Effect of maternal HFD on offspring response to MCD diet challenge.

<p><b>(A)</b> Body weight of offspring from control and HFD-fed dams. Offspring were provided either Con diet (<b>CC</b>, n = 11 or <b>HC</b>, n = 11) or MCD (<b>C-MCD</b>, n = 9 or <b>H-MCD</b>, n = 9) for 25 days starting at 7 wk of age. <b>(B)</b> Relative liver weights and <b>(C)</b> Serum alanine aminotransferase (ALT) concentrations in offspring. <b>(D)</b> Kleiner scores for steatosis, lobular inflammation, ballooning and fibrosis compared between C-MCD and H-MCD groups. <b>(E)</b> Photomicrographs of H&E stained liver sections from offspring. Data are expressed as means ± SE. Statistical differences in body weight were determined using two-way ANOVA to examine the main effects of maternal and post-weaning MCD diet, followed by Student-Newman-Keuls <i>post hoc</i> analyses. Statistical differences pathology scores were assessed using Students t-test. (*p<0.05, **p<0.01, ***p<0.001).</p

Offspring characteristics and hepatic transcriptome analysis.

<p><b>(A)</b> Body weight and <b>(B)</b> Relative liver weights of offspring from control and HFD-fed dams weaned onto either Con (<b>CC</b>, n = 10; <b>HC</b>, n = 7) or HFD (<b>CH</b>, n = 11; <b>HH</b>, n = 7). Offspring were provided <i>ad libitum</i> access to Con or HF diet for 14 wk after weaning. <b>(C)</b> Photomicrographs of H&E stained liver sections from offspring. <b>(D)</b> Principal component analysis of global gene expression profiles showing unsupervised clustering of samples based on maternal and post-weaning HFD. <b>(E)</b> Venn diagram showing the number of differentially expressed transcripts due to maternal and offspring HFD (± 2.0-fold change, p<0.05, corrected for multiple testing). <b>(F)</b> Analysis of GO biological process terms via BiNGO and <b>(G)</b> Clustering using ClueGO, among differentially expressed genes showing enrichment of immune response, inflammatory pathways and fibroblasts activation. <b>(H)</b> Real-time RT-PCR based confirmation of mRNA expression of genes differentially expressed in the combination of maternal obesity and offspring HFD. Data are expressed as means ± SE. Statistical differences in body weight were determined using two-way ANOVA to examine the main effects of maternal and post-weaning HFD diet, followed by Student-Newman-Keuls <i>post hoc</i> analyses. Statistical differences in gene expression were determined using a one-way ANOVA. (*p<0.05, **p<0.01).</p

Maternal HFD increases pro-fibrogenic response in offspring liver following MCD diets.

<p><b>(A)</b> Photomicrographs of Picro-Sirius red stain showing peri-cellular fibrosis. <b>(B)</b> Real-time PCR quantification of the genes involved in fibrosis, steatosis and inflammation (<i>Col1a1</i>, <i>Mmp9</i>, <i>Mmp2</i>, <i>α-SMA</i>, <i>Mmp13</i>, <i>Timp1</i>, <i>Casp1 and Tgfβ1</i>). Data are expressed as means ± SE. Statistical differences in gene expression were determined using a 2-way ANOVA to examine the main effects of maternal HFD and offspring MCD diet, followed by Student-Newman-Keuls post hoc analyses (*p<0.05, **p<0.01).</p

DNA methylation changes in livers of offspring from lean and obese dams challenged with MCD diet.

<p>Genome-scale DNA methylation was assessed using RRBS. <b>(A)</b> Percent frequency distribution of methylation status of informative promoters (TSS), promoters containing CGI (TSS_CGI), all CGIs, in offspring from lean and HFD dams challenged with control or MCD diet. Methylation status of features is binned into 5 categories (0%–20%, 20%–40%, and so on). <b>(B)</b> Genomic localization of differentially methylated regions (DMRs). <b>(C)</b> Δ_me (difference in average methylation between groups) for each DMR showing both hypo and hypermethylated regions. See full list in Supplementary tables. Each group was compared against CC offspring.</p

Effect of maternal HF and offspring MCD diet challenge on cecal microbiota.

<p><b>(A)</b> α-diversity (OTU abundance) between treatments (CC, C-MCD, HC and H-MCD) or as main effects of maternal HFD and offspring MCD diet. <b>(B)</b> PCoA of gut microbiota composition based on unweighted UniFrac distances in offspring from control and HFD dams fed control or MCD diet (n = 9–11 mice per group) <b>(C)</b> Pie charts showing relative composition of gut microbial phyla in offspring groups. <b>(D)</b> Hierarchical clustering of family-level OTUs using STAMP showing predominant separation by offspring (MCD) diets. <b>(E)</b> Effect of maternal HFD on at least genera i.e. <i>Ruminococcus</i>, <i>Turibacter</i> and <i>Oscillospria</i>. <b>(F)</b> Abundance of specific microbial families in CC, C-MCD, HC and H-MCD groups (n = 9–11 mice per group). Data are expressed as means ± SE. Statistical differences in OTU abundance were determined using a one-way ANOVA followed by Student-Newman-Keuls post hoc analyses (*p<0.05, ***p<0.001). <b>(G)</b> Cladogram from LEfSe analysis showing taxa enriched in microbiota from mice fed MCD diets.</p

Changes in DNA methylation with either maternal HFD (HC vs CC) or maternal HFD and offspring MCD diet (H-MCD vs CC).

<p>Scatter plots of average methylation of DMRs showing altered methylation with <b>(A)</b> maternal HFD, or <b>(B)</b> the combination of maternal HFD and offspring MCD diet are presented along with annotation of key genes. <b>(C-D)</b> Enrichment of GO biological process terms of DMRs in proximity of the genes in <b>(C)</b> CC vs HC and <b>(D)</b> CC vs H-MCD comparisons. Maternal HFD influences methylation of regions proximal to <b>(E)</b> <i>Ppargc1β</i> and <i>Fgf21</i>. The combination of maternal HFD and offspring MCD diet feeding influences methylation of regions close to <i>Ephb2</i> and <i>Vwf</i>. Average methylation of the DMR is depicted on the histograms in the two lower tracks.</p