Maternal high-fat diet alters methylation and gene expression of dopamine and opioid-related genes

Abstract

Maternal obesity during pregnancy increases the risk of obesity in the offspring. Obesity, arising from an imbalance of energy intake and expenditure, can be driven by the ingestion of palatable [high fat (HF), high sugar], energy-dense foods. Dopamine and opioid circuitry are neural substrates associated with reward that can affect animals' preference for palatable foods. Using a mouse model, the long-term effect of maternal consumption of a HF diet on dopamine and opioid gene expression within the mesocorticolimbic reward circuitry and hypothalamus of the offspring was investigated. Mice from dams fed a HF diet during pregnancy and lactation showed an increased preference for sucrose and fat. Gene expression, measured using quantitative real-time PCR, revealed a significant approximately 3- to 10-fold up-regulation of dopamine reuptake transporter (DAT) in the ventral tegmental area, nucleus accumbens, and prefrontal cortex and a down-regulation of DAT in the hypothalamus. Additionally, expression of both μ-opioid receptor (MOR) and preproenkephalin (PENK) was increased in nucleus accumbens, prefrontal cortex, and hypothalamus of mice from dams that consumed the HF diet. Epigenetic mechanisms have been associated with long-term programming of gene expression after various in utero insults. We observed global and gene-specific (DAT, MOR, and PENK) promoter DNA hypomethylation in the brains of offspring from dams that consumed the HF diet. These data demonstrate that maternal consumption of a HF diet can change the offsprings' epigenetic marks (DNA hypomethylation) in association with long-term alterations in gene expression (dopamine and opioids) and behavior (preference for palatable foods).

Figure 1

Preference for 4% sucrose solution (top) or 60% fat diet (bottom) was evaluated using preference tests in control offspring (gray bar) and offspring from HF-fed dams (black bar; n = 6–8 per group). Offspring from HF-fed dams have increased sucrose and fat preference compared with controls. Values represent mean ± sem. *, P < 0.05.

Figure 2

Dopamine-related gene expression. Quantitative RT-PCR was used to examine mRNA expression levels of dopamine pathway-related genes TH, DAT, D1, D2, and DARPP-32 in the CNS of adult male mice from dams fed either the control diet (control; gray bars) or HF diet (black bars). Mice from HF-fed dams demonstrate significant differences in multiple dopamine-related genes. Expression level of each gene examined was normalized to GAPDH levels and expressed relative to the control group set to 1. Values are mean ± sem. *, P values < 0.05 (n = 5 per group). HYP, Hypothalamus.

Figure 3

Opioid ligand and receptor gene expression. mRNA expression levels of the opioid prepropeptides (left) was examined in the PFC, NAc, and hypothalamus (HYP) of offspring from HF-fed dams (black bars) and control mice (gray bars) using qRT-PCR. PENK (NAc, PFC, and hypothalamus) and POMC (hypothalamus) were significantly up-regulated in mice from dams fed the HF diet. mRNA expression levels of opioid receptor genes OPRD1 (δ), OPRK1 (κ), and OPRM1 (μ) in the NAc, PFC, and hypothalamus of offspring from HF-fed dams (black bars) and control mice (gray bars) was determined by qRT-PCR (right). Mice from HF-fed dams demonstrate a significant up-regulation of μ-opioid receptor. Expression level of each gene examined was normalized to the GAPDH and β-actin levels and expressed relative to the control group set to 1. Values are mean ± sem. *, P values <0.05 (n = 6 per group).

Figure 4

Global DNA methylation in brain regions of adult animals. Global methylation was analyzed using Methylamp kit (Epigentek) in animals from control (gray bars) or HF-fed (black bars) dams. Values are mean ± sem. Two-way ANOVA revealed a main effect for group (*, P < 0.0001), with offspring from HF-fed dams demonstrating a significant reduction in global DNA methylation. Furthermore, global methylation levels in the VTA were significantly lower than in all other brain regions (a, P < 0.008), and global methylation in PFC was significantly lower than in the hypothalamus (b, P < 0.008) (n = 5 per group).

Figure 5

DNA methylation changes at regulatory regions of reward-associated genes in offspring from HF-fed dams. Genomic DNA was isolated, sheared, and immunoprecipitated with 5-methylcytosine-specific antibody from brain regions from offspring of control (gray bars) or HF-fed (black bars) dams. The enrichment of DNA methylation relative to input genomic DNA at proximal promoter regions of TH, DAT, PENK, μ-opioid receptor (MOR), and GAPDH was quantified by qRT-PCR. Significant hypomethylation was observed in DAT, MOR, and PENK in offspring from HF-fed dams, which paralleled the increased expression of these genes. Values are mean ± sem. *, P values < 0.05 (n = 6 per group, two-tailed t test).