Phenotypic and metabolic characteristics of the mice after 24 weeks on the CD and HFD, for the F1LM (CD1, OP1 and OR1) and F2OM (CD2, OP2 and OR2) mice.

<p>The values presented are the means±SEM. For weight and caloric intake, <i>n</i> = 50 to 70, for plasma hormone and metabolite determinations, <i>n</i> = 10 to 15, for FA level determination, <i>n</i> = 7, and for Piximus analysis and organ weights, <i>n</i> = 5.</p>a<p><i>p</i><0.05 for comparison with control,</p>b<p><i>p</i><0.05 for comparison between OP and OR mice,</p>c<p><i>p</i><0.05 for comparison between the F1LM and F2OM mice, assessed by Kruskal-Wallis tests followed by Dunn’s <i>post hoc</i> tests.</p

Hierarchical clustering heat map representing increases (red) and decreases (red) in intensity of peaks significantly different in mild <i>vs</i> severe patients.

<p>Peak masses are represented in the vertical axis, and individuals in the horizontal axis. Data were log-transformed, obtained after clustering of peaks (vertical axis) and individuals (horizontal axis), analyzed by the Ward Agglomeration Algorithm, and filtered for mild <i>vs</i> severe patients comparison. The green trace delimits the data corresponding to the cluster of individuals that show consistent changes in lipid content.</p

Schematic diagram summarizing the transcriptional data obtained for the liver, muscle and adipose tissue and the results of epigenetic studies in OP1, OP2 and OR2 mice.

<p>Blue arrows represent potential lipid fluxes. In the hyperphagic OP1 and OP2 mice, lipid ingestion was much greater than in OR2 mice, in which caloric intake was normalized. In OP mice, excess lipids were initially stored in the adipose tissue, leading to adipocyte hypertrophy in OP1 mice and hyperplasia in OP2 mice, as a function of the level of expression of <i>Lep</i> and <i>Peg1</i>. In OR2 mice, lipids were stored in the adipose tissue without adipocyte abnormalities or ectopic storage, as in mice supplied with limited amounts of lipid. In OP1 mice, the excess lipids were stored in the liver, contributing to hepatic hypertrophy. Transcriptomic data indicated that <i>de novo</i> lipogenesis was activated in OP1 mice and that insulin signaling was greatly disturbed. In OP2 mice, genes related to insulin signaling were less affected, whereas genes involved in fatty acid oxidation were globally upregulated. Changes to hepatic metabolism, together with the probable redirection of lipids to muscle thus spared the liver from lipid accumulation. Finally, in OR2 mice, lipid metabolism as a whole was downregulated, whereas thyroid hormone signaling was upregulated. The HFD response was also associated, in OP1 mice, with an upregulation of potassium channels and serotonin receptors, subsequently reversed in both OP2 and OR2 mice. Changes in DNA methylation were observed in the livers of OP1 mice and the muscle of OP2 mice. In the liver, <i>Set7/9</i> expression was decreased by the HFD in mice born to either lean or obese/diabetic mothers (F1LM and F2OM), whether OP or OR. In the livers of OP1 mice, DNA hypomethylation was associated with an upregulation of <i>Suv39h1</i> and <i>Suv39h2</i> expression, whereas, in both OR2 and OP2 mice, normal DNA methylation was associated with a decrease in <i>Jhdm2a</i> expression and an increase in the level of <i>Dnmt2</i> mRNA. In muscle, normal DNA methylation was associated with an upregulation of <i>Suv39h1, Set7/9</i> and <i>Dnmt2</i> in OP1 and OR2 mice, contrasting with the lower level of expression of the <i>Set7/9</i> and <i>Dnmt2</i> genes in the muscle of OP2 mice presenting DNA hypomethylation.</p

Analysis, by RT-qPCR, of the expression of genes encoding histone-modifying enzymes in the liver and muscle of mice fed the HFD, for both lean and obese mothers.

<p>The values shown are the ratios between OP1 or OR1 and CD1 and between OP2 or OR2 and CD2. They are expressed as the mean±SEM, <i>n</i> = 8 per group. ^a<i>p</i><0.05 for comparison with control CD, ^b<i>p</i><0.05 for comparison between OP and OR mice, ^c<i>p</i><0.05 for comparison between the F1LM and F2OM (maternal effect: lean versus obese mother), assessed by Kruskal-Wallis tests and <i>post hoc</i> Dunn’s tests.</p

Analysis of mRNA levels for key adipogenic genes by RT-qPCR on the adipose tissue of mice fed either the CD or the HFD, born to either lean or obese/diabetic mothers (F1LM and F2OM).

<p>The values shown are the ratios between OP1 or OR1 and CD1 and between OP2 or OR2 and CD2 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066816#pone-0066816-g009" target="_blank">Figures 9</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066816#pone-0066816-g010" target="_blank">10</a>). They are expressed as the mean±SEM, <i>n</i> = 6 per group. ^a<i>p</i><0.05 for comparison with control CD, ^b<i>p</i><0.05 for comparison between OP and OR mice, ^c<i>p</i><0.05 for comparison between F1LM and F2OM, assessed by Kruskal-Wallis tests followed by <i>post hoc</i> Dunn’s tests.</p

Heat map construction representing the differential expression of genes involved in metabolic function and neurotransmission.

<p>The heat map shows changes in the hepatic expression of genes encoding proteins involved in neurotransmission and genes encoding energy homeostasis-related proteins. Red, green and white squares represent upregulated, downregulated and unmodified genes, respectively.</p

Box plots corresponding to four peaks differentially displayed.

<p>Two sphingomyelin (m/z 489.3 and 725.5, upper panels) and two phosphatidylcholine (m/z 782.6 and 810.6, lower panels) species are represented. Relative intensity corresponds to the area of each peak of the spectrum related to the total ion count. Box plots are constructed from log-transformed relative intensity data.</p

Peaks differentially displayed in either healthy controls <i>vs</i> CF patients (*) or in CF mild <i>vs</i> CF severe patients (**) with p<0.05 after T-test analysis.

<p>All peaks are [M+H]⁺ except 489.27 and 725.53, which correspond to [M+Na]⁺. # Theoretical identity (not confirmed by MSⁿ).</p

Resistance to the obesogenic effects of the HFD: Major networks identified by IPA analysis of the genes involved in the response and adaptation to HFD of OR2 mice.

<p>(A) These networks were built from the 142 genes differentially expressed between OR2 and CD2 mice (direct comparison OR2/CD2). Only the first two networks are represented in (B) and (C). The node color indicates the level of expression of the genes: red, upregulated; green, downregulated.</p

Analysis, by RT-qPCR, of the expression of genes encoding DNA methyltransferase enzymes in the liver and muscle of mice fed the HFD, born to either lean or obese/diabetic mothers (F1LM and F2OM).

<p>The values shown are the ratios between OP1 or OR1 and CD1 and between OP2 or OR2 and CD2. They are expressed as the mean±SEM, <i>n</i> = 8 per group. ^a<i>p</i><0.05 for comparison with control CD, ^b<i>p</i><0.05 for comparison between OP and OR mice, ^c<i>p</i><0.05 for comparison between the F1LM and F2OM born to lean and obese/diabetic mothers, respectively, assessed by Kruskal-Wallis tests and <i>post hoc</i> Dunn’s tests.</p