Chronic high-fat diet drives postnatal epigenetic regulation of μ-opioid receptor in the brain

Abstract

Opioid system dysregulation has been observed in both genetic and high-fat diet (HFD)-induced models of obesity. An understanding of the molecular mechanisms of MOR transcriptional regulation, particularly within an in vivo context, is lacking. Using a diet-induced model of obesity (DIO), mice were fed a high-fat diet (60% calories from fat) from weaning to >18 weeks of age. Compared with mice fed the control diet, DIO mice had a decreased preference for sucrose. MOR mRNA expression was decreased in reward-related circuitry (ventral tegmental area (VTA), nucleus accumbens (NAc), and prefrontal cortex (PFC)) but not the hypothalamus, important in the homeostatic regulation of feeding. DNA methylation is an epigenetic modification that links environmental exposures to altered gene expression. We found a significant increase in DNA methylation in the MOR promoter region within the reward-related brain regions. Methyl CpG-binding protein 2 (MeCP2) can bind methylated DNA and repress transcription, and DIO mice showed increased binding of MeCP2 to the MOR promoter in reward-related regions of the brain. Finally, using ChIP assays we examined H3K9 methylation (inactive chromatin) and H3 acetylation (active chromatin) within the MOR promoter region and found increased H3K9 methylation and decreased H3 acetylation. These data are the first to identify DNA methylation, MeCP2 recruitment, and chromatin remodeling as mechanisms leading to transcriptional repression of MOR in the brains of mice fed a high-fat diet.

Figures

Figure 1

Long-term exposure to high-fat diet (HFD) induces obesity in mice, and decreases preference for sucrose. Mice had continuous access to control or 60% HFD from weaning at 3 weeks until 18–20 weeks of age. (a) Dietary-induced obese mice (DIO) were 27% heavier than controls at 20 weeks. (b) Preference for 4% sucrose solution was evaluated using 48-h two-bottle preference test in control mice (white bar) and DIO mice (black bar). DIO mice showed a decreased preference for sucrose compared with control-diet fed animals. Values represent mean±SEM. *p<0.05, n=6–8/group.

Figure 2

Expression of endogenous opioid ligand precursors in control and obese mice. RT-qPCR was used to assay mRNA levels of prodynorphin (PDYN), preproenkephalin (PENK) and pro-opiomelanocortin (POMC) mRNA in nucleus accumbens (NAc), prefrontal cortex (PFC), and HYP of control (gray bars) and obese mice (Dietary-induced obese mice (DIO), black bars). PENK levels were significantly increased in DIO mice in all CNS regions tested. Mean±SEM. **p<0.01, #p<0.005.

Figure 3

Expression of opioid receptors in control and obese mice. RT-qPCR was used to assay mRNA levels of delta (DOR), kappa (KOR) and mu-opioid (MOR) receptors in ventral tegmental area (VTA), nucleus accumbens (NAc), prefrontal cortex (PFC) and HYP of control (Control; gray bars) and obese mice (Dietary-induced obese mice (DIO), black bars). DOR was significantly increased in VTA, whereas MOR levels were significantly reduced in VTA, PFC and NAc of DIO mice. KOR levels displayed a trend toward decreased levels in the VTA (p<0.05, Bonferroni corrected p-value=0.0165) Values are mean±SEM. **p<0.01, #p<0.005.

Figure 4

DNA methylation status of μ-opioid receptor (MOR) promoter in dietary-induced obese mice (DIO) mice. Genomic DNA was isolated from dissected ventral tegmental area (VTA), nucleus accumbens (NAc), prefrontal cortex (PFC), and HYP of control (gray bars) and obese (DIO) mice, sheared by sonication and immunoprecipitated with 5-methylcytosine antibody. The enrichment of DNA methylation relative to input genomic DNA at the proximal promoter region of MOR was quantified by qPCR. DIO mice displayed hypermethylation of MOR. GAPDH methylation was not altered in DIO mice. Values are mean±SEM. *p<0.05, **p<0.01, n=6/group, Two-tailed t-test.

Figure 5

Association of methyl CpG-binding protein 2 (MeCP2) on μ-opioid receptor (MOR) promoter in central nervous system (CNS) of dietary-induced obese mice (DIO) mice. Chromatin was isolated from dissected ventral tegmental area (VTA), nucleus accumbens (NAc), prefrontal cortex (PFC), and HYP of control (gray bars) and DIO mice (black bars), enzymatically digested and immunoprecipitated with IgG and anti- MeCP2. The enrichment of genomic DNA associated with immunoprecipitated chromatin around proximal promoter (−0.5 and +0.5 kb) and 2 kb distal of MOR was quantified by qPCR. DIO mice displayed increased association of MeCP2 only in the −0.5-kb region of the MOR promoter in NAc and PFC, and decreased MeCP2 association in the hypothalamus. There was no difference in MeCP2 binding either +0.5 or −2.0 kb from the MOR promoter and no differential binding to GAPDH. Values are mean±SEM. *p<0.05, n=3/group, Two-tailed t-test.

Figure 6

Histone modifications on μ-opioid receptor (MOR) promoter in central nervous system (CNS) of dietary-induced obese mice (DIO) mice. Chromatin was isolated from dissected ventral tegmental area (VTA), nucleus accumbens (NAc), prefrontal cortex (PFC), and HYP of control (gray bars) and DIO mice (black bars), enzymatically digested and immunoprecipitated with IgG, anti di-methyl-H3K9 (inactive chromatin) and anti-acetyl-H3 antibody (active chromatin). The enrichment of genomic DNA associated with immunoprecipitated chromatin at proximal promoter regions of the MOR was quantified by qPCR as described for MeDIP. DIO mice displayed increased amount of methylated histone on the MOR promoter, whereas the amount of acetyl-histone associated DNA was reduced in NAc and PFC of DIO mice. Changes in histone modifications were not observed on GAPDH promoter in DIO mice. This suggests an epigeneticlly repressed state of the MOR promoter in DIO mice. Values are mean±SEM. *p<0.05, **p<0.01, n=6/group, Two-tailed t-test.