A Western-like fat diet is sufficient to induce a gradual enhancement in fat mass over generations[S]

      The prevalence of obesity has steadily increased over the last few decades. During this time, populations of industrialized countries have been exposed to diets rich in fat with a high content of linoleic acid and a low content of α-linolenic acid compared with recommended intake. To assess the contribution of dietary fatty acids, male and female mice fed a high-fat diet (35% energy as fat, linoleic acid:α-linolenic acid ratio of 28) were mated randomly and maintained after breeding on the same diet for successive generations. Offspring showed, over four generations, a gradual enhancement in fat mass due to combined hyperplasia and hypertrophy with no change in food intake. Transgenerational alterations in adipokine levels were accompanied by hyperinsulinemia. Gene expression analyses of the stromal vascular fraction of adipose tissue, over generations, revealed discrete and steady changes in certain important players, such as CSF3 and Nocturnin. Thus, under conditions of genome stability and with no change in the regimen over four generations, we show that a Western-like fat diet induces a gradual fat mass enhancement, in accordance with the increasing prevalence of obesity observed in humans.
      The prevalence of obesity and the risk of developing associated diseases have steadily increased across generations over the last few decades. In addition, these events now emerge earlier in life. This epidemic is not attributable to genetic factors as it has occurred relatively recently and is observed in a wide range of human populations. High-fat diets are considered to be obesogenic in that they produce a consistent increase in fat mass that is directly related to the content of the diet and duration of feeding. However, the contribution of dietary fats compared with an excess energy intake in increasing body weight remains controversial, as no major change in the total amount of ingested fats has occurred in the last two decades (
      • Troiano R.P.
      • Briefel R.R.
      • Carroll M.D.
      • Bialostosky K.
      Energy and fat intakes of children and adolescents in the united states: data from the national health and nutrition examination surveys.
      ,
      • Donahoo W.
      • Wyatt H.R.
      • Kriehn J.
      • Stuht J.
      • Dong F.
      • Hosokawa P.
      • Grunwald G.K.
      • Johnson S.L.
      • Peters J.C.
      • Hill J.O.
      Dietary fat increases energy intake across the range of typical consumption in the United States.
      ).
      In addition to caloric excess, a qualitative issue has emerged as a risk factor for obesity in rodents and possibly in humans; i.e., the disequilibrium in polyunsaturated fatty acid (PUFA) metabolism with a high ratio of linoleic acid (C18:2 ω6, LA) versus α-linolenic acid (C18:3 ω3, LNA) (
      • Ailhaud G.
      • Guesnet P.
      • Cunnane S.C.
      An emerging risk factor for obesity: does disequilibrium of polyunsaturated fatty acid metabolism contribute to excessive adipose tissue development?.
      ). Notably, in rodents, reducing this ratio from 59 to 2 under isolipidic, isoenergetic conditions (40% energy as fat) by inclusion of dietary LNA counteracted the enhancing effects of LA on body weight and fat mass, which then became similar to that observed with a chow diet (
      • Massiera F.
      • Saint-Marc P.
      • Seydoux J.
      • Murata T.
      • Kobayashi T.
      • Narumiya S.
      • Guesnet P.
      • Amri E.Z.
      • Negrel R.
      • Ailhaud G.
      Arachidonic acid and prostacyclin signaling promote adipose tissue development: a human health concern?.
      ). ω6 PUFAs were more potent than ω3 PUFAs in promoting adipogenesis (
      • Gaillard D.
      • Negrel R.
      • Lagarde M.
      • Ailhaud G.
      Requirement and role of arachidonic acid in the differentiation of pre-adipose cells.
      ,
      • Vassaux G.
      • Gaillard D.
      • Ailhaud G.
      • Negrel R.
      Prostacyclin is a specific effector of adipose cell differentiation. Its dual role as a cAMP- and Ca(2+)-elevating agent.
      ,
      • Kim H.K.
      • Della-Fera M.
      • Lin J.
      • Baile C.A.
      Docosahexaenoic acid inhibits adipocyte differentiation and induces apoptosis in 3T3–L1 preadipocytes.
      ). When combined with high carbohydrate content, a linoleic acid-enriched diet was found to be pro-adipogenic in vivo through cAMP-dependent signaling (
      • Madsen L.
      • Pedersen L.M.
      • Liaset B.
      • Ma T.
      • Petersen R.K.
      • van den Berg S.
      • Pan J.
      • Muller-Decker K.
      • Dulsner E.D.
      • Kleemann R.
      • et al.
      cAMP-dependent signaling regulates the adipogenic effect of n-6 polyunsaturated fatty acids.
      ). LA acts through arachidonic acid (C20:4 ω6, ARA) and prostacyclin, as pups from mice invalidated for the prostacyclin receptor (IP-R) and fed a LA-rich diet exhibit reduced body weight and fat mass compared with wild-type mice fed the same diet (
      • Massiera F.
      • Saint-Marc P.
      • Seydoux J.
      • Murata T.
      • Kobayashi T.
      • Narumiya S.
      • Guesnet P.
      • Amri E.Z.
      • Negrel R.
      • Ailhaud G.
      Arachidonic acid and prostacyclin signaling promote adipose tissue development: a human health concern?.
      ). Overall, these results emphasize the importance of adipose tissue development in rodents of a high-fat diet combined with a high LA:LNA ratio. Therefore, by analogy to humans, where consumers have been continuously exposed—from in utero to old age—to dietary fats with a high LA content and a low LNA content and in which the prevalence of overweight and obesity has increased within a few generations (
      • Ailhaud G.
      • Massiera F.
      • Weill P.
      • Legrand P.
      • Alessandri J.M.
      • Guesnet P.
      Temporal changes in dietary fats: role of n-6 polyunsaturated fatty acids in excessive adipose tissue development and relationship to obesity.
      ), we decided to set up a nutritional model mimicking a human situation. For this purpose, male and female mice were chronically exposed over four generations to a single Western-like fat diet; i.e., 35% energy as fat with a LA/LNA ratio similar to that found in the most consumed foods. The results show that this condition was sufficient to trigger gradual transgenerational enhancement of the fat mass observed at early and adult ages. In addition to changes in insulin and adipokine circulating levels and to changes in cellularity of adipose tissue, gene expression profiling over generations was used to highlight the major molecular events favoring adipose tissue hyperplasia and metabolic imprinting that lead to an obese phenotype.

      MATERIALS AND METHODS

       Transgenerational mice and diets

      A colony of pure inbred C57BL6/J mice was established by mating four males and four females from the same litter (Charles River, France) that was fed a chow diet. At weaning, pups were either maintained on a chow diet (STD mice) or fed a high-fat diet (ω6HFD) (HF mice). Both diets contained 21.4% proteins, 3.9% fiber, and 5.7% minerals and ash. The fatty acid composition of the ω6HFD (35% energy as fat) and chow diet is detailed in supplementary Table I. The ω6HFD contained a 3.7-fold higher amount of LA (7.9 g per 100 g versus 2.2 g per 100 g) but the same amount of LNA (0.24–0.26 g/100 g) as the chow diet. As shown in Table 1, male and female mice were first fed ω6HFD at weaning and later mated to generate HF0 mice, whereas HF0–HF4 male and female mice were fed ω6HFD. In this way, HF0–HF4 mice were continuously exposed over generations to the isocaloric, isolipidic diet. The whole population of male and female HF0 adults was mated randomly at 10 weeks of age to give HF1, the first generation (Table 1). Among several possibilities, pups could have been derived from a low body fat gainer male crossed with a low body fat gainer female, or from a high body fat gainer male crossed with a high body fat gainer female. Many additional combinations were also possible. Random mating was chosen on purpose to exclude any selection process. After lactation by HF0 dams, HF1 pups were fed at weaning on ω6HFD and mated randomly as described above to generate HF2 mice. The same protocol was used to establish generations HF3 and HF4. Reversion experiments were performed by switching HF1, HF3, and HF4 pups to a chow diet at weaning; these mice were respectively termed revHF1, revHF3, and revHF4 mice. Adult male and female revHF4 mice then fed a chow diet were mated randomly to give birth to the fifth generation of pups that at weaning were fed either the chow diet (termed std5 mice) or ω6HFD (termed hf5 mice) (Table 1). Mice were housed five per cage and body weight was measured weekly for each group of mice from birth to 22 weeks of age. All other measurements were performed with adult mice, i.e., from 8 to 22 weeks of age. At 8 weeks of age, food intake was measured daily for one week. Plasma extraction and adipose tissue dissection were performed as described previously (
      • Massiera F.
      • Seydoux J.
      • Geloen A.
      • Quignard-Boulange A.
      • Turban S.
      • Saint-Marc P.
      • Fukamizu A.
      • Negrel 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 and European Accreditation of Laboratory Animal Care and were approved by the local experimentation committee.
      TABLE 1Experimental protocol to obtain generations of chow diet (STD)- and ω6HFD-fed mice
      STDHF0HF1HF2HF3HF4revHF1revHF3revHF4hf5std5
      Number of generations on a ω6HFD00123413444
      Diet of parents before and during breeding, pregnancy, and lactationChowChowHFDHFDHFDHFDHFDHFDHFDChowChow
      Diet after weaningChowHFDHFDHFDHFDHFDChowChowChowHFDChow
      Abbreviations: HF0–HF4, HFD-fed mice; hf5, ω6HFD-fed fifth-generation offspring of revHF4; HFD, high-fat diet (ω6HFD); revHF1, HF1 pups fed STD diet at weaning; revHF3, HF3 pups fed STD diet at weaning; revHF4, HF4 pups fed STD diet at weaning; STD, chow diet-fed mice; std5, STD-fed fifth-generation offspring of revHF4.

       Lipid analyses of plasma and adipose tissue

      After a 12 h fast, blood was collected from 14- and 22-week-old mice (n = 4). Plasma and adipose tissue were analyzed for the fatty acid content by direct transesterification as described (
      • Lepage G.
      • Roy C.C.
      Specific methylation of plasma nonesterified fatty acids in a one-step reaction.
      ).

       Breast milk

      Indirect measurement was performed by collecting the stomach content of breast-fed HF1 pups (n = 4 for each group). Total lipids of the stomach content were extracted in the presence of 0.02% butylhydroxytoluene before performing gas chromatography (
      • Lepage G.
      • Roy C.C.
      Specific methylation of plasma nonesterified fatty acids in a one-step reaction.
      ).

       Metabolic parameters

      Blood was collected in 22-week-old mice (n ≥ 4) by heart puncture after a 14 h fast. Then plasma leptin, insulin, total adiponectin, interleukin (IL)-6, tumor necrosis factor (TNF)-α, resistin, plasminogen activator inhibitor (PAI)-1, and monocyte chemotactic protein (MCP)-1 concentrations were assessed with a Lincoplex assay (Millipore, St Quentin en Yvelines, France).

       Morphometric analysis of isolated adipocytes

      Epididymal fat pads from 10-week-old mice (n ≥ 3) were isolated and weighed before cell dissociation for 30 min with collagenase type IV (Sigma Chem.). After filtration (250 μm), the infranatant was removed by catheter aspiration. In each condition, three pictures of isolated adipocytes per mouse were taken with a light microscope. Measurement of the cell diameter and number was performed as described previously (
      • Massiera F.
      • Seydoux J.
      • Geloen A.
      • Quignard-Boulange A.
      • Turban S.
      • Saint-Marc P.
      • Fukamizu A.
      • Negrel 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.
      ).

       RNA extraction

      Total RNA was extracted from stromal vascular fraction (SVF) cells prepared from periepididymal adipose tissues of 10-week-old mice (n ≥ 3 per group) using TRI-REAGENT (Euromedex, Souffelweyersheim, France) according to the manufacturer's instructions, and then used for cDNA microarray and quantitative PCR.

       cDNA microarray

      Biological experiments used for expression profiling were independently performed twice. Hybridization was dye balanced to reduce the effects of using two distinct dyes, Cy3 and Cy5. The quality of total RNA was controlled on an Agilent Bioanalyzer 2100 as previously described (
      • Moreilhon C.
      • Gras D.
      • Hologne C.
      • Bajolet O.
      • Cottrez F.
      • Magnone V.
      • Merten M.
      • Groux H.
      • Puchelle E.
      • Barbry P.
      Live Staphylococcus aureus and bacterial soluble factors induce different transcriptional responses in human airway cells.
      ). Pangenomic microarrays were printed using the mouse RNG/MRC oligonucleotide collection (corresponding to 25,299 distinct probes) as previously described (http://www.microarray.fr/). Experimental data and associated microarray designs have been deposited in the National Center for Biotechnology Information (NBCI) Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under series GSE16613 and GSE16636, and under platform record GPL1456. All calculations were performed with the Bioconductor packages (
      • Gentleman R.C.
      • Carey V.J.
      • Bates D.M.
      • Bolstad B.
      • Dettling M.
      • Dudoit S.
      • Ellis B.
      • Gautier L.
      • Ge Y.
      • Gentry J.
      • et al.
      Bioconductor: open software development for computational biology and bioinformatics.
      ) limma (
      • Wettenhall J.M.
      • Smyth G.K.
      limmaGUI: a graphical user interface for linear modeling of microarray data.
      ) and topGO (
      • Alexa A.
      • Rahnenfuhrer J.
      • Lengauer T.
      Improved scoring of functional groups from gene expression data by decorrelating GO graph structure.
      ). Differentially expressed genes were selected using a Benjamini-Hochberg correction of the P value for multiple tests, based on a P value of 0.05 or below. Additional parameters corresponded to an average log2(Signal) above 6, a log2(Fold change) above 0.7 (absolute value) in one of the comparisons.

       Quantitative PCR

      Reverse transcriptase reactions and quantitative RT-PCR assays were performed as described (
      • Zaragosi L.E.
      • Ailhaud G.
      • Dani C.
      Autocrine fibroblast growth factor 2 signaling is critical for self-renewal of human multipotent adipose-derived stem cells.
      ). The expression of selected genes was normalized to that of 18S rRNAs and quantified using the comparative-ΔCt method. Oligonucleotide sequences, designed using Primer Express software (PerkinElmer Life Sciences, Courtaboeuf, France), are available upon request.

       Statistical analysis

      Data are presented as the mean ± SEM. Measurements were compared via the two-tailed t-test or Kruskal and Wallis nonparametric rank-sum test. Body weight curves were compared by ANOVA analysis for repeated measures using XLstat software, followed by either Bonferroni (for pairwise comparison) or Dunnet (for comparison to STD) post hoc analysis. Microarray experiments were analyzed using the limma package from Bioconductor. The scripts used for the analysis are available in the supplementary data.

      RESULTS

       Body weight and fat mass across generations

      As shown in Table 1 and supplementary Table I, four male and four female C57BL/6J mice from the same litter were mated randomly. At weaning, their pups were fed an LA-enriched diet [termed ω6 high-fat diet (ω6HFD)], which contained 35% energy as fat with a LA:LNA ratio ∼28 (see supplementary data). From weaning up to 8 weeks old, body weight was not affected by the diet. However, when the adult male and female HF0 mice on the ω6HFD were mated randomly and produced HF1 pups, the body weight of the male mice at weaning became significantly higher (10.9 ± 0.2 versus 9.6 ± 0.6 g, P < 0.05; n ≥ 6 for each generation) than that of the HF0 mice; this weight difference persisted at the adult age. Suckling HF1 pups from both sexes were fed the ω6HFD at weaning and then mated as above. The difference in body weight both at weaning and at 8 weeks was further increased in HF2 male pups and adults compared with HF1 mice, although no significant weight difference persisted between HF1 and HF2 mice at the adult age (supplementary Fig. IA).
      Regarding fat mass, a large difference in the weight of the epididymal fat pad was already observed across generations at 8 weeks for HF0, HF1, and HF2 mice (Fig. 1A; n ≥ 6 for each generation). This difference became more evident when comparing HF0 and HF2 mice at 14 (Fig. 1B) and 22 weeks (Fig. 1C), consistent with the obvious difference in the morphological appearance of STD and HF4 mice at 22 weeks (Fig. 1D). Most importantly, the number of pups per litter was not statistically different: 5.5 ± 0.6, 5.6 ± 0.5, 5.0 ± 0.4, 4.5 ± 0.5, and 4.7 ± 0.4 for STD/HF0, HF1, HF2, HF3, and HF4 mice, respectively. This finding showed that the ω6HFD had no impact upon reproduction, thus excluding the possibility that litter size could explain an indirect effect of the diet across generations by increasing the energy intake of suckling pups. When assessed at 8 weeks, no significant difference in the caloric intake could be observed among STD, HF0, HF1, HF2, and HF4 mice (n ≥ 5; supplementary Table II). Moreover, when HF4 pups were transferred at weaning to a chow diet (Table 1), the food intake of revHF4 mice (n = 3) remained similar to that of HF4 and previous generations. Thus, these results exclude the possibility that changes in food and fat intake—across generations early and late in life—explain the transgenerational increase in body weight and fat mass observed at the adult age, suggesting that other mechanisms are implicated. Interestingly, body weight at birth appeared to decrease across generations, but this trend was not statistically significant (supplementary Fig. IB). The major effect of the ω6HFD on body weight of STD/HF0 versus HF3 mice took place between the second and third week, suggesting a similar differential effect on fat mass that was not accurately measurable at this very early age (supplementary Fig. IC).
      Figure thumbnail gr1
      Fig. 1(A–C) Epididymal fat mass as a function of age. Data are means ± SEM. P < 0.05 versus STD; ∗∗P < 0.01 versus HF0; n > 6 for each generation. (D) Morphology of representative male STD after four generations and HF4 mice at 22 weeks old. STD, chow diet-fed mice; HF0–HF4, HFD-fed mice; HFD, high-fat diet (ω6HFD).

       Lipid profile across generations

      PUFA metabolism in the mothers' milk lipids was altered in response to the linoleic acid-enriched diet but then remained similar across generations. The ω6HFD altered the PUFA composition of milk lipids of HF0 dams. It strongly increased the content of LA and to a lesser extent that of ARA (supplementary Fig. IIA, B; n = 4 for each group). In contrast, it decreased significantly the content of long-chain polyunsaturated fatty acids (LC-PUFA) of the ω3 series, i.e., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), thus increasing the ARA:DHA and ARA:DHA+EPA ratio from 1.3 to 2.2 and from 1.0 to 1.5, respectively (supplementary Fig. IIA, C). As for mothers' milk, the ω6HFD induced an increase in the ω6 content (LA and ARA) and a decrease in LNA, EPA, and DHA in plasma and adipose tissue lipids at 14 weeks (Fig. 2 and supplementary Table III). All these observations are in agreement with human studies showing that an increase in LA intake leads to stimulation of ARA and/or inhibition of EPA and DHA synthesis (
      • 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 postnatal changes in term infants fed breast milk or formula.
      ). The PUFA profile was not different between HF0 and HF4 mice (n = 4), showing that no further changes occurred once STD-fed mice had been switched to the ω6HFD (Fig. 2). Taken together, these results show that the PUFA profile was altered by the diet fat content and that a steady state was then observed across generations. As expected, changes in the PUFA content induced by the ω6HFD were reversed in a single generation of mice fed the chow diet (revHF4; n = 3). Palmitoleate (C16:1 ω7) has recently been reported to be an insulin-sensitizing, adipose tissue-derived hormone that improves glucose metabolism (
      • Cao H.
      • Gerhold K.
      • Mayers J.R.
      • Wiest M.M.
      • Watkins S.M.
      • Hotamisligil G.S.
      Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism.
      ). Palmitoleate levels were regulated in plasma and adipose tissue with a 2- to 3-fold reduction upon initial ω6HFD exposure, but no further change occurred across generations (supplementary Table III). The decrease in the ω3 LC-PUFA status in mice fed the ω6HFD had no impact on the DHA concentration in brain phospholipids (supplementary Table III). This finding excludes an essential fatty acid deficiency arising across generations, which would have altered various physiological functions and subsequently affected white adipose tissue (WAT) development.
      Figure thumbnail gr2
      Fig. 2ARA and DHA content of plasma and adipose tissue lipids across generations (see supplementary Table III for details). Significant difference versus STD at P < 0.05; ¤No significant difference versus HF0. ARA, arachidonic acid; DHA, docosahexaenoic acid; STD, chow diet-fed mice; HF0–HF4, HFD-fed mice; HFD, high-fat diet (ω6HFD).

       The adipose phenotype can be partially reversed at later generations

      To assess whether further transgenerational effects of the ω6HFD could be reversed, HF4 mice were switched at weaning to the chow diet. In plasma, the percentage of ω6 PUFAs of revHF4 mice was also similar at 14 weeks old to that of STD mice. The ARA:EPA+DHA ratio increased more than 2-fold in HF4 mice and reversed completely in revHF4 mice. A reversible pattern was also observed for the ω6 and ω3 PUFA composition of adipose tissue lipids (Fig. 2 and supplementary Table III). Under these conditions, the body weight of revHF4 mice returned 5 weeks later to that of HF0 mice (Fig. 3A; n ≥ 4). Importantly, a similar but incomplete reversal was observed for the epididymal fatpad weight (Fig. 3B; n ≥ 6). Therefore adult male and female revHF4 mice were subsequently mated randomly to give birth to the 5th generation of pups (Table 1). When the litters were fed at weaning either the chow diet (std5 mice) or the ω6HFD (hf5 mice; n = 5 for each group), not only were std5 mice heavier at weaning than STD mice, but a large difference in the rate of weight gain could be observed in hf5 mice compared with std5, as early as the first week after weaning and maintained thereafter (Fig. 3C). It is striking that such a difference was not observed for HF0 mice for which the body weight was similar to that of STD mice from weaning until 8 weeks (supplementary Fig. IA). The incomplete reversal of the adipose phenotype at later generations suggested that some transgenerational memory had been acquired, allowing revHF4 mice to respond more rapidly than STD mice to the ω6HFD. This point was further examined at the physiological and cellular levels.
      Figure thumbnail gr3
      Fig. 3Reversion of the adipose phenotype. Body (A) and fat pad weight (B) at 8 weeks old. Data are means ± SEM. P < 0.05 versus STD; ∗∗P < 0.01 versus HF0. Differential response of std5 and hf5 weaned pups in response to the ω6HFD (C). For each generation, n ≥ 4, (A); n ≥ 6 (B); and n = 5 (C). Data are means ± SEM. P < 0.05 for hf5 versus std5/HF0. HF0–HF4, HFD-fed mice; HFD, high-fat diet (ω6HFD); hf5, ω6HFD-fed fifth-generation offspring of revHF4; revHF4, HF4 pups fed STD diet at weaning; STD, chow diet-fed mice; std5, STD-fed fifth-generation offspring of revHF4.

       Metabolic changes in plasma across generations

      Compared with mice fed a chow diet, those fed the ω6HFD for 19 weeks after weaning exhibited an increase in the plasma level of most parameters traditionally associated with the metabolic syndrome (i.e., TNFα, resistin, insulin, leptin, and MCP-1) in the first, second, and third generations, while adiponectin and IL-6 levels remained rather constant. Surprisingly, this inflammatory signature was almost reversed at the fourth generation. TNFα, resistin, IL-6, MCP-1, and leptin levels became dramatically reduced compared with those observed in HF3 mice and became similar to those of STD mice (Fig. 4; n ≥ 4). Fasting insulin levels followed a pattern similar to that of cytokines but remained significantly higher than those of HF0 mice. These observations indicate that, despite the fact that glycemia in HF4 mice appeared normal at 22 weeks old (151 ± 30 mg/dl for HF4 versus 170 ± 30 mg/dl for STD mice), continuous exposure to the ω6HFD led to a sustained increase in plasma insulin levels, which strongly suggests the emergence of insulin resistance of adult animals at later generations.
      Figure thumbnail gr4
      Fig. 4Changes in insulin and adipokine circulating levels across generations at 22 weeks. Values are means ± SEM. P < 0.05 versus STD; ∗∗P < 0.01 versus STD; ¤P < 0.05 versus HF0; ¤¤P < 0.05 versus HF1; ¤¤¤P < 0.05 versus HF3 (n ≥ 4). HFD, high-fat diet (ω6HFD); HF0–HF4, HFD-fed mice; IL, interleukin; MCP, monocyte chemotactic protein; PAI, plasminogen activator inhibitor; STD, chow diet-fed mice; TNF, tumor necrosis factor.

       Cellularity and cell subpopulations of adipose tissue across generations

      Exposing pups at weaning to the ω6HFD led within seven weeks to changes in adipose tissue cellularity (Fig. 5; n ≥ 3). HF0 mice exhibited an increase in the percentage of very small adipocytes (∼20 μm) and a slight increase in that of large adipocytes (40–70 μm). This finding suggests recruitment of adipocyte precursors from the SVF and a trend toward adipocyte hypertrophy. The percentage of small adipocytes (20–30 μm) increased in HF1 mice, suggesting further recruitment of adipocyte precursors. However, in HF3 mice, a substantial change in cell hypertrophy occurred, with a shift to large-sized adipocytes (50–70 μm) and the emergence of a population of severely hypertrophied adipocytes (80–100 μm). Cell hypertrophy became dramatic for HF4 mice, in which two additional populations of large-size adipocytes, 50–70 and 70–130 μm, could be observed. A fairly large proportion of small adipocytes (20–40 μm) remained detectable in HF4 compared with HF0 mice, suggesting that adipocyte recruitment was still taking place at the fourth generation.
      Figure thumbnail gr5
      Fig. 5Adipocyte size distribution across generations. The frequency of distribution was analyzed using the adipocyte fraction of pooled fat pads from at least three mice at 10 weeks old as described in “Materials and Methods.” HFD, high-fat diet (ω6HFD); HF0–HF4, HFD-fed mice; revHF4, HF4 pups fed STD diet at weaning; STD, chow diet-fed mice.
      When HF4 mice were switched at weaning to a chow diet, the pattern of adipocyte size distribution of revHF4 mice was dramatically altered after seven weeks as both populations of large-size adipocytes could no longer be observed. The values for STD, HF0, HF4, and revHF4 mice were 35.4 ± 15.4, 35.8 ± 17.9, 48.8 ± 29.1 and 41 ± 11.5 µm for the mean adipocyte size, whereas the percentage of cells above 50 µm in diameter was 12%, 20%, 40%, and 22%, respectively, suggesting “remnant” adipocyte hypertrophy.
      To gain insight into recruitment of adipocyte precursor cells, we analyzed by qRT-PCR the expression levels of preadipocyte markers. No significant enrichment was found in the stromal vascular fraction of adipose tissue of HF0–HF3 mice compared with STD mice with respect to Pref-1, PPARβ/δ, C/EBPβ, FAT/CD36, or aP2/FABP4 (not shown). These results suggest that the hyperplastic growth of fat tissue implicated progenitor cells were more immature than preadipocytes and able to replenish the pool of preadipocytes. To characterize progenitor cell populations that could ultimately contribute to the formation of new adipocytes, a comparative FACS analysis of stromal vascular cells of epididymal fat tissue was carried out in STD, HF0, HF1, and HF2 mice (n = 8) at 4, 8, and 22 weeks of age by using cell surface-specific markers of various cell lineages known to be present in this cell fraction. No difference could be found in the proportion of Sca1+/CD34+ cells representative of stem cells and immature progenitors or in that of CD45 and CD31 cells representative of the hematopoietic and endothelial lineages, respectively (not shown). Clearly, we cannot exclude changes in the proportion of the minor subpopulation of CD34+/Sca1+/CD24+ cells, which have been shown very recently to behave in vivo as early adipocyte progenitors (
      • Rodeheffer M.S.
      • Birsoy K.
      • Friedman J.M.
      Identification of white adipocyte progenitor cells in vivo.
      ).

       Gene expression analysis of stromal vascular cells

      Expression profiles of the SVF of adipose tissue were established for the different generations of mice fed the ω6HFD (n ≥ 3 per group). Two independent experiments were carried out, and the identified probes were kept for subsequent analysis. To rule out possible contamination by adjacent tissues, a preliminary experiment was performed to directly compare the profile of SVF with those of testis and epidydimis. This led to a list of 2,366 distinct SVF-specific probes, which was subsequently used for further analysis. We then selected from this list about 108 transcripts divided into four groups (supplementary Table IV), based on a linear model proposed by Smyth in the limma package from Bioconductor (
      • Smyth G.K.
      Linear models and empirical bayes methods for assessing differential expression in microarray experiments.
      ). Probes were selected based on average log2(Signal) above 6, a log2(Fold change) above 0.7 (absolute value), and an adjusted P value below 0.05 for the following comparisons: HF3 versus HF0, and HF4 versus HF0. This initial analysis identified 46 transcripts (groups 1 and 2) that varied after three or four generations fed the ω6HFD. Because we felt that such a criterion was too restrictive and may not identify genes involved in the partial reversion of the phenotype (Fig. 3, Fig. 4, Fig. 5), we selected additional transcripts for which the expression differed due to the change in regimen from the chow diet to the ω6HFD and vice versa. The final list was established based on the following comparisons: (HF0+HF1+HF3+HF4) versus (STD+revHF1+revHF3+ revHF4). Such transcripts were expected to correspond more precisely to transcripts affected negatively or positively by the change in regimen (groups 3 and 4).
      The four lists of genes that varied along generations or after modification of the regimen were then inspected using Ingenuity Pathways (http://www.ingenuity.com) to reveal any significant enrichment of functional networks. These genes were then further directly analyzed to identify transcripts with known functions in adipocyte biology as discussed below (Table 2). Genes showing a significant difference but with no published evidence for involvement in adipose biology were not pursued further.
      TABLE 2Genes displaying adiposity status, impairment of angiogenesis, and inflammation
      Gene Identification NumberGene SymbolDescriptionModeFold Change
      NM_008337Ifnginterferon γTg0.61
      NM_009971Csf3colony stimulating factor 3 (granulocyte)Tg2.12
      NM_031168Il6interleukin 6Tg2.56
      NM_024435NtsneurotensinTg2.00
      NM_011347Selpselectin, plateletTg2.05
      NM_009605Adipoqadiponectin, C1Q and collagen domain containingRg0.48
      NM_009627AdmadrenomedullinRg0.80
      NM_011784Agtrl1angiotensin receptor-like 1Rg0.58
      NM_019764Amotl2angiomotin like 2Rg0.66
      NM_007643Cd36CD36 antigenRg0.45
      NM_010228Flt1FMS-like tyrosine kinase 1Rg0.72
      NM_026730Gpihbp1GPI-anchored HDL-binding protein 1Rg0.56
      NM_010612Kdrkinase insert domain protein receptorRg0.48
      NM_013599Mmp9matrix metallopeptidase 9Rg0.58
      NM_009221Sncasynuclein, α (Snca), transcript variant 1Rg0.76
      NM_011587Tie1tyrosine kinase receptor 1Rg0.61
      NM_008215Has1hyaluronan synthase1Rg2.04
      NM_031167Il1rninterleukin 1 receptor antagonist (Il1rn), transcript variant 1Rg1.71
      NM_011111Serpinb2serine (or cysteine) peptidase inhibitor, clade B, member 2 (Serpinb2) / PAI-2Rg1.43
      NM_016687Sfrp4secreted frizzled-related protein 4Rg2.16
      NM_009311Tac1tachykinin 1Rg1.72
      NM_009421Traf1Tnf receptor-associated factor 1Rg1.48
      Abbreviations: Rg, genes regulated by the ω6HFD Tg, genes regulated across generations; Tnf, tumor necrosis factor.

       Genes regulated across generations

      As hyperplasia of adipose tissue took place over generations, we anticipated a generation-dependent increase in the expression of growth factors. Colony-stimulating factors (CSF) are proteins necessary for survival, proliferation, and differentiation of hematopoietic progenitors, but the specificity of these growth factors is rather promiscuous. For instance, overexpression of CSF-1 (MCSF) in vivo led to a dramatic increase in adipose tissue growth in rabbits through WAT hyperplasia (
      • Levine J.A.
      • Jensen M.D.
      • Eberhardt N.L.
      • O'Brien T.
      Adipocyte macrophage colony-stimulating factor is a mediator of adipose tissue growth.
      ), whereas invalidation of the CSF-2 (GM-CSF) gene reduces inflammation in adipose tissue of C57 BL/6J mice in response to a high-fat diet (
      • Kim D.H.
      • Sandoval D.
      • Reed J.A.
      • Matter E.K.
      • Tolod E.G.
      • Woods S.C.
      • Seeley R.J.
      The role of GM-CSF in adipose tissue inflammation.
      ). Of interest, our data show that expression of CSF-3 (G-CSF) increased over generations in mice fed the ω6HFD (validation by qRT-PCR, supplementary Fig. III). These results strongly suggest a role for CSF-3 in stimulating growth of adipocyte progenitors and in enhancing macrophage recruitment. Despite the fact that upregulation of Nocturnin (Ccrn41) did not reach statistical significance, the phenotype of the ω6HFD-fed mice led us to assess its expression by qRT-PCR. As shown in supplementary Fig. III, this circadian deadenylase, the deletion of which confers resistance to diet-induced obesity, showed increased expression over generations, suggesting a pro-adipogenic role in fat tissue formation by promoting nuclear transport of PPARγ (Ref. (
      • Green C.B.
      • Douris N.
      • Kojima S.
      • Strayer C.A.
      • Fogerty J.
      • Lourim D.
      • Keller S.R.
      • Besharse J.C.
      Loss of Nocturnin, a circadian deadenylase, confers resistance to hepatic steatosis and diet-induced obesity.
      ) and unpublished observations). There was a sustained, decreased expression of interferon γ (Ifng) over generations, suggesting that the ω6HFD altered the number of interferon γ-producing T cells, which should in turn decrease the expression of inflammatory genes (
      • Rocha V.Z.
      • Folco E.J.
      • Sukhova G.
      • Shimizu K.
      • Gotsman I.
      • Vernon A.H.
      • Libby P.
      Interferon-gamma, a Th1 cytokine, regulates fat inflammation: a role for adaptive immunity in obesity.
      ). However, upregulation of other genes favoring recruitment of neutrophils and monocytes was noted. Expression of the P-selectin (Selp) gene, reported to be upregulated in HFD-induced obese mice, increased with generations, suggesting local platelet activation and formation of monocyte-platelet conjugates. These cell-cell interactions could then contribute to local inflammatory processes (
      • Nishimura S.
      • Manabe I.
      • Nagasaki M.
      • Seo K.
      • Yamashita H.
      • Hosoya Y.
      • Ohsugi M.
      • Tobe K.
      • Kadowaki T.
      • Nagai R.
      • et al.
      In vivo imaging in mice reveals local cell dynamics and inflammation in obese adipose tissue.
      ). The tendency toward additional proinflammatory pathways was also suggested by enhanced expression over generations of neurotensin (Nts), which is known to participate in the activation of the NF-κB signaling pathway. Although its expression is increased over generations, the contribution of interleukin 6 (IL-6) secreted from stromal vascular cells should be low compared with that of adipocytes then recovered in blood (Fig. 4), but its local involvement in developing insulin resistance cannot be excluded.

       Genes regulated by regimen

      Among the genes affected by diet, genes encoding the VEGF receptor-1 (Flt1) and angiopoetin-1 receptor (Tie1), the expression of which is mainly present in the SVF of adipose tissue, were downregulated. Both receptors bind ligands that are known to promote angiogenesis during excessive WAT development in different strains of mice fed a high-fat diet (
      • Dallabrida S.M.
      • Zurakowski D.
      • Shih S.C.
      • Smith L.E.
      • Folkman J.
      • Moulton K.S.
      • Rupnick M.A.
      Adipose tissue growth and regression are regulated by angiopoietin-1.
      ,
      • Voros G.
      • Maquoi E.
      • Demeulemeester D.
      • Clerx N.
      • Collen D.
      • Lijnen H.R.
      Modulation of angiogenesis during adipose tissue development in murine models of obesity.
      ). The VEGF receptor-2 (KDR) gene, encoding an early marker of endothelial cell differentiation, was also decreased (
      • Madonna R.
      • De Caterina R.
      In vitro neovasculogenic potential of resident adipose tissue precursors.
      ). Interestingly, the APJ receptor (Agtrl1), which is part of the apelin/APJ-R signaling system that plays a critical role in the development of a functional network in WAT (
      • Kunduzova O.
      • Alet N.
      • Delesque-Touchard N.
      • Millet L.
      • Castan-Laurell I.
      • Muller C.
      • Dray C.
      • Schaeffer P.
      • Herault J.P.
      • Savi P.
      • et al.
      Apelin/APJ signaling system: a potential link between adipose tissue and endothelial angiogenic processes.
      ), is equally decreased in the ω6HFD-fed mice. Last, but not least, expression of adiponectin (Adipoq), which has been reported to promote proliferation and function of endothelial cells (
      • Shibata R.
      • Skurk C.
      • Ouchi N.
      • Galasso G.
      • Kondo K.
      • Ohashi T.
      • Shimano M.
      • Kihara S.
      • Murohara T.
      • Walsh K.
      Adiponectin promotes endothelial progenitor cell number and function.
      ), was also decreased. Unexpectedly, the glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (Gpihbp1), a gene highly expressed in WAT and involved in the lipolytic processing of chylomicrons, appeared downregulated by the regimen (
      • Beigneux A.P.
      • Davies B.S.
      • Gin P.
      • Weinstein M.M.
      • Farber E.
      • Qiao X.
      • Peale F.
      • Bunting S.
      • Walzem R.L.
      • Wong J.S.
      • et al.
      Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 plays a critical role in the lipolytic processing of chylomicrons.
      ). In all these cases, the variation was equivalent between HF0 and HF3 or HF4, indicating that these modifications were caused by the regimen. Collectively, these data strongly suggest that hypoxia should prevail under such conditions.
      In accordance with these observations, which favor the possibility that decreased gene expression on the ω6HFD may impair angiogenesis, the genes encoding angiomotin-like 2 (Amolt2), which is assumed to regulate, like angiomotin, the migration of endothelial cells (
      • Aase K.
      • Ernkvist M.
      • Ebarasi L.
      • Jakobsson L.
      • Majumdar A.
      • Yi C.
      • Birot O.
      • Ming Y.
      • Kvanta A.
      • Edholm D.
      • et al.
      Angiomotin regulates endothelial cell migration during embryonic angiogenesis.
      ) and adrenomedullin (Adm), a potent vasodilator and antioxidative peptide, were downregulated (
      • Nambu T.
      • Arai H.
      • Komatsu Y.
      • Yasoda A.
      • Moriyama K.
      • Kanamoto N.
      • Itoh H.
      • Nakao K.
      Expression of the adrenomedullin gene in adipose tissue.
      ). In addition to adrenomedullin, downregulation of the α-synuclein (Snca) and CD36 genes, which may prevent excessive reactive oxygen species (ROS) production (
      • Klivenyi P.
      • Siwek D.
      • Gardian G.
      • Yang L.
      • Starkov A.
      • Cleren C.
      • Ferrante R.J.
      • Kowall N.W.
      • Abeliovich A.
      • Beal M.F.
      Mice lacking alpha-synuclein are resistant to mitochondrial toxins.
      ,
      • Okamura D.M.
      • Pennathur S.
      • Pasichnyk K.
      • Lopez-Guisa J.M.
      • Collins S.
      • Febbraio M.
      • Heinecke J.
      • Eddy A.A.
      CD36 regulates oxidative stress and inflammation in hypercholesterolemic CKD.
      ), was observed. The modulation of extracellular matrix components via the production of metalloproteinases is an important regulator of fat tissue remodeling. The metalloproteinase 9 (Mmp-9) gene, known to be expressed at higher levels in preadipocytes than in adipocytes, decreased over generations in accordance with its downregulation in nutritionally induced obesity of C57BL/6J mice (
      • Maquoi E.
      • Munaut C.
      • Colige A.
      • Collen D.
      • Lijnen H.R.
      Modulation of adipose tissue expression of murine matrix metalloproteinases and their tissue inhibitors with obesity.
      ).
      As expected, chronic exposure to the ω6HFD led to upregulation of genes implicated in the hyperplastic/ hypertrophic development of adipose tissue. The glycosaminoglycan hyaluronan is an essential component of the extracellular matrix that increases adhesion and retention of monocytes in adipose tissue in diet-induced obese C57BL/6J mice (
      • Han C.Y.
      • Subramanian S.
      • Chan C.K.
      • Omer M.
      • Chiba T.
      • Wight T.N.
      • Chait A.
      Adipocyte-derived serum amyloid A3 and hyaluronan play a role in monocyte recruitment and adhesion.
      ). Accordingly, the expression of hyaluronan synthase 1 (Has1), known to be present in adipocytes and preadipocytes, increased with the ω6HFD. Of note, the increased expression of the precursor Tachykinin 1 (Tac1) suggests enhanced production of substance P, which has been reported to promote proliferation of preadipocytes (
      • Gross K.
      • Karagiannides I.
      • Thomou T.
      • Koon H.W.
      • Bowe C.
      • Kim H.
      • Giorgadze N.
      • Tchkonia T.
      • Pirtskhalava T.
      • Kirkland J.L.
      • et al.
      Substance P promotes expansion of human mesenteric preadipocytes through proliferative and antiapoptotic pathways.
      ). Regarding adipocyte hypertrophy, the overexpression of PAI-2 (Serpinb2) over generations is consistent with the fact that PAI-2−/− mice fed a high-fat diet exhibit a lower adipocyte size and higher adipocyte number than wild-type mice (
      • Lijnen H.R.
      • Frederix L.
      • Scroyen I.
      Deficiency of plasminogen activator inhibitor-2 impairs nutritionally induced murine adipose tissue development.
      ), and suggests that PAI-2 overexpression promotes excessive WAT development. Reports have implicated Wnt signaling pathway components as proteins that maintain the preadipocyte compartment and inhibit adipogenesis. Interestingly, the gene expression of secreted frizzled-related protein 4 (sFRP4) became elevated with the ω6HFD (validation by qRT-PCR, supplementary Fig. III). As sFRP4 antagonizes the Wnt signaling pathway (
      • Christodoulides C.
      • Lagathu C.
      • Sethi J.K.
      • Vidal-Puig A.
      Adipogenesis and WNT signalling.
      ), this strongly suggests a stimulating effect on WAT development. Adrenomedullin, the expression of which decreased over generations, has been proposed to be antiadipogenic (
      • Harmancey R.
      • Senard J.M.
      • Rouet P.
      • Pathak A.
      • Smih F.
      Adrenomedullin inhibits adipogenesis under transcriptional control of insulin.
      ). If it were so in vivo, this will also favor adipogenesis. With respect to the emergence of insulin resistance, upregulation of interleukin-1 receptor antagonist (Il1rn) and TNFα receptor-associated factor 1 (Traf1) with a regimen appears to be important as the respective blockade of Il-1 and TNFα signaling pathways improved insulin sensitivity in diet-induced obesity (
      • Somm E.
      • Cettour-Rose P.
      • Asensio C.
      • Charollais A.
      • Klein M.
      • Theander-Carrillo C.
      • Juge-Aubry C.E.
      • Dayer J.M.
      • Nicklin M.J.
      • Meda P.
      • et al.
      Interleukin-1 receptor antagonist is upregulated during diet-induced obesity and regulates insulin sensitivity in rodents.
      ,
      • Liang H.
      • Yin B.
      • Zhang H.
      • Zhang S.
      • Zeng Q.
      • Wang J.
      • Jiang X.
      • Yuan L.
      • Wang C.Y.
      • Li Z.
      Blockade of tumor necrosis factor (TNF) receptor type 1-mediated TNF-alpha signaling protected Wistar rats from diet-induced obesity and insulin resistance.
      ).

      DISCUSSION

      We and others have pointed out the detrimental effects in humans caused by a dramatic change in the fatty acid composition of dietary fats at a time when the quantitative changes in fat consumption were not observed. Notably, at a time where overweightness and obesity have steadily increased over generations in most industrialized countries, consumption of LA and ARA has increased. In France, an increase of 250% and 230%, respectively, occurred from 1960 to 2000 (
      • Ailhaud G.
      • Massiera F.
      • Weill P.
      • Legrand P.
      • Alessandri J.M.
      • Guesnet P.
      Temporal changes in dietary fats: role of n-6 polyunsaturated fatty acids in excessive adipose tissue development and relationship to obesity.
      ). During the same period, consumption of LNA has decreased by 40%, resulting in a 4.2-fold increase in the LA:LNA ratio. A similar increase in LA intake has been observed in other countries of the Western world, with a LA:LNA ratio ranging from 10 to 41 for most foods consumed in the US (
      • Taber L.
      • Chiu C.H.
      • Whelan J.
      Assessment of the arachidonic acid content in foods commonly consumed in the American diet.
      ). Under isolipidic, isoenergetic conditions, an LA-enriched diet was shown in elderly humans to increase body weight, and the rate of LA accumulation in adipose tissue was associated positively with gain in body weight and, presumably, fat mass (
      • Dayton S.
      • Hashimoto S.
      • Dixon W.
      • Pearce M.L.
      Composition of lipids in human serum and adipose tissue during prolonged feeding of a diet high in unsaturated fat.
      ). In our experiments, LA represented 18% and LNA 0.6% of the total energy intake compared with 5–7% and 0.8–1%, respectively, recommended for humans by expert committees on nutrition. Unfortunately, a significantly higher LA intake and much lower LNA intake are frequently observed in most industrialized countries (
      • Ailhaud G.
      • Guesnet P.
      • Cunnane S.C.
      An emerging risk factor for obesity: does disequilibrium of polyunsaturated fatty acid metabolism contribute to excessive adipose tissue development?.
      ,
      • Ailhaud G.
      • Massiera F.
      • Weill P.
      • Legrand P.
      • Alessandri J.M.
      • Guesnet P.
      Temporal changes in dietary fats: role of n-6 polyunsaturated fatty acids in excessive adipose tissue development and relationship to obesity.
      ). Our results show that, in a situation of genome stability that is reminiscent of augmentation in the prevalence of obesity observed worldwide, a gradual transgenerational increase in adiposity can occur in mice fed a Western-like fat diet.
      So far, studies have been mainly focused on the relationships between diet-induced obesity in female rodents and the development of adult offspring adiposity and associated diseases (
      • Cottrell E.C.
      • Ozanne S.E.
      Early life programming of obesity and metabolic disease.
      ,
      • Samuelsson A.M.
      • Matthews P.A.
      • Argenton M.
      • Christie M.R.
      • McConnell J.M.
      • Jansen E.H.
      • Piersma A.H.
      • Ozanne S.E.
      • Twinn D.F.
      • Remacle C.
      • et al.
      Diet-induced obesity in female mice leads to offspring hyperphagia, adiposity, hypertension, and insulin resistance: a novel murine model of developmental programming.
      ). Herein we chose purposely ad libitum conditions to (i) expose both male and female mice across several generations to a Western-like diet, i.e., 35% energy as fat, whereas in most studies of diet-induced obesity in rodents these figures ranged from 30% to 80% energy as fat and (ii) use at-random breeding of all pups from the same litter, thus excluding, in contrast to a recent report (
      • Koza R.A.
      • Nikonova L.
      • Hogan J.
      • Rim J.S.
      • Mendoza T.
      • Faulk C.
      • Skaf J.
      • Kozak L.P.
      Changes in gene expression foreshadow diet-induced obesity in genetically identical mice.
      ), any selection of high or low body-fat gainers, which would have put emphasis on genetic issues and would have been less relevant to humans.
      The results show clearly, under conditions minimizing an imbalance between fetal and postnatal environments, a transgenerational increase in body weight and fat mass that was already apparent in pups at weaning and was maintained in adulthood. With respect to adipose tissue development, at any given generation, triggering events associated with the diet can be postulated, as it is known that in utero placenta selectively extracts ARA and DHA from maternal blood. It is thus conceivable that the ω6HFD will not increase LA levels in fetal blood but rather will increase ARA levels in the maternal and subsequently in the fetal blood (
      • Ghebremeskel K.
      • Crawford M.A.
      • Lowy C.
      • Min Y.
      • Thomas B.
      • Golfetto I.
      • Bitsanis D.
      • Costeloe K.
      Arachidonic and docosahexaenoic acids are strongly associated in maternal and neonatal blood.
      ). According to our data on the fatty acid composition of milk, adipose tissue, and plasma lipids, we assumed after birth a similar contribution of LA in enhancing adipose tissue development owing to the adipogenic role played by its metabolite ARA via prostacyclin at the time where the content of the anti-adipogenic EPA and DHA decreased (
      • Massiera F.
      • Saint-Marc P.
      • Seydoux J.
      • Murata T.
      • Kobayashi T.
      • Narumiya S.
      • Guesnet P.
      • Amri E.Z.
      • Negrel R.
      • Ailhaud G.
      Arachidonic acid and prostacyclin signaling promote adipose tissue development: a human health concern?.
      ,
      • Kim H.K.
      • Della-Fera M.
      • Lin J.
      • Baile C.A.
      Docosahexaenoic acid inhibits adipocyte differentiation and induces apoptosis in 3T3–L1 preadipocytes.
      ).
      Noteworthy, enhanced adiposity occurs over generations through hyperplasia and hypertrophy despite no significant change in food intake in pups and adult mice. Clearly, a progressive decrease in energy expenditure cannot be excluded, although the resistance of C57BL/6J mice to diet-induced thermogenesis has been reported (
      • Madsen L.
      • Pedersen L.M.
      • Liaset B.
      • Ma T.
      • Petersen R.K.
      • van den Berg S.
      • Pan J.
      • Muller-Decker K.
      • Dulsner E.D.
      • Kleemann R.
      • et al.
      cAMP-dependent signaling regulates the adipogenic effect of n-6 polyunsaturated fatty acids.
      ). Actually, we observed a trend (not statistically significant) toward higher expression of UCP1 in brown adipose tissue, suggesting an increase in energy expenditure over generations. It cannot be excluded that an increase of subcutaneous adiposity contributes to decreased thermogenicity over generations as heat loss is known to increase basal thermogenesis and resistance to diet-induced obesity (
      • Sampath H.
      • Flowers M.T.
      • Liu X.
      • Paton C.M.
      • Sullivan R.
      • Chu K.
      • Zhao M.
      • Ntambi J.M.
      Skin-specific deletion of stearoyl-CoA desaturase-1 alters skin lipid composition and protects mice from high fat diet-induced obesity.
      ). Regarding the transgenerational hyperplasia of adipose tissue, it may be due to the increased expression of the CSF3 gene across generations that enhanced the proliferation of adipose progenitors. In addition, we suggest that enhanced Nocturnin expression across generations modulates hypertrophy and associated metabolic events through subtle alterations in glucose homeostasis and lipid-partitioning between various organs including adipose tissue, as HF4 mice appear to be a striking mirror image of Nocturnin-null mice fed a HFD. Indeed, compared with wild-type mice, Nocturnin-null mice exhibited similar locomotor activity but an abnormality in weight regulation despite equivalent caloric intake and less heat production. Importantly, these knockout mice also showed improved insulin sensitivity, i.e., similar glucose and lower insulin levels (
      • Green C.B.
      • Douris N.
      • Kojima S.
      • Strayer C.A.
      • Fogerty J.
      • Lourim D.
      • Keller S.R.
      • Besharse J.C.
      Loss of Nocturnin, a circadian deadenylase, confers resistance to hepatic steatosis and diet-induced obesity.
      ).
      We observed also impairment in the expression of genes promoting angiogenesis, suggesting increased hypoxic and even highly hypoxic conditions in adipocytes distant from the vasculature, which lead in turn to alterations in adipokine secretion (
      • Trayhurn P.
      • Wang B.
      • Wood I.S.
      Hypoxia in adipose tissue: a basis for the dysregulation of tissue function in obesity?.
      ) and insulin sensitivity (
      • Regazzetti C.
      • Peraldi P.
      • Gremeaux T.
      • Najem-Lendom R.
      • Ben-Sahra I.
      • Cormont M.
      • Bost F.
      • Le Marchand-Brustel Y.
      • Tanti J.F.
      • Giorgetti-Peraldi S.
      Hypoxia decreases insulin signaling pathways in adipocytes.
      ). Surprisingly, after a rise in TNF-α, resistin, leptin, and MPC-1 in the first generation, normalization of circulating levels of adipokines occurred in HF4 mice compared with STD mice. Despite fat tissue expansion across generations, leptin levels returned to normal. This suggests that hypothalamic leptin sensitivity had somehow been redefined through transgenerational exposure to the ω6HFD. Moreover, the decreased levels of inflammatory adipokines could be regarded as adaptive evolution of the autonomic nervous system output. The most persistent dysregulation was that of insulin. In the absence of hyperglycemia, this strongly suggests insulin resistance, which is consistent with a progressive loss in sympathetic inhibition of insulin release over generations. Thus it looks as if an enhanced fat mass was an adaptive response to prevent aggravation of a metabolic syndrome.
      Collectively, our data show that continuous exposure to a high-fat diet combined with a high LA:LNA ratio from fetal to adult age over generations triggers a discrete and steady increase in inflammatory stimuli accompanied by enhancement of fat mass that was already observed a few weeks after birth. Most interestingly, in humans, the recent Framingham Heart study pointed out that nonobese offspring with two obese parents had higher C-reactive protein levels compared with offspring with one or no obese parent. Thus offspring with a high risk of developing obesity are characterized at birth by a proinflammatory state (
      • Lieb W.
      • Pencina M.J.
      • Lanier K.J.
      • Tofler G.H.
      • Levy D.
      • Fox C.S.
      • Wang T.J.
      • D'Agostino Sr., R.B.
      • Vasan R.S.
      Association of parental obesity with concentrations of select systemic biomarkers in nonobese offspring: the Framingham Heart Study.
      ).
      Our results on the incomplete reversal of adipocyte hypertrophy suggest epigenetic changes. To our knowledge, there is no direct evidence that fatty acid nutrition affects epigenetic gene regulation and that epigenetic mechanisms determining overweightness/obesity in an obesogenic environment have any relationship with those observed in malnourished fetuses (
      • Jimenez-Chillaron J.C.
      • Isganaitis E.
      • Charalambous M.
      • Gesta S.
      • Pentinat-Pelegrin T.
      • Faucette R.R.
      • Otis J.P.
      • Chow A.
      • Diaz R.
      • Ferguson-Smith A.
      • et al.
      Intergenerational transmission of glucose intolerance and obesity by in utero undernutrition in mice.
      ). Among candidate genes, only sFRP4, a member of the Wnt signaling pathway known to exhibit epigenetic regulation through methylation, may be relevant to this observation (
      • Dhir M.
      • Montgomery E.A.
      • Glockner S.C.
      • Schuebel K.E.
      • Hooker C.M.
      • Herman J.G.
      • Baylin S.B.
      • Gearhart S.L.
      • Ahuja N.
      Epigenetic regulation of WNT signaling pathway genes in inflammatory bowel disease (IBD) associated neoplasia.
      ). We speculate that its increased expression over generations should be responsible, at least in part, for the fast “rebound” in body weight observed in hf5 mice compared with std5 mice on a high-fat diet. Further studies will indicate whether histone modification rather than methylation is involved in epigenetic changes on continuous exposure to the ω6HFD.

      Acknowledgments

      The authors thank Drs. N. Combe and C. Vaysse (Université de Bordeaux, ITERG, Département de Nutrition) for performing analysis of the fatty acid composition of plasma and adipose tissue lipids. The authors wish to thank Dr. C. Dani (IBDC, CNRS, UMR 6543) for his continuous support and helpful discussion. The authors are grateful to the skilled assistance of Virginie Virolle for performing initial microarray experiments. We are grateful for the skilled technical assistance of Mansour Djedaini and the skillful secretarial assistance of Geneviève Oillaux. We are grateful to the Nice-Sophia Antipolis Transcriptome Platform of the Marseille-Nice Genopole where the microarray experiments were performed. We thank Dr. Christiane Brahimi-Horn (http://cbrahimihorn.free.fr) for editorial correction of the manuscript.

      Supplementary Material

      REFERENCES

        • Troiano R.P.
        • Briefel R.R.
        • Carroll M.D.
        • 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-1353S
        • Donahoo W.
        • Wyatt H.R.
        • Kriehn J.
        • Stuht J.
        • Dong F.
        • Hosokawa P.
        • Grunwald G.K.
        • Johnson S.L.
        • Peters J.C.
        • Hill J.O.
        Dietary fat increases energy intake across the range of typical consumption in the United States.
        Obesity (Silver Spring). 2008; 16: 64-69
        • Ailhaud G.
        • Guesnet P.
        • Cunnane S.C.
        An emerging risk factor for obesity: does disequilibrium of polyunsaturated fatty acid metabolism contribute to excessive adipose tissue development?.
        Br. J. Nutr. 2008; 100: 461-470
        • Massiera F.
        • Saint-Marc P.
        • Seydoux J.
        • Murata T.
        • Kobayashi T.
        • Narumiya S.
        • Guesnet P.
        • Amri E.Z.
        • Negrel R.
        • Ailhaud G.
        Arachidonic acid and prostacyclin signaling promote adipose tissue development: a human health concern?.
        J. Lipid Res. 2003; 44: 271-279
        • Gaillard D.
        • Negrel R.
        • Lagarde M.
        • Ailhaud G.
        Requirement and role of arachidonic acid in the differentiation of pre-adipose cells.
        Biochem. J. 1989; 257: 389-397
        • Vassaux G.
        • Gaillard D.
        • Ailhaud G.
        • Negrel R.
        Prostacyclin is a specific effector of adipose cell differentiation. Its dual role as a cAMP- and Ca(2+)-elevating agent.
        J. Biol. Chem. 1992; 267: 11092-11097
        • Kim H.K.
        • Della-Fera M.
        • Lin J.
        • Baile C.A.
        Docosahexaenoic acid inhibits adipocyte differentiation and induces apoptosis in 3T3–L1 preadipocytes.
        J. Nutr. 2006; 136: 2965-2969
        • Madsen L.
        • Pedersen L.M.
        • Liaset B.
        • Ma T.
        • Petersen R.K.
        • van den Berg S.
        • Pan J.
        • Muller-Decker K.
        • Dulsner E.D.
        • Kleemann R.
        • et al.
        cAMP-dependent signaling regulates the adipogenic effect of n-6 polyunsaturated fatty acids.
        J. Biol. Chem. 2008; 283: 7196-7205
        • Ailhaud G.
        • Massiera F.
        • Weill P.
        • Legrand P.
        • Alessandri J.M.
        • Guesnet P.
        Temporal changes in dietary fats: role of n-6 polyunsaturated fatty acids in excessive adipose tissue development and relationship to obesity.
        Prog. Lipid Res. 2006; 45: 203-236
        • Massiera F.
        • Seydoux J.
        • Geloen A.
        • Quignard-Boulange A.
        • Turban S.
        • Saint-Marc P.
        • Fukamizu A.
        • Negrel 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: 5220-5225
        • Lepage G.
        • Roy C.C.
        Specific methylation of plasma nonesterified fatty acids in a one-step reaction.
        J. Lipid Res. 1988; 29: 227-235
        • Moreilhon C.
        • Gras D.
        • Hologne C.
        • Bajolet O.
        • Cottrez F.
        • Magnone V.
        • Merten M.
        • Groux H.
        • Puchelle E.
        • Barbry P.
        Live Staphylococcus aureus and bacterial soluble factors induce different transcriptional responses in human airway cells.
        Physiol. Genomics. 2005; 20: 244-255
        • Gentleman R.C.
        • Carey V.J.
        • Bates D.M.
        • Bolstad B.
        • Dettling M.
        • Dudoit S.
        • Ellis B.
        • Gautier L.
        • Ge Y.
        • Gentry J.
        • et al.
        Bioconductor: open software development for computational biology and bioinformatics.
        Genome Biol. 2004; 5: R80
        • Wettenhall J.M.
        • Smyth G.K.
        limmaGUI: a graphical user interface for linear modeling of microarray data.
        Bioinformatics. 2004; 20: 3705-3706
        • Alexa A.
        • Rahnenfuhrer J.
        • Lengauer T.
        Improved scoring of functional groups from gene expression data by decorrelating GO graph structure.
        Bioinformatics. 2006; 22: 1600-1607
        • Zaragosi L.E.
        • Ailhaud G.
        • Dani C.
        Autocrine fibroblast growth factor 2 signaling is critical for self-renewal of human multipotent adipose-derived stem cells.
        Stem Cells. 2006; 24: 2412-2419
        • 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 postnatal changes in term infants fed breast milk or formula.
        Am. J. Clin. Nutr. 1999; 70: 292-298
        • Cao H.
        • Gerhold K.
        • Mayers J.R.
        • Wiest M.M.
        • Watkins S.M.
        • Hotamisligil G.S.
        Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism.
        Cell. 2008; 134: 933-944
        • Rodeheffer M.S.
        • Birsoy K.
        • Friedman J.M.
        Identification of white adipocyte progenitor cells in vivo.
        Cell. 2008; 135: 240-249
        • Smyth G.K.
        Linear models and empirical bayes methods for assessing differential expression in microarray experiments.
        Stat. Appl. Genet. Mol. Biol. 2004; 3 (Article 3.)
        • Levine J.A.
        • Jensen M.D.
        • Eberhardt N.L.
        • O'Brien T.
        Adipocyte macrophage colony-stimulating factor is a mediator of adipose tissue growth.
        J. Clin. Invest. 1998; 101: 1557-1564
        • Kim D.H.
        • Sandoval D.
        • Reed J.A.
        • Matter E.K.
        • Tolod E.G.
        • Woods S.C.
        • Seeley R.J.
        The role of GM-CSF in adipose tissue inflammation.
        Am. J. Physiol. Endocrinol. Metab. 2008; 295: E1038-E1046
        • Green C.B.
        • Douris N.
        • Kojima S.
        • Strayer C.A.
        • Fogerty J.
        • Lourim D.
        • Keller S.R.
        • Besharse J.C.
        Loss of Nocturnin, a circadian deadenylase, confers resistance to hepatic steatosis and diet-induced obesity.
        Proc. Natl. Acad. Sci. USA. 2007; 104: 9888-9893
        • Rocha V.Z.
        • Folco E.J.
        • Sukhova G.
        • Shimizu K.
        • Gotsman I.
        • Vernon A.H.
        • Libby P.
        Interferon-gamma, a Th1 cytokine, regulates fat inflammation: a role for adaptive immunity in obesity.
        Circ. Res. 2008; 103: 467-476
        • Nishimura S.
        • Manabe I.
        • Nagasaki M.
        • Seo K.
        • Yamashita H.
        • Hosoya Y.
        • Ohsugi M.
        • Tobe K.
        • Kadowaki T.
        • Nagai R.
        • et al.
        In vivo imaging in mice reveals local cell dynamics and inflammation in obese adipose tissue.
        J. Clin. Invest. 2008; 118: 710-721
        • Dallabrida S.M.
        • Zurakowski D.
        • Shih S.C.
        • Smith L.E.
        • Folkman J.
        • Moulton K.S.
        • Rupnick M.A.
        Adipose tissue growth and regression are regulated by angiopoietin-1.
        Biochem. Biophys. Res. Commun. 2003; 311: 563-571
        • Voros G.
        • Maquoi E.
        • Demeulemeester D.
        • Clerx N.
        • Collen D.
        • Lijnen H.R.
        Modulation of angiogenesis during adipose tissue development in murine models of obesity.
        Endocrinology. 2005; 146: 4545-4554
        • Madonna R.
        • De Caterina R.
        In vitro neovasculogenic potential of resident adipose tissue precursors.
        Am. J. Physiol. Cell Physiol. 2008; 295: C1271-C1280
        • Kunduzova O.
        • Alet N.
        • Delesque-Touchard N.
        • Millet L.
        • Castan-Laurell I.
        • Muller C.
        • Dray C.
        • Schaeffer P.
        • Herault J.P.
        • Savi P.
        • et al.
        Apelin/APJ signaling system: a potential link between adipose tissue and endothelial angiogenic processes.
        FASEB J. 2008; 22: 4146-4153
        • Shibata R.
        • Skurk C.
        • Ouchi N.
        • Galasso G.
        • Kondo K.
        • Ohashi T.
        • Shimano M.
        • Kihara S.
        • Murohara T.
        • Walsh K.
        Adiponectin promotes endothelial progenitor cell number and function.
        FEBS Lett. 2008; 582: 1607-1612
        • Beigneux A.P.
        • Davies B.S.
        • Gin P.
        • Weinstein M.M.
        • Farber E.
        • Qiao X.
        • Peale F.
        • Bunting S.
        • Walzem R.L.
        • Wong J.S.
        • et al.
        Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 plays a critical role in the lipolytic processing of chylomicrons.
        Cell Metab. 2007; 5: 279-291
        • Aase K.
        • Ernkvist M.
        • Ebarasi L.
        • Jakobsson L.
        • Majumdar A.
        • Yi C.
        • Birot O.
        • Ming Y.
        • Kvanta A.
        • Edholm D.
        • et al.
        Angiomotin regulates endothelial cell migration during embryonic angiogenesis.
        Genes Dev. 2007; 21: 2055-2068
        • Nambu T.
        • Arai H.
        • Komatsu Y.
        • Yasoda A.
        • Moriyama K.
        • Kanamoto N.
        • Itoh H.
        • Nakao K.
        Expression of the adrenomedullin gene in adipose tissue.
        Regul. Pept. 2005; 132: 17-22
        • Klivenyi P.
        • Siwek D.
        • Gardian G.
        • Yang L.
        • Starkov A.
        • Cleren C.
        • Ferrante R.J.
        • Kowall N.W.
        • Abeliovich A.
        • Beal M.F.
        Mice lacking alpha-synuclein are resistant to mitochondrial toxins.
        Neurobiol. Dis. 2006; 21: 541-548
        • Okamura D.M.
        • Pennathur S.
        • Pasichnyk K.
        • Lopez-Guisa J.M.
        • Collins S.
        • Febbraio M.
        • Heinecke J.
        • Eddy A.A.
        CD36 regulates oxidative stress and inflammation in hypercholesterolemic CKD.
        J. Am. Soc. Nephrol. 2009; 20: 495-505
        • Maquoi E.
        • Munaut C.
        • Colige A.
        • Collen D.
        • Lijnen H.R.
        Modulation of adipose tissue expression of murine matrix metalloproteinases and their tissue inhibitors with obesity.
        Diabetes. 2002; 51: 1093-1101
        • Han C.Y.
        • Subramanian S.
        • Chan C.K.
        • Omer M.
        • Chiba T.
        • Wight T.N.
        • Chait A.
        Adipocyte-derived serum amyloid A3 and hyaluronan play a role in monocyte recruitment and adhesion.
        Diabetes. 2007; 56: 2260-2273
        • Gross K.
        • Karagiannides I.
        • Thomou T.
        • Koon H.W.
        • Bowe C.
        • Kim H.
        • Giorgadze N.
        • Tchkonia T.
        • Pirtskhalava T.
        • Kirkland J.L.
        • et al.
        Substance P promotes expansion of human mesenteric preadipocytes through proliferative and antiapoptotic pathways.
        Am. J. Physiol. Gastrointest. Liver Physiol. 2009; 296: G1012-G1019
        • Lijnen H.R.
        • Frederix L.
        • Scroyen I.
        Deficiency of plasminogen activator inhibitor-2 impairs nutritionally induced murine adipose tissue development.
        J. Thromb. Haemost. 2007; 5: 2259-2265
        • Christodoulides C.
        • Lagathu C.
        • Sethi J.K.
        • Vidal-Puig A.
        Adipogenesis and WNT signalling.
        Trends Endocrinol. Metab. 2009; 20: 16-24
        • Harmancey R.
        • Senard J.M.
        • Rouet P.
        • Pathak A.
        • Smih F.
        Adrenomedullin inhibits adipogenesis under transcriptional control of insulin.
        Diabetes. 2007; 56: 553-563
        • Somm E.
        • Cettour-Rose P.
        • Asensio C.
        • Charollais A.
        • Klein M.
        • Theander-Carrillo C.
        • Juge-Aubry C.E.
        • Dayer J.M.
        • Nicklin M.J.
        • Meda P.
        • et al.
        Interleukin-1 receptor antagonist is upregulated during diet-induced obesity and regulates insulin sensitivity in rodents.
        Diabetologia. 2006; 49: 387-393
        • Liang H.
        • Yin B.
        • Zhang H.
        • Zhang S.
        • Zeng Q.
        • Wang J.
        • Jiang X.
        • Yuan L.
        • Wang C.Y.
        • Li Z.
        Blockade of tumor necrosis factor (TNF) receptor type 1-mediated TNF-alpha signaling protected Wistar rats from diet-induced obesity and insulin resistance.
        Endocrinology. 2008; 149: 2943-2951
        • Taber L.
        • Chiu C.H.
        • Whelan J.
        Assessment of the arachidonic acid content in foods commonly consumed in the American diet.
        Lipids. 1998; 33: 1151-1157
        • Dayton S.
        • Hashimoto S.
        • Dixon W.
        • Pearce M.L.
        Composition of lipids in human serum and adipose tissue during prolonged feeding of a diet high in unsaturated fat.
        J. Lipid Res. 1966; 7: 103-111
        • Cottrell E.C.
        • Ozanne S.E.
        Early life programming of obesity and metabolic disease.
        Physiol. Behav. 2008; 94: 17-28
        • Samuelsson A.M.
        • Matthews P.A.
        • Argenton M.
        • Christie M.R.
        • McConnell J.M.
        • Jansen E.H.
        • Piersma A.H.
        • Ozanne S.E.
        • Twinn D.F.
        • Remacle C.
        • et al.
        Diet-induced obesity in female mice leads to offspring hyperphagia, adiposity, hypertension, and insulin resistance: a novel murine model of developmental programming.
        Hypertension. 2008; 51: 383-392
        • Koza R.A.
        • Nikonova L.
        • Hogan J.
        • Rim J.S.
        • Mendoza T.
        • Faulk C.
        • Skaf J.
        • Kozak L.P.
        Changes in gene expression foreshadow diet-induced obesity in genetically identical mice.
        PLoS Genet. 2006; 2: e81
        • Ghebremeskel K.
        • Crawford M.A.
        • Lowy C.
        • Min Y.
        • Thomas B.
        • Golfetto I.
        • Bitsanis D.
        • Costeloe K.
        Arachidonic and docosahexaenoic acids are strongly associated in maternal and neonatal blood.
        Eur. J. Clin. Nutr. 2000; 54: 50-56
        • Sampath H.
        • Flowers M.T.
        • Liu X.
        • Paton C.M.
        • Sullivan R.
        • Chu K.
        • Zhao M.
        • Ntambi J.M.
        Skin-specific deletion of stearoyl-CoA desaturase-1 alters skin lipid composition and protects mice from high fat diet-induced obesity.
        J. Biol. Chem. 2009; 284: 19961-19973
        • Trayhurn P.
        • Wang B.
        • Wood I.S.
        Hypoxia in adipose tissue: a basis for the dysregulation of tissue function in obesity?.
        Br. J. Nutr. 2008; 100: 227-235
        • Regazzetti C.
        • Peraldi P.
        • Gremeaux T.
        • Najem-Lendom R.
        • Ben-Sahra I.
        • Cormont M.
        • Bost F.
        • Le Marchand-Brustel Y.
        • Tanti J.F.
        • Giorgetti-Peraldi S.
        Hypoxia decreases insulin signaling pathways in adipocytes.
        Diabetes. 2009; 58: 95-103
        • Lieb W.
        • Pencina M.J.
        • Lanier K.J.
        • Tofler G.H.
        • Levy D.
        • Fox C.S.
        • Wang T.J.
        • D'Agostino Sr., R.B.
        • Vasan R.S.
        Association of parental obesity with concentrations of select systemic biomarkers in nonobese offspring: the Framingham Heart Study.
        Diabetes. 2009; 58: 134-137
        • Jimenez-Chillaron J.C.
        • Isganaitis E.
        • Charalambous M.
        • Gesta S.
        • Pentinat-Pelegrin T.
        • Faucette R.R.
        • Otis J.P.
        • Chow A.
        • Diaz R.
        • Ferguson-Smith A.
        • et al.
        Intergenerational transmission of glucose intolerance and obesity by in utero undernutrition in mice.
        Diabetes. 2009; 58: 460-468
        • Dhir M.
        • Montgomery E.A.
        • Glockner S.C.
        • Schuebel K.E.
        • Hooker C.M.
        • Herman J.G.
        • Baylin S.B.
        • Gearhart S.L.
        • Ahuja N.
        Epigenetic regulation of WNT signaling pathway genes in inflammatory bowel disease (IBD) associated neoplasia.
        J. Gastrointest. Surg. 2008; 12: 1745-1753