Long-term effects of pro-opiomelanocortin methylation induced in food-restricted dams on metabolic phenotypes in male rat offspring

Abstract

Objective: Maternal malnutrition affects the growth and metabolic health of the offspring. Little is known about the long-term effect on metabolic indices of epigenetic changes in the brain caused by maternal diet. Thus, we explored the effect of maternal food restriction during pregnancy on metabolic profiles of the offspring, by evaluating the DNA methylation of hypothalamic appetite regulators at 3 weeks of age.

Methods: Sprague-Dawley rats were divided into 2 groups: a control group and a group with a 50% food-restricted (FR) diet during pregnancy. Methylation and expression of appetite regulator genes were measured in 3-week-old offspring using pyrosequencing, real-time polymerase chain reaction, and western blotting analyses. We analyzed the relationship between DNA methylation and metabolic profiles by Pearson's correlation analysis.

Results: The expression of pro-opiomelanocortin (POMC) decreased, whereas DNA methylation significantly increased in male offspring of the FR dams, compared to the male offspring of control dams. Hypermethylation of POMC was positively correlated with the levels of high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol in 3-week-old male offspring. In addition, there were significant positive correlations between hypermethylation of POMC and the levels of triglycerides, HDL-C, and leptin in 6-month-old male offspring.

Conclusion: Our findings suggest that maternal food restriction during pregnancy influences the expression of hypothalamic appetite regulators via epigenetic changes, leading to the development of metabolic disorders in the offspring.

Keywords: Appetite regulation; DNA methylation; Malnutrition; Metabolic syndrome; Pro-opiomelanocortin.

Fig. 1. Experimental design. Eight-week-old male and female Sprague-Dawley (SD) rats (n=8/group) were used in this study. At day 10 of gestation, pregnant SD rats were divided into 2 groups: 1) a control group, fed a normal diet throughout the whole experimental period and 2) a food-restriction (FR) group, fed a 50% FR diet during pregnancy and a normal diet after delivery.

Fig. 2. Relative mRNA and protein expression levels of pro-opiomelanocortin (POMC), neuropeptide Y (NPY), and melanocortin 4 receptor (MC4R) in the hypothalamus of 3-week-old offspring. (A) The mRNA expression levels of POMC, NPY, and MC4R were analyzed using real-time PCR in the food-restricted (FR) and control (C) groups (n=9 males, 9 females/group). Data are presented as means±standard deviation (SD). (B) The protein expression levels of POMC, NPY, and MC4R were analyzed using western blotting in the FR and C groups (n=9 males, 9 females/group). Data are presented as means±SD.

GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ^a)P<0.05; ^b)P<0.01; ^c)P<0.001.

Fig. 3. Hypothalamic pro-opiomelanocortin (POMC) methylation in male (A) and female (B) offspring at 3 weeks of age (n=9 males, 9 females/group). POMC methylation was measured by pyrosequencing in offspring of the food-restricted (FR) and control (C) dams. Data are presented as means±standard deviation.

^a)P<0.05.

Fig. 4. Hypothalamic neuropeptide Y (NPY) methylation in male and female offspring at 3 weeks of age (n=9 males, 9 females/group). (A) Methylation of promoter region 1 and (B) promoter region 2 of the NPY gene. Data are presented as means±standard deviation.

C, control; FR, food-restricted.

Fig. 5. Hypothalamic melanocortin-4 receptor (MC4R) methylation in male and female offspring at 3 weeks of age (n=9 males, 9 females/group). (A) DNA methylation of promoter region 1 in and (B) promoter region 2 of the MC4R gene. Values are presented as means±standard deviation.

C, control; FR, food-restricted.