Additional file 4 of Placental transcriptome co-expression analysis reveals conserved regulatory programs across gestation

Predicted transcription factos for each co-expression module. Comma-seperated values file Top 10 predicted transcription factors for each co-expression module. (XLSX 62 kb

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

High Fat Diet and <i>In Utero</i> Exposure to Maternal Obesity Disrupts Circadian Rhythm and Leads to Metabolic Programming of Liver in Rat Offspring

<div><p>The risk of obesity in adulthood is subject to programming beginning at conception. In animal models, exposure to maternal obesity and high fat diets influences the risk of obesity in the offspring. Among other long-term changes, offspring from obese rats develop hyperinsulinemia, hepatic steatosis, and lipogenic gene expression in the liver at weaning. However, the precise underlying mechanisms leading to metabolic dysregulation in the offspring remains unclear. Using a rat model of overfeeding-induced obesity, we previously demonstrated that exposure to maternal obesity from pre-conception to birth, is sufficient to program increased obesity risk in the offspring. Offspring of obese rat dams gain greater body weight and fat mass when fed high fat diet (HFD) as compared to lean dam. Since, disruptions of diurnal circadian rhythm are known to detrimentally impact metabolically active tissues such as liver, we examined the hypothesis that maternal obesity leads to perturbations of core clock components and thus energy metabolism in offspring liver. Offspring from lean and obese dams were examined at post-natal day 35, following a short (2 wk) HFD challenge. Hepatic mRNA expression of circadian (CLOCK, BMAL1, REV-ERBα, CRY, PER) and metabolic (PPARα, SIRT1) genes were strongly suppressed in offspring exposed to both maternal obesity and HFD. Using a mathematical model, we identified two distinct biological mechanisms that modulate PPARα mRNA expression: i) decreased mRNA synthesis rates; and ii) increased non-specific mRNA degradation rate. Moreover, our findings demonstrate that changes in PPARα transcription were associated with epigenomic alterations in H3K4me3 and H3K27me3 histone marks near the PPARα transcription start site. Our findings indicated that offspring from obese rat dams have detrimental alternations to circadian machinery that may contribute to impaired liver metabolism in response to HFD, specifically via reduced PPARα expression prior to obesity development.</p></div

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

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

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

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

H3K27me3 enrichment on the PPARα promoter.

<p>Chromatin immunoprecipitation for H3K27me3 was carried out and A) upstream (−500 base pairs) and B) downstream (+500 base pairs from TSS) regions of the PPAR-α gene were amplified in pools of liver samples (each pool represents 3–5 separate animals) from offspring of lean or obese dams fed either control or HFD (run in triplicate). Enrichment was determined by real time RT-PCR and normalized to input levels. Data are presented as mean ± SEM. Statistical differences were determined using a two-way ANOVA to examine the effects of maternal obesity and post-weaning HFD (P<0.05). Significant interactions were followed by one way ANOVA and Student-Newman-Keuls <i>post hoc</i> analyses (P<0.05). Bold values represent significant main effects and interactions and values with different letter superscripts are significantly different from each other (P<0.05).</p

Circadian expression of EZH2 and SIRT1 mRNA.

<p>Hepatic mRNA expression of EZH2 in A) Control-fed and B) HFD-fed offspring of lean and obese dams at PND35 (N = 3–5 animals per group, Lean-Con 2AM and Obese-HFD 6AM (N = 5 per group), Lean-Con 10AM and Obese-Con 10PM (N = 3 per group), and all the remaining groups (N = 4)). C) Hepatic mRNA expression of SIRT1 in Control-fed and D) HFD-fed offspring of lean and obese dams at PND35 (N = 3–5 animals per group, Lean-Con 2AM and Obese-HFD 6AM (N = 5 per group), Lean-Con 10AM and Obese-Con 10PM (N = 3 per group), and all the remaining groups (N = 4)). Data are presented as mean ± SEM. Statistical differences were determined using a Student's <i>t</i> test. * denotes significance, P<0.05.</p