1984; 48: 415-424Google Scholar, 4Romieu I. Willett W.C. Stampfer M.J. Colditz G.A. Sampson L. Rosner B. Hennckens C.H. Speizer F.E. Energy intake and other determinants of relative weight.Am. J. Clin. Nutr. 1988; 74: 406-412Google Scholar, 5Nguyen T. Larson D.E. Johnson R.K. Goran M.I. Fat intake and adiposity in children of lean and obese parents.Am. J. Clin. Nutr. 1996; 63: 507-513Google Scholar). However, the lack of evidence of a general increase in energy intake as fat among youths, despite a striking increase in the prevalence of obesity in industrial and developing countries, may be due in part to decreased physical activity and nonexercise activity thermogenesis (6Levine J.A. Eberhardt N.L. Jensen M.D. Role of nonexercise activity thermogenesis in resistance to fat gain in humans.Science. 1999; 283: 212-214Google Scholar), but also to the composition of food intake in early life. The long-term relationship between the fatty acid composition of dietary fats and the development of adipose tissue in humans is difficult to assess in contrast to animals. When mother rats were fed a high-fat diet rich in linoleic acid (C18:2, ω-6) or saturated fatty acids, suckling pups at 17 days of age exhibited hyperplasia or hypertrophy of white adipose tissue, respectively (7Clearly M.P. Philips F.C. Morton A.A. Genotype and diet effects in lean and obese Zucker rats fed either safflower or coconut oil diets.Proc. Soc. Exp. Biol. Med. 1999; 220: 153-161Google Scholar). Moreover, fish oil rich in eicosapentaenoic acid (C20:5, ω-3, EPA) and docosahexaenoic acid (C22:6, ω-3, DHA) prevents obesity in rats (8Parrish C.C. Pathy D.A. Angel A. Dietary fish oils limit adipose tissue hypertrophy in rats.Metabolism. 1990; 39: 217-219Google Scholar, 9Raclot T. Groscolas R. Langin D. Ferré P. Site-specific regulation of gene expression by n-3 polyunsaturated fatty acids in rat white adipose tissues.J. Lipid Res. 1997; 38: 1963-1972Google Scholar), as well as feeding rats after weaning with dietary fats rich in α-linolenic acid (C18:3, ω-3), the precursor of EPA and DHA, prevents excessive growth of adipose tissue (10Okuno M. Kajiwara K. Imai S. Kobayashi T. Honma N. Maki T. Suruga K. Goda T. Takase S. Muto Y. Moriwaki H. Perilla oil prevents the excessive growth of visceral adipose tissue in rats by down-regulating adipocyte differentiation.J. Nutr. 1997; 127: 1752-1757Google Scholar). The mechanisms underlying the differential adipogenic effect of ω-6 versus ω-3 polyunsaturated fatty acids suggest differences between fatty acids and/or fatty acid metabolites in promoting differentiation of adipose precursor cells into adipocytes. In vitro, at the preadipocyte stage, a member of the peroxisome proliferator-activated receptor (PPAR) family, i.e., PPARδ, and two members of the CCAAT-enhancer binding protein family, i.e., C/EBPβ and C/EBPδ, act concomitantly to upregulate the subsequent and critical expression of PPARγ leading to adipogenesis (11Barak Y. Nelson M.C. Ong E.S. Jones Y.Z. Ruiz-Lozano P. Chien K.R. Kader A. Evans R.M. PPARγ is required for placental, cardiac, and adipose tissue development.Mol. Cell. 1999; 4: 585-595Google Scholar, 12Kubota N. Terauchi Y. Miki H. Tamemoto H. Yamauchi T. Komeda K. Satoh S. Nakano R. Ishii C. Sugiyama T. Eto K. Tsubamoto Y. Okuno A. Murakami K. Sekihara H. Hasegawa G. Naito M. Toyoshima Y. Tanaka S. Shiota K. Kitamura T. Fujita T. Ezaki O. Aizawa S. Nagai R. Tobe K. Kimura S. Kadowaki T. PPARγ mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance.Mol. Cell. 1999; 4: 597-609Google Scholar, 13Rosen E.D. Sarraf P. Troy A.E. Bradwin G. Moore K. Milstone D.S. Spiegelman B.M. Mortensen R.M. PPARγ is required for the differentiation of adipose tissue in vivo and in vitro.Cell. 1999; 4: 611-617Google Scholar, 14Ren D. Collingwood T.N. Rebar E.J. Wolffe A.P. Camp H.S. PPARγ knockdown by engineered transcription factors: exogenous PPARγ2 but not PPARγ1 reactivates adipogenesis.Genes Dev. 2002; 16: 27-32Google Scholar, 15Rosen E.D. Hsu C.H. Wang X. Sakai S. Freeman M.W. Gonzalez F.J. Spiegelman B.M. C/EBPα induces adipogenesis through PPARγ: a unified pathway.Genes Dev. 2002; 16: 22-26Google Scholar). Natural long-chain fatty acids act in preadipocytes as adipogenic hormones, participate as transcriptional regulators of the expression of various lipid-related genes, and promote adipogenesis (16Amri E. Ailhaud G. Grimaldi P. Fatty acids as signal transducing molecules: involvement in the differentiation of preadipose to adipose cells.J. Lipid Res. 1994; 35: 930-937Google Scholar). These effects implicate PPARs that bind long-chain fatty acids and fatty acid metabolites (17Xu H.E. Lambert M.H. Montana V.G. Parks D.J. Blanchard S.G. Brown P.J. Sternbach D.D. Lehmann J.M. Wisely G.B. Willson T.M. Kliewer S.A. Milburn M.V. Molecular recognition of fatty acids by peroxisome proliferator-activated receptors.Mol. Cell. 1999; 3: 397-403Google Scholar). Among fatty acids, arachidonic acid (C20:4, ω-6, ARA), a precursor of prostaglandin I2 (prostacyclin), synthesized and released from preadipocytes, has been identified as one of the main adipogenic components of serum. Arachidonic acid induces a rapid cAMP production. Both this effect and its long-term adipogenic effect are impaired by cyclooxygenase inhibitors such as aspirin and indomethacin (18Gaillard D. Négrel R. Lagarde M. Ailhaud G. Requirement and role of arachidonic acid in the differentiation of preadipose cells.Biochem. J. 1989; 257: 389-397Google Scholar). Consistent with an autocrine-paracrine mechanism via released prostacyclin, antibodies directed against this prostanoid and added externally decrease by half the adipogenic effect of arachidonic acid (19Catalioto R.M. Gaillard D. Maclouf J. Ailhaud G. Négrel R. Autocrine control of adipose cell differentiation by prostacyclin and PGF2α.Biochim. Biophys. Acta. 1991; 1091: 364-369Google Scholar). Also consistent with a role of prostacyclin acting as a ligand at the cell surface, it has been shown that i) prostacyclin and its stable analog carbacyclin mimic the effects of arachidonic acid (20Négrel R. Gaillard D. Ailhaud G. Prostacyclin as a potent effector of adipose cell differentiation.Biochem. J. 1989; 257: 399-405Google Scholar) and also promote adipogenesis of clonal mouse preadipocytes and primary preadipocytes from rat and human (21Vassaux G. Gaillard D. Ailhaud G. Négrel R. Prostacyclin is a specific effector of adipose cell differentiation: its dual role as a cAMP- and Ca2+-elevating agent.J. Biol. Chem. 1992; 267: 11092-11097Google Scholar), and ii) prostacyclin binding to its cell surface receptor (IP-R) activates in preadipocytes the protein kinase A (PKA) pathway (21Vassaux G. Gaillard D. Ailhaud G. Négrel R. Prostacyclin is a specific effector of adipose cell differentiation: its dual role as a cAMP- and Ca2+-elevating agent.J. Biol. Chem. 1992; 267: 11092-11097Google Scholar) and upregulates the early expression of the C/EBPβ and C/EBPδ (22Belmonte N. Phillips B.W. Massiéra F. Villageois P. Wdziekonski B. Saint-Marc P. Nichols J. Aubert J. Saeki K. Yuo A. Narumiya S. Ailhaud G. Dani C. Activation of extracellular signal-regulated kinases and CREB/ATF-1 mediate the expression of C/EBPβ and C/EBPδ in preadipocytes.Mol. Endocrinol. 2001; 15: 2037-2049Google Scholar). Circumstantial evidence favors the possibility that prostacyclin also binds like carbacyclin to PPARδ (23Forman B.M. Chen J. Evans R.M. Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors α and δ.Proc. Natl. Acad. Sci. USA. 1997; 94: 4312-4317Google Scholar), and this has been recently supported by studies on stromal cells surrounding the implanting blastocysts (24Lim H. Gupta R.A. Ma W. Paria B.C. Moller D.E. Morrow J.D. DuBois R.N. Trzaskos J.M. Dey S.K. Cyclo-oxygenase-2-derived prostacyclin mediates embryo implantation in the mouse via PPARδ.Genes Dev. 1999; 13: 1561-1574Google Scholar). In vivo, invalidation of C/EBPβ and C/EBPδ genes impairs severely but does not abolish adipose tissue formation (25Tanaka T. Yoshida N. Kishimoto T. Akira S. Defective adipocyte differentiation in mice lacking the C/EBPβ and/or C/EBPδ gene.EMBO J. 1997; 16: 7432-7443Google Scholar), whereas invalidation of PPARδ gene leads to a controversial disproportionate decrease in fat mass (26Peters J.M. Lee S.S.T. Li W. Ward J.M. Gavrilova O. Everett C. Reitman M.L. Hudson L.D. Gonzalez F.J. Growth, adipose, brain and skin alterations resulting from targeted disruption of the mouse peroxisome proliferator-activated receptor β (δ).Mol. Cell. Biol. 2000; 20: 5119-5128Google Scholar, 27Barak Y. Liao D. He W. Ong F.S. Nelson M.C. Olefsky J.M. Boland R. Evans R.M. Effects of peroxisome proliferator-activated receptor delta on placentation, adiposity and colorectal cancer.Proc. Natl. Acad. Sci. USA. 2002; 99: 303-308Google Scholar). This suggests that prostacyclin signaling, arising from arachidonic metabolism, may play a more important adipogenic role through C/EBPβ and C/EBPδ than through PPARδ in up-regulating PPARγ expression. In order to estimate the relative importance of the two regulatory pathways, we have taken advantage of the recent availability of specific PPAR agonists and the generation of prostacyclin receptor-null (ip-r−/−) mice (28Murata T. Ushikubi F. Matsuoka T. Hirata M. Yamasaki A. Sugimoto Y. Ichikawa A. Aze Y. Tanaka T. Yoshida N. Oh-ishi S. Narumiya S. Altered pain perception and inflammatory response in mice lacking prostacyclin receptor.Nature. 1997; 388: 678-682Google Scholar). Our results with wild-type and ip-r−/− mice show that polyunsaturated fatty acids of the ω-6 and ω-3 series are not equipotent in promoting adipogenesis both in vitro and in vivo, and that arachidonic acid and prostacyclin signaling favor this process. In infants, given the relative enrichment of various foods in linoleic acid as precursor of arachidonic acid, its excessive consumption at a time where adipose tissue is in a dynamic phase of its development may favor childhood obesity. The ip-r-null mice were established by gene targeting and backcrossed with C57/BL6J mice for at least 10 generations (28Murata T. Ushikubi F. Matsuoka T. Hirata M. Yamasaki A. Sugimoto Y. Ichikawa A. Aze Y. Tanaka T. Yoshida N. Oh-ishi S. Narumiya S. Altered pain perception and inflammatory response in mice lacking prostacyclin receptor.Nature. 1997; 388: 678-682Google Scholar), then ip-r−/− males and ip-r−/− females were bred to generate further generations. Both ip-r−/− and C57/BL6J control mice were maintained on a light/dark cycle with light from 6 AM to 6 PM at 25°C. The female mice designated to be mothers were fed either a standard diet which consisted (by energy) of 7% fat, 66% carbohydrates, and 27% proteins, or a high-fat diet containing 15% corn oil (LO diet) or a mixture of 10% corn oil and 5% perilla oil (LO/LL diet). Both high-fat diets consisted (by energy) of 40% fat, 35% carbohydrates, and 25% proteins, and were supplemented with 0.04% vitamin C and 0.02% vitamin E (UAR, Carbon Blanc, France). Corn oil contained, expressed in g/100 g of total fatty acids, 13% saturated, 27% monounsaturated, 59% ω-6 polyunsaturated, and 1% ω-3 polyunsaturated fatty acids. The mixture of corn oil and perilla oil contained 10.9% saturated, 22.9% monounsaturated, 44.3% ω-6 polyunsaturated, and 21.9% ω-3 polyunsaturated fatty acids. At 8 weeks of age, female mice fed the same diet since weaning were bred to male mice and maintained throughout mating, pregnancy, and lactation on the same diet. At 18 days of age, male pups were weaned onto the same diets that their mothers had consumed and maintained thereafter. Food intake, body weight, body composition, and cellularity measurements of epididymal fat pad were performed as described previously (29Massiéra F. Seydoux J. Geloen A. Quignard-Boulangé A. Turban S. Saint-Marc P. Fukamizu A. Négrel R. Ailhaud G. Teboul M. Angiotensinogen-deficient mice exhibit impairment of diet-induced weight gain with alteration in adipose tissue development and increased locomotor activity.Endocrinology. 2001; 142: 5220-5225Google Scholar). All experimental animal protocols were performed in accordance with the recommendations of the French Accreditation of Laboratory Animal Care. Embryos from C57 BL/6J wild-type and ip-r−/− mice at 14.5 day postcoitus were used to prepare fibroblasts after removing head, heart, and legs. Mouse embryo fibroblasts were then differentiated into adipocytes as previously described (22Belmonte N. Phillips B.W. Massiéra F. Villageois P. Wdziekonski B. Saint-Marc P. Nichols J. Aubert J. Saeki K. Yuo A. Narumiya S. Ailhaud G. Dani C. Activation of extracellular signal-regulated kinases and CREB/ATF-1 mediate the expression of C/EBPβ and C/EBPδ in preadipocytes.Mol. Endocrinol. 2001; 15: 2037-2049Google Scholar). Stock cultures of Ob1771 cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Cergy-Pontoise, France) supplemented with biotin, pantothenate, antibiotics, and 8% (v/v) fetal bovine serum as previously described (18Gaillard D. Négrel R. Lagarde M. Ailhaud G. Requirement and role of arachidonic acid in the differentiation of preadipose cells.Biochem. J. 1989; 257: 389-397Google Scholar). Experiments were performed after growth and differentiation of confluent cells in serum-free medium as previously described (18Gaillard D. Négrel R. Lagarde M. Ailhaud G. Requirement and role of arachidonic acid in the differentiation of preadipose cells.Biochem. J. 1989; 257: 389-397Google Scholar). Fatty acids, prostaglandins (Cayman Chemicals, Montluçon, France), GW2433, and BRL49653 were dissolved in ethanol and added at a 1:100 dilution into culture media. Ethanol concentration, which did not exceed 1%, had no effect on either adipose conversion or cyclic AMP production. Oil-Red O staining was performed as described previously (18Gaillard D. Négrel R. Lagarde M. Ailhaud G. Requirement and role of arachidonic acid in the differentiation of preadipose cells.Biochem. J. 1989; 257: 389-397Google Scholar). Glycerol-3-phosphate dehydrogenase assays were carried out in duplicate at day 7 after confluence (18Gaillard D. Négrel R. Lagarde M. Ailhaud G. Requirement and role of arachidonic acid in the differentiation of preadipose cells.Biochem. J. 1989; 257: 389-397Google Scholar). Intracellular cyclic AMP was determined with a commercial kit by radioimmunoassay, according to the manufacturer’s instructions (Amersham). Before assays, cyclic AMP was extracted with 1.2 ml of ice-cold ethanol-5 mM-EDTA (2:1, v/v). After scraping the cell monolayer and centrifugation, 1 ml of the supernatant was dried in a Speed-Vac evaporator. Duplicate samples were solubilized before assays in 150 μl of 50 mM Tris-HCl buffer, pH 7.5, containing 4 mM EDTA and assayed at least in duplicate. Statistical comparisons were performed on absolute values by Fisher's PLSD using the STATVIEW software package. We performed experiments in confluent preadipocytes exposed for 7 days to serum-free medium in the absence (Fig. 1A)or the presence of various effectors. Differentiation was enhanced in cells exposed to the naturally abundant ω-6 ARA (Fig. 1B) compared with ω-3 ARA, which is only present at trace amounts in dietary fat sources (Fig. 1C). Differentiation in the presence of ω-6 ARA was severely impaired when aspirin was included as a cyclooxygenase inhibitor (Fig. 1D). Another polyunsaturated fatty acid of the ω-3 series, DHA, behaved similarly to ω-3 ARA (Fig. 1E). In agreement with our previous observations (20Négrel R. Gaillard D. Ailhaud G. Prostacyclin as a potent effector of adipose cell differentiation.Biochem. J. 1989; 257: 399-405Google Scholar), carbacyclin, a stable prostacyclin analog that binds to the cell surface prostacyclin receptor IP-R, was highly adipogenic (Fig. 1F), exhibiting greater activity than GW2433, a specific PPARδ agonist (17Xu H.E. Lambert M.H. Montana V.G. Parks D.J. Blanchard S.G. Brown P.J. Sternbach D.D. Lehmann J.M. Wisely G.B. Willson T.M. Kliewer S.A. Milburn M.V. Molecular recognition of fatty acids by peroxisome proliferator-activated receptors.Mol. Cell. 1999; 3: 397-403Google Scholar) (Fig. 1G), or a combination of GW2433 and BRL49663, a specific PPARγ agonist (30Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ).J. Biol. Chem. 1995; 270: 12953-12956Google Scholar) (Fig. 1H). In order to delineate at which step ω-6 ARA was active in the differentiation process, we first studied adipose conversion in the presence of maximally effective concentrations of PPARδ and/or PPARγ agonist using glycerol-3-phosphate dehydrogenase activity, an adipocyte indicator. The sequential addition of PPAR agonists was based upon several considerations. i) Early markers are expressed in PPARγ−/− embryonic stem cells that can be committed into the adipose lineage, i.e., lipoprotein lipase and PPARδ but not the adipocyte lipid binding protein (ALBP or aP2) (31Vernochet C. Milstone D.S. Iehlé C. Belmonte N. Phillips B. Wdziekonski B. Villageois P. Amri E.Z. E. O’Donnell P. Mortensen R.M. Ailhaud G. Dani C. PPARγ-dependent and PPARγ-independent effects on the development of adipose cells from embryonic stem cells.FEBS Lett. 2002; 510: 94-98Google Scholar); ii) PPARδ is expressed at near maximal levels in early confluent Ob1771 cells, in contrast to PPARγ, which is highly expressed later during adipogenesis (32Amri E.Z. Bonino F. Ailhaud G. Abumrad N. Grimaldi P.A. Cloning of a protein that mediates transcriptional effects of fatty acids in preadipocytes. Homology to peroxisome proliferator-activated receptors.J. Biol. Chem. 1995; 270: 2367-2371Google Scholar); and iii) activation of PPARδ by activators-ligands is required to upregulate the expression of PPARγ (33Bastié C. Luquet S. Holst D. Jehl-Pietri C. Grimaldi P. Expression of peroxisome proliferator-activated receptor PPARδ promotes induction of PPARγ and adipocyte differentiation in 3T3C2 fibroblasts.J. Biol. Chem. 1999; 274: 21920-21925Google Scholar, 34Hansen J.B. Zhang H. Rasmussen T.H. Petersen R.K. Flindt E.N. Kristiansen K. Peroxisome proliferator-activated receptor δ (PPARδ)-mediated regulation of preadipocyte proliferation and gene expression is dependent on cAMP signaling.J. Biol. Chem. 2001; 276: 3175-3182Google Scholar). Consistent with these observations, addition of the PPARδ agonist from day 0 to day 3, followed by its removal and addition of the PPARγ agonist from day 3 to day 7, proved to be optimal. Adipogenesis was similar to that observed in the presence of both agonists between day 0 and day 7. In contrast, discontinuous or continuous exposure to either agonist for 7 days was less efficient in promoting adipogenesis (Table 1, part A). Of note, when the PPARγ agonist was present from day 3 to day 7, the adipogenic potency of ω-6 ARA added during the first 3 days was 3-fold higher than that of the PPARδ agonist and this effect was severely reduced after cyclooxygenase inhibition (Table 1, part A). In contrast to ω-6 ARA and carbacyclin, the adipogenic potencies of ω-3 ARA and PPARδ agonist were similar. Interestingly, in the presence of the PPARδ agonist between day 0 and day 3, ω-6 and ω-3 ARA exhibited an adipogenic potency similar to that of the PPARγ agonist (Table 1, part A). Altogether, these results show that the substantial effect of ω-6 ARA takes place at early step(s) of the differentiation process, in accordance with the fact that we did not observe prostacyclin synthesis and response through IP-R in differentiating, PPARγ-expressing cells (21Vassaux G. Gaillard D. Ailhaud G. Négrel R. Prostacyclin is a specific effector of adipose cell differentiation: its dual role as a cAMP- and Ca2+-elevating agent.J. Biol. Chem. 1992; 267: 11092-11097Google Scholar).TABLE 1Comparative stimulation of adipogenesis by PPAR agonists and long-chain fatty acidsAddition from Day 0 to Day 3Addition from Day 3 to Day 7GPDH Activity (Fold Increase)Part ANoneNone1.0a30 ± 3 mU/mg of protein.Noneγ agonist2.4 ± 0.6δ agonistNone3.6 ± 0.8bP < 0.05 versus untreated cells.δ agonistγ agonist5.7 ± 1.6cP < 0.01 versus untreated cells.δ agonistδ agonist2.1 ± 1.0γ agonistγ agonist5.2 ± 1.0cP < 0.01 versus untreated cells.δ + γ agonistsδ + γ agonists6.6 ± 2.1cP < 0.01 versus untreated cells.5 μM ω-6 ARAγ agonist9.3 ± 2.1cP < 0.01 versus untreated cells.10 μM ω-6 ARAγ agonist17.5 ± 6.2cP < 0.01 versus untreated cells.5 μM ω-6 ARA + 100 μM aspirinγ agonist4.4 ± 1.4bP < 0.05 versus untreated cells.5 μM ω-3 ARAγ agonist2.6 ± 0.7bP < 0.05 versus untreated cells.10 μM ω-3 ARAγ agonist5.5 ± 1.2cP < 0.01 versus untreated cells.5 μM ω-3 ARA + 100 μM aspirinγ agonist1.5 ± 0.3δ agonist5 μM ω−6 ARA4.9 ± 2.3bP < 0.05 versus untreated cells.δ agonist5 μM ω−3 ARA5.8 ± 1.2cP < 0.01 versus untreated cells.Carbacyclinγ agonist17.3 ± 3.0cP < 0.01 versus untreated cells.Part B5 μM Palmitic acidγ agonist1.9 ± 0.610 μM Palmitic acidγ agonist2.3 ± 0.5bP < 0.05 versus untreated cells.5 μM Palmitoleic acidγ agonist3.0 ± 0.6bP < 0.05 versus untreated cells.10 μM Palmitoleic acidγ agonist3.5 ± 0.8bP < 0.05 versus untreated cells.5 μM Oleic acidγ agonist2.7 ± 0.6bP < 0.05 versus untreated cells.10 μM Oleic acidγ agonist2.2 ± 0.4bP < 0.05 versus untreated cells.5 μM EPAd10 μM EPA proved to be cytotoxic on a long-term basis.γ agonist3.5 ± 0.9bP < 0.05 versus untreated cells.5 μM DHAγ agonist4.1 ± 1.210 μM DHAγ agonist5.5 ± 0.9bP < 0.05 versus untreated cells.ARA, arachidonic acid; δ agonist, GW2433 (1 μM); γ agonist, BRL49653 (0.5 μM); carbacyclin (0.2 μM). Values are expressed as mean ± SEM of experiments performed on 3–10 independent series of cells.a 30 ± 3 mU/mg of protein.b P < 0.05 versus untreated cells.c P < 0.01 versus untreated cells.d 10 μM EPA proved to be cytotoxic on a long-term basis. Open table in a new tab ARA, arachidonic acid; δ agonist, GW2433 (1 μM); γ agonist, BRL49653 (0.5 μM); carbacyclin (0.2 μM). Values are expressed as mean ± SEM of experiments performed on 3–10 independent series of cells. In order to gain further insights into the role of IP-R, adipogenesis of mouse embryo fibroblasts from wild-type and ip-r−/− mice (22Belmonte N. Phillips B.W. Massiéra F. Villageois P. Wdziekonski B. Saint-Marc P. Nichols J. Aubert J. Saeki K. Yuo A. Narumiya S. Ailhaud G. Dani C. Activation of extracellular signal-regulated kinases and CREB/ATF-1 mediate the expression of C/EBPβ and C/EBPδ in preadipocytes.Mol. Endocrinol. 2001; 15: 2037-2049Google Scholar) was compared upon stimulation by a combination of BMY45778, a specific agonist of IP-R like carbacyclin but unable to activate PPARδ (35Aubert J. Saint-Marc P. Belmonte N. Dani C. Négrel R. Ailhaud G. Prostacyclin IP receptor up-regulates the early expression of C/EBPβ and C/EBPδ in preadipose cells.Mol. Cell. Endocrinol. 2000; 160: 149-156Google Scholar), and the PPARγ agonist. Adipogenesis was decreased 2-fold in ip-r−/− fibroblasts compared with that of wild-type fibroblasts while exposure to a combination of BMY45778 and PPARγ agonist did not enhance adipogenesis above that observed with the PPARγ agonist alone (data not shown). As no expression of C/EBPβ and C/EBPδ was observed in ip-r−/− mouse embryo fibroblasts in response to carbacyclin (22Belmonte N. Phillips B.W. Massiéra F. Villageois P. Wdziekonski B. Saint-Marc P. Nichols J. Aubert J. Saeki K. Yuo A. Narumiya S. Ailhaud G. Dani C. Activation of extracellular signal-regulated kinases and CREB/ATF-1 mediate the expression of C/EBPβ and C/EBPδ in preadipocytes.Mol. Endocrinol. 2001; 15: 2037-2049Google Scholar), it appears that the remarkable adipogenic potency of carbacyclin was likely due to its dual role, first as a ligand of IP-R and activation of the PKA pathway leading in turn to C/EBPβ and C/EBPδ expression and up-regulation of PPARγ expression (36Wu Z. Bucher N.L. Farmer S.R. Induction of peroxisome proliferator-activated receptor gamma during the conversion of 3T3 fibroblasts into adipocytes is mediated by C/EBPbeta, C/EBPdelta and glucocorticoids.Mol. Cell. Biol. 1996; 16: 4128-4136Google Scholar), and second as a ligand of PPARδ. The potency of various long-chain fatty acids to stimulate early events of differentiation was next examined in preadipocytes exposed subsequently to the PPARγ agonist (Table 1, part B). A saturated fatty acid (palmitate) or monounsaturated fatty acids (palmitoleate and oleate) was poorly adipogenic compared with ω-6 ARA. Furthermore, two ω-3 polyunsaturated fatty acids, EPA and DHA, were also poorly adipogenic. The higher adipogenic activity of ω-6 ARA compared with other fatty acids cannot be ascribed to its higher affinity for PPARδ, as the latter binds arachidonic acid, saturated, monounsaturated, and ω-3 polyunsaturated fatty acids with similar affinity (17Xu H.E. Lambert M.H. Montana V.G. Parks D.J. Blanchard S.G. Brown P.J. Sternbach D.D. Lehmann J.M. Wisely G.B. Willson T.M. Kliewer S.A. Milburn M.V. Molecular recognition of fatty acids by peroxisome proliferator-activated receptors.Mol. Cell. 1999; 3: 397-403Google Scholar). As ω-6 ARA was unique among natural fatty acids in promoting extensive adipocyte differentiation, we compared cyclic AMP production after a very short exposure of preadipocytes to concentrations of the various long-chain fatty acids (Table 2). ω-6 ARA increased cyclic AMP production by 15-fold in 5 min in confluent preadipocytes, and this effect was abolished by indo
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