Folic acid supplementation during the juvenile-pubertal period in rats modifies the phenotype and epigenotype induced by prenatal nutrition.

Prenatal nutritional constraint is associated with increased risk of metabolic dysregulation in adulthood contingent on adult diet. In rats, folic acid supplementation of a protein-restricted (PR) diet during pregnancy prevents altered phenotype and epigenotype in the offspring induced by the PR diet. We hypothesized that increasing folic acid intake during the juvenile-pubertal (JP) period would reverse the effects of a maternal PR diet on the offspring. Rats were fed a control (C) or PR diet during pregnancy and AIN93G during lactation. Offspring were weaned on d 28 onto diets containing 1 mg [adequate folate (AF)] or 5 mg [folic acid-supplemented (FS)] folic acid/kg feed. After 28 d, all offspring were fed a high-fat (18% wt:wt) diet and killed on d 84. As expected, offspring of PR dams fed the AF diet had increased fasting plasma triglyceride (TAG) and beta-hydroxybutyrate (betaHB) concentrations. The FS diet induced increased weight gain, a lower plasma betaHB concentration, and increased hepatic and plasma TAG concentration compared with AF offspring irrespective of maternal diet. PPARalpha and glucocorticoid receptor promoter methylation increased in liver and insulin receptor promoter methylation decreased in liver and adipose tissue in FS compared with AF offspring, with reciprocal changes in mRNA expression irrespective of maternal diet. These findings show that increased folic acid intake during the JP period did not simply reverse the phenotype induced by the maternal diet. This may represent a period of plasticity when specific nutrient intakes may alter the phenotype of the offspring through epigenetic changes in specific genes.

Feeding pregnant rats a protein-restricted diet persistently alters the methylation of specific cytosines in the hepatic PPAR alpha promoter of the offspring.

Induction of an altered phenotype by prenatal under-nutrition involves changes in the epigenetic regulation of specific genes. We investigated the effect of feeding pregnant rats a protein-restricted (PR) diet with different amounts of folic acid on the methylation of individual CpG dinucleotides in the hepatic PPAR alpha promoter in juvenile offspring, and the effect of the maternal PR diet on CpG methylation in adult offspring. Pregnant rats (five per group) were fed 180 g/kg casein (control) or 90 g/kg casein with 1 mg/kg folic acid (PR), or 90 g/kg casein and 5 mg/kg folic acid (PRF). Offspring were killed on postnatal day 34 (five males and females per group) and day 80 (five males per group). Methylation of sixteen CpG dinucleotides in the PPAR alpha promoter was measured by pyrosequencing. Mean PPAR alpha promoter methylation in the PR offspring (4.5 %) was 26 % lower than controls (6.1 %) due to specific reduction at CpG dinucleotides 2 (40 %), 3 (43 %), 4 (33 %) and 16 (48 %) (P < 0.05). There was no significant difference in methylation at these CpG between control and PRF offspring. Methylation of CpG 5 and 8 was higher (47 and 63 %, respectively, P < 0.05) in the PRF offspring than control or PR offspring. The methylation pattern in day 80 PR offspring was comparable to day 34 PR offspring. These data show for the first time that prenatal nutrition induces differential changes to the methylation of individual CpG dinucleotides in juvenile rats which persist in adults.

Dietary protein restriction of pregnant rats in the F0 generation induces altered methylation of hepatic gene promoters in the adult male offspring in the F1 and F2 generations.

Epidemiological studies and experimental models show that maternal nutritional constraint during pregnancy alters the metabolic phenotype of the offspring and that this can be passed to subsequent generations. In the rat, induction of an altered metabolic phenotype in the liver of the F1 generation by feeding a protein-restricted diet (PRD) during pregnancy involves the altered methylation of specific gene promoters. We therefore investigated whether the altered methylation of PPARalpha and glucocorticoid receptor (GR) promoters was passed to the F2 generation. Females rats (F0) were fed a reference diet (180 g/kg protein) or PRD (90 g/kg protein) throughout gestation, and AIN-76A during lactation. The F1 offspring were weaned onto AIN-76A. F1 females were mated and fed AIN-76A throughout pregnancy and lactation. F1 and F2 males were killed on postnatal day 80. Hepatic PPARalpha and GR promoter methylation was significantly (P<0 x 05) lower in the PRD group in the F1 (PPARalpha 8 %, GR 10 %) and F2 (PPARalpha 11 %, GR 8 %) generations. There were trends (P<0 x 1) towards a higher expression of PPARalpha, GR, acyl-CoA oxidase and phosphoenolpyruvate carboxykinase (PEPCK) in the F1 and F2 males, although this was significant only for PEPCK. These data show for the first time that the altered methylation of gene promoters induced in the F1 generation by maternal protein restriction during pregnancy is transmitted to the F2 generation. This may represent a mechanism for the transmission of induced phenotypes between generations

Metabolic plasticity during mammalian development is directionally dependent on early nutritional status.

Developmental plasticity in response to environmental cues can take the form of polyphenism, as for the discrete morphs of some insects, or of an apparently continuous spectrum of phenotype, as for most mammalian traits. The metabolic phenotype of adult rats, including the propensity to obesity, hyperinsulinemia, and hyperphagia, shows plasticity in response to prenatal nutrition and to neonatal administration of the adipokine leptin. Here, we report that the effects of neonatal leptin on hepatic gene expression and epigenetic status in adulthood are directionally dependent on the animal's nutritional status in utero. These results demonstrate that, during mammalian development, the direction of the response to one cue can be determined by previous exposure to another, suggesting the potential for a discontinuous distribution of environmentally induced phenotypes, analogous to the phenomenon of polyphenism.

Dietary protein restriction of pregnant rats induces and folic acid supplementation prevents epigenetic modification of hepatic gene expression in the offspring.

Environmental constraints during early life result in phenotypic changes that can be associated with increased disease risk in later life. This suggests persistent alteration of gene transcription. DNA methylation, which is largely established in utero, provides a causal mechanism by which unbalanced prenatal nutrition results in such altered gene expression. We investigated the effect of unbalanced maternal nutrition on the methylation status and expression of the glucocorticoid receptor (GR) and peroxisomal proliferator-activated receptor (PPAR) genes in rat offspring after weaning. Dams were fed a control protein (C; 180 g/kg protein plus 1 mg/kg folic acid), restricted protein (R; 90 g/kg casein plus 1 mg/kg folic acid), or restricted protein plus 5 mg/kg folic acid (RF) diet throughout pregnancy. Pups were killed 6 d after weaning (n = 10 per group). Gene methylation was determined by methylation-sensitive PCR and mRNA expression by semiquantitative RT-PCR. PPARalpha gene methylation was 20.6% lower (P < 0.001) and expression 10.5-fold higher in R compared with C pups. GR gene methylation was 22.8% lower (P < 0.05) and expression 200% higher (P < 0.01) in R pups than in C pups. The RF diet prevented these changes. PPARgamma methylation status and expression did not differ among the groups. Acyl-CoA oxidase expression followed that of PPARalpha. These results show that unbalanced prenatal nutrition induces persistent, gene-specific epigenetic changes that alter mRNA expression. Epigenetic regulation of gene transcription provides a strong candidate mechanism for fetal programming.

The expression of the developmentally regulated proto-oncogene Pax-3 is modulated by N-Myc.

N-Myc is a member of the Myc family of transcription factors that have been shown to play a pivotal role in cell proliferation and differentiation. In this report, we have investigated the relationship between N-Myc and the developmental control gene Pax-3. Using transient transfection assays, we show that the Pax-3 promoter is activated by both N-Myc-Max and c-Myc-Max. Moreover, we show that Myc regulation of Pax-3 promoter activity is dependent upon a noncanonical E box site in the 5' promoter region of Pax-3. In addition, we show that ectopic expression of both N-Myc and c-Myc leads to increased expression of Pax-3 mRNA. Furthermore, we show that Pax-3 mRNA expression is cell cycle-regulated and that the 5' promoter region of Pax-3 (bp -1578 to +56) can direct cell cycle-dependent gene expression with kinetics similar to that of the endogenous transcript. Site-directed mutagenesis of the E box site within the Pax-3 promoter significantly altered the pattern of expression through the cell cycle. These results suggest that the Myc family of transcription factors may modulate Pax-3 expression in vivo.