High folate gestational and post-weaning diets alter hypothalamic feeding pathways by DNA methylation in Wistar rat offspring

Abstract

Excess vitamins, especially folate, are consumed during pregnancy but later-life effects on the offspring are unknown. High multivitamin (10-fold AIN-93G, HV) gestational diets increase characteristics of metabolic syndrome in Wistar rat offspring. We hypothesized that folate, the vitamin active in DNA methylation, accounts for these effects through epigenetic modification of food intake regulatory genes. Male offspring of dams fed 10-fold folate (HFol) diet during pregnancy and weaned to recommended vitamin (RV) or HFol diets were compared with those born to RV dams and weaned to RV diet for 29 weeks. Food intake and body weight were highest in offspring of HFol dams fed the RV diet. In contrast, the HFol pup diet in offspring of HFol dams reduced food intake (7%, p = 0.02), body weight (9%, p = 0.03) and glucose response to a glucose load (21%, p = 0.02), and improved glucose response to an insulin load (20%, p = 0.009). HFol alone in either gestational or pup diet modified gene expression of feeding-related neuropeptides. Hypomethylation of the pro-opiomelanocortin (POMC) promoter occurred with the HFol pup diet. POMC-specific methylation was positively associated with glucose response to a glucose load (r = 0.7, p = 0.03). In conclusion, the obesogenic phenotype of offspring from dams fed the HFol gestational diet can be corrected by feeding them a HFol diet. Our work is novel in showing post-weaning epigenetic plasticity of the hypothalamus and that in utero programming by vitamin gestational diets can be modified by vitamin content of the pup diet.

Keywords: DNA methylation; folate; gene expression; gestation; glucose response; hypothalamus; obesity; post-weaning.

Figure 1. Food intake over 72 h of male offspring from 0–29 weeks post-weaning. Diet abbreviations: RV, the AIN-93G diet with the recommended vitamins; HFol, RV+10-fold the folate content. Gestational and pup diets denoted before and after the dash line, respectively. Diet (p = 0.02), Time (p < 0.0001), Diet*Time (p = 1.0). ^ab Significantly different by PROC MIXED model repeated measures. Values are mean ± SEM, n = 10–12/group.

Figure 2. Body weight of male offspring from 0–29 weeks post-weaning. Diet abbreviations: RV, the AIN-93G diet with the recommended vitamins; HFol, RV+10-fold the folate content. Gestational and pup diets denoted before and after the dash line, respectively. Weight Gain: Diet (p = 0.03), Time (p < 0.0001), Diet*Time (p = 0.7). ^ab Significantly different by PROC MIXED model repeated measures. Values are mean ± SEM, n = 11–12/group.

Figure 3. Blood glucose response to an insulin load (0.75 U of insulin per kg of body weight) as net area under the curve (netAUC) of male offspring at 7, 12 and 16 weeks post-weaning. Diet abbreviations: RV, the AIN-93G diet with the recommended vitamins; HFol, RV+10-fold the folate content. Gestational and pup diets denoted before and after the dash line, respectively. ^ab Significantly different by one-way ANOVA followed by Tukey’s post-hoc test. Values are mean ± SEM, n = 9–12/group.

Figure 4. Blood glucose response to a glucose load (5 g of glucose per kg of body weight) as incremental area under the curve (iAUC) of male offspring at 10, 14 and 18 weeks post-weaning. Diet abbreviations: RV, the AIN-93G diet with the recommended vitamins; HFol, RV+10-fold the folate content. Gestational and pup diets denoted before and after the dash line, respectively. ^ab Significantly different by one-way ANOVA followed by Tukey’s post-hoc test. Values are mean ± SEM, n = 9–12/group.

Figure 5. Hypothalamic gene expression of neuropeptide Y (NPY), pro-opiomelanocortin (POMC), brain-derived neurotrophic factor (BNDF) and serotonin receptors (5-HTR1A/2A/2C) of right hypothalamus in male offspring at 29 weeks post-weaning. Diet abbreviations: RV, the AIN-93G diet with the recommended vitamins; HFol, RV+10-fold the folate content. Gestational and pup diets denoted before and after the dash line, respectively. ^ab Significantly different by one-way ANOVA followed by Tukey’s post-hoc test. Values are mean ± SEM, n = 4–7/group.

Figure 6. DNA methylation at (A) global and (B) pro-opiomelanocortin (POMC)-specific of left hypothalamus in male offspring at 29 weeks post-weaning. Diet abbreviations: RV, the AIN-93G diet with the recommended vitamins; HFol, RV+10-fold the folate content. Gestational and pup diets denoted before and after the dash line, respectively. Global DNA methylation not significant, n = 5/group. ^ab POMC-specific methylation p = 0.0001 by one-way ANOVA followed by Tukey’s post-hoc test, n = 4–6/group. Values are mean ± SEM.

Figure 7. Correlation between pro-opiomelanocortin (POMC)-specific methylation and glucose response to a glucose load at 10 weeks post-weaning by Spearman’s correlation coefficient, n = 9.

Figure 8. Folate concentrations in the (A) brain and (B) plasma of male offspring at 29 weeks post-weaning. Diet abbreviations: RV, the AIN-93G diet with the recommended vitamins; HFol, RV+10-fold the folate content. Gestational and pup diets denoted before and after the dash line, respectively. ^ab Brain folate: p = 0.002, n = 9–11 and plasma folate: p = 0.004, n = 13–15 by one-way ANOVA followed by Tukey’s post-hoc test. Values are mean ± SEM.

Figure 9. Schematic sketch of the study (A) design and (B) protocol for male offspring from 0–29 weeks post-weaning. Diet abbreviations: RV, the AIN-93G diet with the recommended vitamins; HFol, RV+10-fold the folate content. Gestational and pup diets denoted before and after the dash line, respectively.

Figure 10. Location of 5 cytosine-phosphate-guanine (CpG) sites in the pro-opiomelanocortin (POMC) gene promoter shown by the shadowed areas.