Gene expression and DNA methylation as mechanisms of disturbed metabolism in offspring after exposure to a prenatal HF diet

Abstract

Exposure to a prenatal high-fat (HF) diet leads to an impaired metabolic phenotype in mouse offspring. The underlying mechanisms, however, are not yet fully understood. Therefore, this study investigated whether the impaired metabolic phenotype may be mediated through altered hepatic DNA methylation and gene expression. We showed that exposure to a prenatal HF diet altered the offspring's hepatic gene expression of pathways involved in lipid synthesis and uptake (SREBP), oxidative stress response [nuclear factor (erythroid-derived 2)-like 2 (Nrf2)], and cell proliferation. The downregulation of the SREBP pathway related to previously reported decreased hepatic lipid uptake and postprandial hypertriglyceridemia in the offspring exposed to the prenatal HF diet. The upregulation of the Nrf2 pathway was associated with increased oxidative stress levels in offspring livers. The prenatal HF diet also induced hypermethylation of transcription factor (TF) binding sites upstream of lipin 1 (Lpin1), a gene involved in lipid metabolism. Furthermore, DNA methylation of Lpin1 TF binding sites correlated with mRNA expression of Lpin1 These findings suggest that the effect of a prenatal HF diet on the adult offspring's metabolic phenotype are regulated by changes in hepatic gene expression and DNA methylation.

Keywords: deoxyribonucleic acid; development; diet and dietary lipids; epigenetics; high-fat diet; in utero; lipid metabolism; liver; microarrays; obesity; oxidative stress; pregnancy.

Fig. 1.

Overview of study design and workflow. Parent mice received a HF or LF diet starting 6 weeks before mating and continuing during pregnancy and lactation. Offspring were weaned onto a HF diet, thereby creating the experimental groups LF/HF and HF/HF, with the first two letters indicating the parental diet and the last two letters indicating the offspring diet. At 12 and 28 weeks of age, offspring livers were collected and analyzed. Gene expression was assessed using microarrays and GSEA. Selected differentially expressed (DE) genes were confirmed with real-time qPCR. DNA methylation of TF binding sites upstream of differentially expressed genes was measured using bisulfite pyrosequencing. Oxidative stress was measured with the GSH/GSSG assay.

Fig. 2.

Identification of TF binding regions and selection of CpG sites for pyrosequencing. A1: Regions with a high density of TF binding sites were identified <2,000 bp upstream of the TSS of a selected gene, based on GTRD. A2: CpG sites within the selected TF binding regions were selected for DNA methylation analysis. B: Genomic positions of TF binding regions (indicated by black horizontal lines) and CpGs within these regions (indicated by vertical lines), which were selected for DNA methylation analysis. Arrows mark TSS.

Fig. 3.

GSH and GSSG concentrations and the ratio between GSH and GSSG concentrations in 12-week-old (n = 7 for LF and n = 6 for HF) and 28-week-old (n = 9 per group) offspring livers after both parent mice received either a prenatal LF or HF diet. Boxplots represent minimum, first quartile, median, second quartile, and maximum. *P < 0.05, based on two-tailed independent samples t-test (GSH and GSH/GSSG) or two-tailed Mann-Whitney U test (GSSG).

Fig. 4.

Effect of prenatal HF diet on hepatic mRNA expression of Cd163, Hmgcr, Aacs, Lpin1, Pla2g16, Saa1, and Il1r1 in 12-week-old (n = 7 per group) and 28-week-old offspring (n = 9 per group) analyzed by microarray and real-time qPCR. Expression was normalized against Actb and Gapdh and presented relative to control (LF/HF) expression (controls have Log2-fold change of 0). Bars indicate mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, based on two-tailed paired intensity-based moderated t-statistics (microarray) or two-tailed independent samples t-test (real-time qPCR).

Fig. 5.

Effect of prenatal HF diet on offspring hepatic DNA methylation of Lpin1 TF binding region 2 (Chr12: 16,590,890–16,591,130 bp) at 12 weeks (n = 7 per group) and 28 weeks of age (n = 9 per group). Lines and error bars indicate mean ± SEM. **FDR q-value <0.01, based on two-tailed independent samples t-test followed by correction for multiple testing using the Benjamini-Hochberg procedure.