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Arachidonic acid and prostacyclin signaling promote adipose tissue development: a human health concern?

Open AccessPublished:November 04, 2002DOI:https://doi.org/10.1194/jlr.M200346-JLR200
      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 (
      • Ailhaud G.
      • Hauner H.
      Development of white adipose tissue.
      ). High dietary fat intake is now recognized to be associated with a gain of fat mass in animals and humans at all ages (
      • Troiano R.P.
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      Energy and fat intakes of children and adolescents in the United States: data from the National Health and Nutrition examination surveys.
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      ). 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 (
      • Levine J.A.
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      Role of nonexercise activity thermogenesis in resistance to fat gain in humans.
      ), 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 (
      • Clearly M.P.
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      • Morton A.A.
      Genotype and diet effects in lean and obese Zucker rats fed either safflower or coconut oil diets.
      ). Moreover, fish oil rich in eicosapentaenoic acid (C20:5, ω-3, EPA) and docosahexaenoic acid (C22:6, ω-3, DHA) prevents obesity in rats (
      • Parrish C.C.
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      • Angel A.
      Dietary fish oils limit adipose tissue hypertrophy in rats.
      ,
      • Raclot T.
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      • Ferré P.
      Site-specific regulation of gene expression by n-3 polyunsaturated fatty acids in rat white adipose tissues.
      ), 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 (
      • Okuno 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.
      ).
      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 (
      • Barak 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.
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      • Naito M.
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      • 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.
      ,
      • Rosen 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.
      ,
      • Ren 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.
      ,
      • Rosen 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.
      ). 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 (
      • Amri E.
      • Ailhaud G.
      • Grimaldi P.
      Fatty acids as signal transducing molecules: involvement in the differentiation of preadipose to adipose cells.
      ). These effects implicate PPARs that bind long-chain fatty acids and fatty acid metabolites (
      • Xu 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.
      ). 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 (
      • Gaillard D.
      • Négrel R.
      • Lagarde M.
      • Ailhaud G.
      Requirement and role of arachidonic acid in the differentiation of preadipose cells.
      ). 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 (
      • Catalioto R.M.
      • Gaillard D.
      • Maclouf J.
      • Ailhaud G.
      • Négrel R.
      Autocrine control of adipose cell differentiation by prostacyclin and PGF.
      ). 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 (
      • Négrel R.
      • Gaillard D.
      • Ailhaud G.
      Prostacyclin as a potent effector of adipose cell differentiation.
      ) and also promote adipogenesis of clonal mouse preadipocytes and primary preadipocytes from rat and human (
      • Vassaux 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.
      ), and ii) prostacyclin binding to its cell surface receptor (IP-R) activates in preadipocytes the protein kinase A (PKA) pathway (
      • Vassaux 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.
      ) and upregulates the early expression of the C/EBPβ and C/EBPδ (
      • Belmonte 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.
      ). Circumstantial evidence favors the possibility that prostacyclin also binds like carbacyclin to PPARδ (
      • Forman B.M.
      • Chen J.
      • Evans R.M.
      Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors α and δ.
      ), and this has been recently supported by studies on stromal cells surrounding the implanting blastocysts (
      • Lim 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.
      ).
      In vivo, invalidation of C/EBPβ and C/EBPδ genes impairs severely but does not abolish adipose tissue formation (
      • Tanaka T.
      • Yoshida N.
      • Kishimoto T.
      • Akira S.
      Defective adipocyte differentiation in mice lacking the C/EBPβ and/or C/EBPδ gene.
      ), whereas invalidation of PPARδ gene leads to a controversial disproportionate decrease in fat mass (
      • Peters 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).
      ,
      • Barak 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.
      ). 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 (
      • Murata 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.
      ).
      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.

      MATERIALS AND METHODS

      Mice

      The ip-r-null mice were established by gene targeting and backcrossed with C57/BL6J mice for at least 10 generations (
      • Murata 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.
      ), 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 (
      • Massiéra 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.
      ). All experimental animal protocols were performed in accordance with the recommendations of the French Accreditation of Laboratory Animal Care.

      Fibroblast culture

      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 (
      • Belmonte 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.
      ).

      Preadipocyte culture

      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 (
      • Gaillard D.
      • Négrel R.
      • Lagarde M.
      • Ailhaud G.
      Requirement and role of arachidonic acid in the differentiation of preadipose cells.
      ). Experiments were performed after growth and differentiation of confluent cells in serum-free medium as previously described (
      • Gaillard D.
      • Négrel R.
      • Lagarde M.
      • Ailhaud G.
      Requirement and role of arachidonic acid in the differentiation of preadipose cells.
      ). 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 (
      • Gaillard D.
      • Négrel R.
      • Lagarde M.
      • Ailhaud G.
      Requirement and role of arachidonic acid in the differentiation of preadipose cells.
      ).

      Biochemical assays

      Glycerol-3-phosphate dehydrogenase assays were carried out in duplicate at day 7 after confluence (
      • Gaillard D.
      • Négrel R.
      • Lagarde M.
      • Ailhaud G.
      Requirement and role of arachidonic acid in the differentiation of preadipose cells.
      ). 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 analysis

      Statistical comparisons were performed on absolute values by Fisher's PLSD using the STATVIEW software package.

      RESULTS

      ω-6 ARA but not ω-3 ARA promotes adipogenesis

      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 (
      • Négrel R.
      • Gaillard D.
      • Ailhaud G.
      Prostacyclin as a potent effector of adipose cell differentiation.
      ), 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 (
      • Xu 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.
      ) (Fig. 1G), or a combination of GW2433 and BRL49663, a specific PPARγ agonist (
      • Lehmann 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).
      ) (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.
      Figure thumbnail gr1
      Fig. 1Photomicrographs of confluent Ob1771 preadipocytes maintained for 7 days and stained with Oil Red-O in serum-free medium (A) or in serum-free medium supplemented with 10 μM ω-6 arachidonic acid (ARA) (B), 10 μM ω-3 ARA (C), 10 μM ω-6 ARA and 100 μM aspirin (D), 10 μM docosahexaenoic acid (DHA) (E), 1 μM carbacyclin (F), 1 μM GW2433 as peroxisome proliferator-activated receptor (PPAR)δ agonist (G), 1 μM GW2433, and 0.5 μM of BRL49653 as PPARγ agonist (H). Magnification, 600-fold.
      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) (
      • Vernochet 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.
      ); ii) PPARδ is expressed at near maximal levels in early confluent Ob1771 cells, in contrast to PPARγ, which is highly expressed later during adipogenesis (
      • Amri 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.
      ); and iii) activation of PPARδ by activators-ligands is required to upregulate the expression of PPARγ (
      • Bastié 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.
      ,
      • Hansen 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.
      ).
      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 (
      • Vassaux 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.
      ).
      TABLE 1Comparative stimulation of adipogenesis by PPAR agonists and long-chain fatty acids
      Addition from Day 0 to Day 3Addition from Day 3 to Day 7GPDH Activity (Fold Increase)
      Part A
      NoneNone1.0
      30 ± 3 mU/mg of protein.
      Noneγ agonist2.4 ± 0.6
      δ agonistNone3.6 ± 0.8
      P < 0.05 versus untreated cells.
      δ agonistγ agonist5.7 ± 1.6
      P < 0.01 versus untreated cells.
      δ agonistδ agonist2.1 ± 1.0
      γ agonistγ agonist5.2 ± 1.0
      P < 0.01 versus untreated cells.
      δ + γ agonistsδ + γ agonists6.6 ± 2.1
      P < 0.01 versus untreated cells.
      5 μM ω-6 ARAγ agonist9.3 ± 2.1
      P < 0.01 versus untreated cells.
      10 μM ω-6 ARAγ agonist17.5 ± 6.2
      P < 0.01 versus untreated cells.
      5 μM ω-6 ARA + 100 μM aspirinγ agonist4.4 ± 1.4
      P < 0.05 versus untreated cells.
      5 μM ω-3 ARAγ agonist2.6 ± 0.7
      P < 0.05 versus untreated cells.
      10 μM ω-3 ARAγ agonist5.5 ± 1.2
      P < 0.01 versus untreated cells.
      5 μM ω-3 ARA + 100 μM aspirinγ agonist1.5 ± 0.3
      δ agonist5 μM ω−6 ARA4.9 ± 2.3
      P < 0.05 versus untreated cells.
      δ agonist5 μM ω−3 ARA5.8 ± 1.2
      P < 0.01 versus untreated cells.
      Carbacyclinγ agonist17.3 ± 3.0
      P < 0.01 versus untreated cells.
      Part B
      5 μM Palmitic acidγ agonist1.9 ± 0.6
      10 μM Palmitic acidγ agonist2.3 ± 0.5
      P < 0.05 versus untreated cells.
      5 μM Palmitoleic acidγ agonist3.0 ± 0.6
      P < 0.05 versus untreated cells.
      10 μM Palmitoleic acidγ agonist3.5 ± 0.8
      P < 0.05 versus untreated cells.
      5 μM Oleic acidγ agonist2.7 ± 0.6
      P < 0.05 versus untreated cells.
      10 μM Oleic acidγ agonist2.2 ± 0.4
      P < 0.05 versus untreated cells.
      5 μM EPA
      10 μM EPA proved to be cytotoxic on a long-term basis.
      γ agonist3.5 ± 0.9
      P < 0.05 versus untreated cells.
      5 μM DHAγ agonist4.1 ± 1.2
      10 μM DHAγ agonist5.5 ± 0.9
      P < 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.
      In order to gain further insights into the role of IP-R, adipogenesis of mouse embryo fibroblasts from wild-type and ip-r−/− mice (
      • Belmonte 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.
      ) was compared upon stimulation by a combination of BMY45778, a specific agonist of IP-R like carbacyclin but unable to activate PPARδ (
      • Aubert 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.
      ), 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 (
      • Belmonte 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.
      ), 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 (
      • Wu 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.
      ), and second as a ligand of PPARδ.

      Only ω-6 ARA promotes extensive adipogenesis and cyclic AMP production

      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 (
      • Xu 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.
      ). 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 indomethacin, another cyclooxygenase inhibitor. Carbacyclin increased cyclic AMP production by 13-fold and, as anticipated, indomethacin had no effect. Compared with control cells, PKA activity was increased 3- and 4-fold in the presence of ω-6 ARA (10 μM) and carbacyclin (1 μM), respectively (data not shown). ω-3 ARA, EPA, DHA, palmitic acid, palmitoleic acid, oleic acid, PPARδ agonist, or PPARγ agonist had no effect on cyclic AMP production. In order to investigate why ω-3 polyunsaturated fatty acids were not potent adipogenic agents, we attempted to inhibit PPARδ and/or PPARγ activity in the presence of ω-3 ARA. Adipogenesis was unaffected compared with that observed in the presence of PPAR agonists only, excluding PPARs as possible targets of ω-3 ARA (data not shown). Thus, we investigated in preadipocytes the possible effect on cyclic AMP production of ω-3 polyunsaturated fatty acids that have been reported to inhibit the cyclic AMP-dependent PKA (
      • Mirnikjoo B.
      • Brown S.E.
      • Seung Kim H.F.
      • Marangell L.B.
      • Sweatt J.D.
      • Weeber E.J.
      Protein kinase inhibition by ω-3 fatty acids.
      ). In the presence of ω-6 ARA (5 μM), addition of ω-3 ARA, EPA, or DHA (10 μM each) inhibited cyclic AMP production by 51%, 91%, and 9% respectively, whereas, in the presence of ω-6 ARA (10 μM), a 33% inhibition of this production was observed by addition of EPA (10 μM) (not shown). These results suggest that ω-3 polyunsaturated fatty acids inhibit in preadipocytes the cyclic AMP-signaling pathways triggered by arachidonic acid at level(s) upstream of PKA.
      TABLE 2Comparative cyclic AMP production by arachidonic acid and various effectors of adipogenesis
      AdditioncAMP Production
      Values are expressed as mean of duplicate values representative of experiments performed on three independent series of cells. Fatty acids and indomethacin (added 15 min prior to fatty acid) were present at 10 μM; δ agonist, GW 2433 (1 μM); γ agonist, BRL 49653 (0.5 μM); carbacyclin (0.2 μM).
      pmol/5 min per well
      None1.0
      ω-6 ARA16.2
      ω-6 ARA + indomethacin1.5
      ω-3 ARA1.8
      ω-3 ARA + indomethacin1.6
      δ agonist1.8
      γ agonist1.2
      Carbacyclin13.9
      Carbacyclin + indomethacin11.0
      Palmitic acid1.9
      Oleic acid1.4
      EPA3.1
      DHA0.2
      a Values are expressed as mean of duplicate values representative of experiments performed on three independent series of cells. Fatty acids and indomethacin (added 15 min prior to fatty acid) were present at 10 μM; δ agonist, GW 2433 (1 μM); γ agonist, BRL 49653 (0.5 μM); carbacyclin (0.2 μM).

      In vivo ω-6 polyunsaturated fatty acids are more adipogenic than ω-3 polyunsaturated fatty acids

      To address whether the fatty acid composition of high-fat diets promotes adipose tissue development, we carried out nutritional studies in wild-type and ip-r−/− mice. We chose a corn oil-supplemented diet rich in linoleic acid, a long-known essential fatty acid precursor of arachidonic acid (LO diet) compared with an isocaloric diet enriched with a mixture of corn oil and perilla oil rich in α-linolenic acid, another essential fatty acid precursor of EPA and DHA (LO/LL diet). The female mice designated to be mothers were fed diets containing either LO diet or LO/LL diet from 4 weeks of age. Four weeks later, these mice were bred to male mice and remained on the same diet. Pups were kept with their mothers until weaning and then maintained on the same respective diets.
      From weaning to 22 weeks of age, body weight of wild-type mice fed LO diet was higher than that of animals fed LO/LL diet and this difference persisted to a large extent at the adult age (Fig. 2)despite a similar food intake (Table 3). Strikingly, the body weight of ip-r−/− mice on either type of diets was similar. As the amount of food intake was also similar despite differences in caloric intake, this suggests increased thermogenesis in ip-r−/− mice fed a high-fat diet. Moreover, body weight of ip-r−/− mice was indistinguishable when fed LO diet or LO/LL diet, demonstrating the critical role of ω-6 polyunsaturated fatty acids in body weight gain during pregnancy and/or the suckling period. No difference was observed in body length between wild-type and ip-r−/− mice fed either type of diets. To evaluate adiposity, we measured total fat mass and epididymal fat pad weight of wild-type and ip-r−/− mice at 8 weeks of age (Table 3). Fat mass of wild-type mice fed standard diet or LO/LL diet was identical, whereas that of animals fed LO diet was increased. In contrast, fat mass was identical in ip-r−/− mice fed either diet. Consistent with these observations, epididymal fat pad weight of wild-type mice was significantly higher in mice fed LO diet than in mice fed LO/LL diet or standard diet. Again, there was no significant difference in the epididymal fat pad weights of ip-r−/− mice fed either type of diets. Adipocyte size of epididymal fat pad was increased 1.9-fold in wild-type mice fed LO diet compared with the two other diets, and this was accompanied by a decrease of adipocyte number (Fig. 3). Compared with wild-type mice, adipocyte size was decreased and adipocyte number was increased in ip-r−/− mice fed standard diet, yet there was no difference in adipocyte size and number of the epididymal fat pad of ip-r−/− mice fed either diet (Fig. 3). Surprisingly, when wild-type mother mice were fed a standard diet and pups were fed after weaning a LO or LO/LL diet, body weight of animals fed LO diet was not significantly higher than that of those fed LO/LL diet, emphasizing the importance of linoleic acid-enriched diet during the pregnancy-lactation period.
      Figure thumbnail gr2
      Fig. 2Body weight of wild-type (A) and ip-r−/− male mice (B). During the pregnancy-lactation period, mother mice were fed a standard diet (diamond), a corn oil-enriched diet (triangle), or a mixture of corn oil and perilla oil-enriched diet (square). From weaning onward, male pups were maintained on the same diet than their mothers had consumed. (n = 20), *P < 0.05 versus wild-type mice fed a standard diet.
      TABLE 3Food intake, fat mass, and epididymal fat pad weight in 8-week-old wild type and ip-r−/− mice fed either a standard diet (Chow) or a high-fat diet enriched with linoleic acid (LO) or enriched with linoleic acid and α-linolenic acids (LO/LL)
      Wild-Typeip-r−/−
      Chow DietLO DietLO/LL DietChow DietLO DietLO/LL Diet
      Food intake   (g/day) (n = 4)4.7 ± 0.54.5 ± 0.64.7 ± 0.64.8 ± 0.94.4 ± 0.44.8 ± 0.6
      Total fat mass   (g) (n = 4)2.3 ± 0.12.7 ± 0.1
      P < 0.08 versus Chow-fed.
      2.3 ± 0.11.8 ± 0.11.6 ± 0.11.8 ± 0.1
      Epididymal fat pads   (mg) (n = 6)307.6 ± 7.5396 ± 15.1
      P < 0.01 versus Chow-fed.
      282 ± 18.2214.5 ± 7.2209.8 ± 3.7227.4 ± 9.8
      a P < 0.08 versus Chow-fed.
      b P < 0.01 versus Chow-fed.
      Figure thumbnail gr3
      Fig. 3Adipocyte number and size in epididymal fat pad of 8-week-old male wild-type and ip-r−/− mice (n = 3 in each group) fed after weaning to a standard diet (SD), LO diet, or LO/LL diet. Values are expressed as mean ± SEM; * P < 0.05 versus wild-type fed a standard diet. O, P < 0.05 versus wild-type fed LO diet; X, P < 0.01 versus wild-type fed LO/LL diet.

      DISCUSSION

      The development of adipose tissue depends on the existence of redundant pathways that ultimately upregulate the expression of PPARγ. This expression relies predominantly on the expression of C/EBPβ and C/EBPδ (
      • Wu 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.
      ) and disruption of both genes severely impairs the formation of adipose tissue (
      • Tanaka T.
      • Yoshida N.
      • Kishimoto T.
      • Akira S.
      Defective adipocyte differentiation in mice lacking the C/EBPβ and/or C/EBPδ gene.
      ). We had identified cues that trigger the expression of C/EBPβ and C/EBPδ in preadipocytes, i.e., leukemia inhibitory factor and its cognate cell surface receptor activating the ERK pathway (
      • Aubert J.
      • Dessolin S.
      • Belmonte N.
      • Li M.
      • McKenzie F.R.
      • Staccini L.
      • Villageois P.
      • Barhanin B.
      • Vernallis A.
      • Smith A.G.
      • Ailhaud G.
      • Dani C.
      Leukemia inhibitory factor and its receptor promote adipocyte differentiation via the mitogen-activated protein kinase cascade.
      ) and the prostacyclin/IP-R system activating the PKA pathway (
      • Belmonte 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.
      ). Our present results show that polyunsaturated fatty acids of the ω-6 series are more adipogenic both in vitro and in vivo compared with their ω-3 counterparts. On one hand, ω-6 ARA is unique via prostacyclin in activating the PKA pathway. On the other hand, ω-3 polyunsaturated fatty acids do not affect this pathway as well as prostaglandin I3, a product synthesized from EPA (not shown). Figure 4summarizes our views on redundant pathways and on the role of arachidonic acid and other long-chain fatty acids in promoting adipogenesis.
      Figure thumbnail gr4
      Fig. 4Redundant pathways and dietary long chain-fatty acids (LCFA) implicated in adipogenesis. This scheme assumes that three ligand/cell surface receptor systems concur to upregulate the expression of C/EBPβ and C/EBPδ, i.e., prostacyclin/IP-R (
      • Belmonte 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.
      ,
      • Raz A.
      • Kamin-Belsky N.
      • Przedecki F.
      • Obukowicz M.G.
      Fish oil inhibits Δ6 desaturase activity in vivo: utility in a dietary paradigm to obtain mice depleted of arachidonic acid.
      ), leukemia inhibitory factor (LIF) receptor (
      • Belmonte 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.
      ,
      • Raz A.
      • Kamin-Belsky N.
      • Przedecki F.
      • Obukowicz M.G.
      Fish oil inhibits Δ6 desaturase activity in vivo: utility in a dietary paradigm to obtain mice depleted of arachidonic acid.
      ), and fibroblast growth factor (FGF)-10 receptor (
      • Sakaue H.
      • Konishi M.
      • Ogawa W.
      • Asaki T.
      • Mori T.
      • Yamasaki M.
      • Takata M.
      • Ueno H.
      • Kato S.
      • Kasuga M.
      • Itoh N.
      Requirement of fibroblast growth factor 10 in development of white adipose tissue.
      ), and to promote adipogenesis. Dietary linoleic acid and/or arachidonic acid favor in preadipocytes prostacyclin synthesis and release. Thus, arachidonic acid via prostacyclin triggers a key event, plays a unique role in activating the protein kinase A pathway by means of IP-R, and enhances the differentiation process. Other dietary LCFA behave as mere activators/ligands of PPARδ and subsequently of PPARγ. Upon terminal differentiation, leukemia inhibitory factor and fibroblast growth factor-10 are no longer produced. Production of prostacyclin and other prostaglandins ceases and is accompanied by reduced expression and loss of functional IP (
      • Vassaux 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.
      ,
      • Börglum J.
      • Richelsen B.
      • Darimont C.
      • Pedersen S.B.
      • Négrel R.
      Expression of the two isoforms of prostaglandin endoperoxide synthase (PGHS-1 and PGHS-2) during adipose cell differentiation.
      ,
      • Vassaux G.
      • Gaillard D.
      • Darimont C.
      • Ailhaud G.
      • Négrel R.
      Differential response of preadipocytes and adipocytes to PGI2 and PGE2: physiological implications.
      ). Therefore, in adipocytes, PPARγ and possibly PPARδ then act as the molecular sensors of all dietary LCFA at a time where endogenous LCFA synthesis (not shown) becomes significant (
      • Ibrahimi A.
      • Teboul L.
      • Gaillard D.
      • Amri E.
      • Ailhaud G.
      • Young P.
      • Cawthorne M.
      • Grimaldi P.
      Evidence for common mechanism of action for fatty acids and thiazolidinedione antidiabetic agents on gene expression in preadipose cells.
      ). Epidermal (keratinocyte) fatty acid binding protein in preadipocytes and adipocytes and adipocyte lipid binding protein in adipocytes are assumed to bind and transport LCFA (
      • Ibrahimi A.
      • Teboul L.
      • Gaillard D.
      • Amri E.
      • Ailhaud G.
      • Young P.
      • Cawthorne M.
      • Grimaldi P.
      Evidence for common mechanism of action for fatty acids and thiazolidinedione antidiabetic agents on gene expression in preadipose cells.
      ,
      • Shaughnessy S.
      • Smith E.R.
      • Kodukula S.
      • Storch J.
      • Fried S.K.
      Adipocyte metabolism in adipocyte fatty acid binding protein knockout (aP2−/−) mice after short-term high-fat feeding: functional compensation by keratinocyte fatty acid binding protein.
      ).
      In light of our results from in vitro studies reported herein, a high-fat diet rich in linoleic acid could stimulate arachidonic acid formation and, through prostacyclin synthesis, activate cyclic AMP-dependent signaling pathways in preadipocytes. This would favor the formation of mature adipocytes whose lipid filling is then increased to cope with the high exogenous levels of fatty acids. In contrast, in wild-type mice fed a high-fat diet containing a mixture of linoleic and α-linolenic acids, the lowered production of cyclic AMP may limit the formation of adipocytes, leading to hyperplasia to accommodate the fatty acid supply. This alteration of cyclic AMP production could be due to a decreased arachidonic acid synthesis from linoleic acid through inhibition of Δ6 desaturase activity by α-linolenic acid and its metabolites (
      • Raz A.
      • Kamin-Belsky N.
      • Przedecki F.
      • Obukowicz M.G.
      Fish oil inhibits Δ6 desaturase activity in vivo: utility in a dietary paradigm to obtain mice depleted of arachidonic acid.
      ). In ip-r−/− mice, no activation of the PKA pathway occurs through the prostacyclin receptor, and the adipogenic effect of ω-6 and ω-3 polyunsaturated fatty acids is similar. Despite prostacyclin synthesis in ip-r−/− mice, no difference in adipose tissue mass was observed between mice fed LO or LO/LL diet, suggesting that the activation of PPARδ by prostacyclin, if any, had no significant impact on the overall differentiation process. Our finding that inclusion of α-linolenic acid in an isocaloric diet rich in linoleic acid prevents the enhancement of fat mass is consistent with our in vitro observations. Varying the proportion of these essential fatty acids thus should alter the proportion of arachidonic acid versus EPA and DHA (
      • Raz A.
      • Kamin-Belsky N.
      • Przedecki F.
      • Obukowicz M.G.
      Fish oil inhibits Δ6 desaturase activity in vivo: utility in a dietary paradigm to obtain mice depleted of arachidonic acid.
      ) and could lead to changes in the pattern of adipose tissue development which occurs during pregnancy and the suckling period.
      Remarkably, although no difference in the litter size was observed in wild-type mice fed either diet, pups from mother mice fed LO diet were, at weaning, 50% heavier than those from mice fed LO/LL diet. This raises an important issue in humans, as adipose tissue is being formed during the third trimester of gestation and sensitive periods of its development take place before 1 year of age (
      • Ailhaud G.
      • Hauner H.
      Development of white adipose tissue.
      ). In this respect, the overweight prevalence among US children of 6 to 11 months of age increased in boys from 4.0% in 1976 through 1980 to 7.5% in 1988 though 1994, and in girls from 6.2% to 10.8% during the same periods of time (
      • Ogden C.L.
      • Troiano R.P.
      • Briefel R.R.
      • Kuczmarski R.J.
      • Flegal K.M.
      • Johnson C.L.
      Prevalence of overweight among preschool children in the United States, 1971 through 1994.
      ). It is of interest to observe that the content of linoleic acid of mature breast milk of US women, a reflection of their fat intake, has increased from 5% to 17% between 1945 and 1995 (r = 0.85, P < 0.001, n = 29). Furthermore, despite the fact that the ratio of linoleic acid to α-linolenic acid in mature breast milk of US and European women is similar, it is also interesting to note that the ratio of arachidonic acid to DHA is 1.8-fold higher in the milk of US women due to its low DHA content (
      • Jensen R.G.
      Fatty acids and related compounds.
      ,
      • Jensen R.G.
      The lipids in human milk.
      ,
      • Jensen R.G.
      Lipids in human milk.
      ). It has been reported that breast feeding may help decrease the prevalence of overweight and obesity in childhood (
      • Von Kries R.
      • Koletzko B.
      • Sauerwald T.
      • von Mutius E.
      • Barnert D.
      • Grunert V.
      • von Voss H.
      Breast feeding and obesity: cross sectional study.
      ,
      • Gillman M.W.
      • Rifas-Shiman S.L.
      • Camargo Jr., C.A.
      • Berkey C.S.
      • Frazier A.L.
      • Rockett H.R.H.
      • Field A.E.
      • Colditz G.A.
      Risk of overweight among adolescents who were breastfed as infants.
      ). Although the enhanced fatness was attributed to the greater energy intake of formula-fed infants (
      • Stunkard A.J.
      • Berkowitz R.I.
      • Stallings V.A.
      • Schoeller D.A.
      Energy intake, not energy output, is a determinant of body size in infants.
      ), the fatty acid composition should be considered, as the percentage of linoleic acid is increased by approximately 50% in infant formula compared with breast milk (
      • Guesnet P.
      • Pugo-Gunsam P.
      • Maurage C.
      • Pinault M.
      • Giraudeau B.
      • Alessandri J-M.
      • Durand G.
      • Antoine J-M.
      • Couet C.
      Blood lipid concentrations of docosahexaenoic and arachidonic acids at birth determine their relative postnal changes in term infants fed breast milk or formula.
      ). We suggest that, at a very early age where energy expenditure appears rather similar between individuals, large amounts of linoleic acid consumed during pregnancy, suckling period, and early infancy are important determinants of physiological events implicated at a time when adipose tissue is in a dynamic phase of its development, and that could lead to childhood obesity.

      Acknowledgments

      This study was supported by grants from Institut Français pour la Nutrition (F.M.) and CERIN (G.A.), by a special grant from the Bristol Myers Squibb Foundation (G.A.), and by a grant from the Swiss National Science Foundation (no. 31-57-129.99 to J.S.). Special thanks are due to G. Oillaux for skillfull secretarial assistance, to C. Vernochet for drawings, and to Dr. R. Arkowitz for critical review of the manuscript. GW2433 and BRL49653 were kind gifts of S. Michel and U. Reichert (Galderma, Sophia Antipolis, France), and BMY 45778 was a kind gift of N. Meanwell (Bristol-Myers Squibb). The authors gratefully acknowledge the generous gift of perilla oil from Dr. Kajiwava (Ajimoto Co., Inc., Suzuki-cho, Japan).

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