Emma S. Phillips

Feeding pregnant rats a protein-restricted diet persistently alters the methylation of specific cytosines in the hepatic PPARα 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 animals 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.

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.

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 ofPax-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.

Dietary protein restriction in the pregnant rat induces altered epigenetic regulation of the glucocorticoid receptor and peroxisomal proliferator-activated receptor alpha in the heart of the offspring which is prevented by folic acid.

In healthy individuals, glucose and fatty acids are substrates for ATP generation in the heart. There is emerging evidence from patients with type 2 diabetes mellitus that preferential use of fatty acid b-oxidation for energy production may be linked to cardiomyopathy (Fink, 2004). PPARa activity is important for regulating fatty acid b-oxidation in the heart and is increased in hearts of rats with experimentally induced diabetes (Fink, 2004). Prenatal undernutrition is related inversely to risk of type 2 diabetes mellitus in man (Poole & Byrne, 2005) and insulin resistance in rats (Bertram & Hanson, 2001). We have shown that maternal dietary protein restriction induces persistent alterations to hepatic and carbohydrate metabolism in the offspring by altering the epigenetic regulation of PPARa and the glucocorticoid receptor (GR) (Lillycrop et al. 2005). Here we have tested the hypothesis that prenatal protein restriction induces hypomethylation of the GR and PPARa promoters in the heart, and that this is prevented by supplementation of the protein-restricted (PR) diet with folic acid.Induction of a modified metabolic phenotype in the offspring by feeding a protein-restricted (PR) diet during pregnancy in the rat involves DNA hypomethylation and altered covalent histone modifications leading to increased expression of specific genes (Lillycrop et al. 2005a,b). Hypomethylation of gene promoters may be achieved by impaired DNA methylation de novo, loss of CpG methylation during mitosis, or active demethylation. Histone modifications which modulate transcription involve binding of methyl CpG binding protein (MeCP)-2 to methylated DNA and recruitment of histone-modifying enzymes (Bird, 2002). We investigated in the offspring the effect of feeding a PR diet during pregnancy on the expression of hepatic DNA methyltransferase (DMNT) 1 which maintains CpG methylation, DNMT 3a and 3b which catalyse DNA methylation de novo and the DNA demethylase MBD2.

Effect of maternal dietary restriction during pregnancy in rats on PPARα-regulated genes in the heart of the male offspring

The demands of the heart for energy are met by a variety of substrates. In the fasting state fatty acids are the main substrate for ATP synthesis, while in the fed state glucose is the preferred substrate. An investigation was conducted into whether prenatal undernutrition constrained future flexibility in the use of substrates for energy production in the heart by measuring the mRNA expression of PPARa and of specific target genes involved in fatty acid metabolism. Rats were fed a control (C; 180 g/kg feed protein) or protein-restricted (PR; 90 g/kg feed protein) diet throughout pregnancy and AIN76G during lactation. Offspring (twelve to seventeen per dietary group) were fed the C diet containing 4 or 10 g fat/100 g from weaning until postnatal day 105 when hearts were collected and frozen in liquid N2. Total RNA was prepared using the AllPrep DNA/ RNA Mini kit (Qiagen Ltd, Crawley, West Sussex, UK). RNA pools were prepared from each dietary group. Total RNA was analysed by an Agilent rat whole-genome oligo microarray (>41 000 genes; Oxford Gene Technology, Oxford, UK). Feature-extracted files were imported into GeneSpring GX (version 7.3.1; Agilent Technologies UK Ltd, Stockport, Ches., UK) and normalised using the Lowess signal-intensity-dependent normalisation method. mRNA expression of PPARa, carnitine palmitoyl transferase (CPT)-1, acyl-CoA oxidase (ACO), diacylglycerol acyltransferase 2 (DGAT2) and lipoprotein lipase (LPL) was measured by real-time RT–PCR. There was no significant effect of post-weaning fat intake and no interaction effect of maternal and post-weaning diet on any of the outcome measures, and so the results from offspring fed the different post-weaning diets were combined. Based on marginal means, RT–PCR analysis showed that the mRNA expression of PPARa and CPT-1 was 12% and 8% lower respectively (P>0.001) in the offspring of the PR dams than C offspring. ACO, DGAT2 and LPL expression was not significantly different between groups. Microarray analysis of pooled samples showed thirty-eight of sixty-two genes associated with the PPARa signaling pathway were altered in the offspring of the PR dams compared with C offspring. Eight genes exhibited up-regulation (40–60% increase), including ACO synthetase long-chain family member 4 and 5, and thirty genes were down regulated (50–280% increase), including ACO synthetase long-chain family member 1. Since PPARa regulates the use of fatty acids for energy production, these findings suggest that prenatal undernutrition may increase capacity for fatty acid uptake, but limits energy production by mitochondrial fatty acid b-oxidation. Since cardiomyopathy involves dysregulation of fatty acid metabolism, one implication of these findings is that nutritional constraint in early life may contribute to risk of heart failure.

Quantitative analysis of the methylation status of the hepatic PPAR-alpha promoter CPG island by pyrosequencing in offspring of rats fed a protein-restricted diet during pregnancy

Objectives: We have previously shown that cyclin G1 expression is reduced in fetal hearts after in utero protein restriction (PR) suggesting reduced cardiac cell cycle. However no difference in cyclin G1 expression was seen in adult offspring hearts. We hypothesised that the hearts of adult PR group should be under greater stress to maintain cardiac output. We therefore measured brain natriuretic peptide (BNP) expression in fetal hearts and left ventricles of adult offspring in the control (C) and PR groups because BNP is a marker of left ventricular dysfunction during volume overload or cardiac fibrosis (Nishikimi et al. Cardiovasc Res. 2006). Methods and results: Pregnant CD1 mice were placed on C (18% casein) or PR (9% casein) diet. Fetal hearts were collected on day 12 of gestation (C, n =11, PR, n =10) and the left ventricles (LV) of adult offspring at 6 months (C, n =17, PR, n =17). Fetal heart BNP mRNA expression relative to unit total RNA as measured by real-time PCR was similar in C and PR (C, 0.858F0.104 vs. PR, 0.761F0.096, p =NS). However, BNP expression in adult LV was greater in the PR than C (C, 7.043F0.68 vs. PR, 11.012F1.54, p =0.04). Conclusion: These results indicate that protein restriction in pregnancy induces cellular changes (indicated by cyclin G1 changes) in the fetal heart which places it under stress in adulthood (elevated BNP production). Because BNP can suppress ventricular remodelling, we are presently investigating cardiac structural changes to assess whether these alterations are adaptive or maladaptive.Objectives: Multiple pregnancy affects size at birth and growth pattern from as early as 8 weeks gestation (Iffy et al., 1983. Am. J. Obstet. Gynecol. 146, 970—972). Male embryos grow at a greater rate than females (Pedersen, 1980. Br. Med. J. 281, 1253). We hypothesised that moderate maternal undernutrion in early gestation will have a greater effect on male offspring growth, particularly if combined with the increased constraint of being a twin. Methods: Welsh Mountain ewes received 100% (C, n =41) or 50% nutrient requirements (U, n =47) from 1 to 31 days gestation (dGA), and 100% thereafter. Ewes were weighed weekly and blood samples were collected at 1, 30, and 65 dGA for cortisol analysis (Immulite analyser, DPC). Results: At day 31, U ewes had gained less weight than C ewes and had a lower plasma cortisol concentration ( p b0.05). During 1—31 dGA, twin bearing ewes gained less weight than singleton bearing ewes. At birth, twins were smaller than singleton lambs ( p b0.05). Weight gained between birth and 12 weeks old and weight at 12 weeks old were greater in U males compared to C males, an effect that was predominantly in twins ( p b0.01). Data were analysed by ANOVA. Conclusion: The increased constraint of being a twin and a male embryo in a nutrient-restricted intrauterine environment induces a phenotype more likely to gain weight in a good postnatal environment. Supported by the British Heart Foundation.

The effect of prenatal under-nutrition on the expression of DNA methyltransferases and methyl CPG binding protein 2 in the liver after weaning

Objectives: We have previously shown that cyclin G1 expression is reduced in fetal hearts after in utero protein restriction (PR) suggesting reduced cardiac cell cycle. However no difference in cyclin G1 expression was seen in adult offspring hearts. We hypothesised that the hearts of adult PR group should be under greater stress to maintain cardiac output. We therefore measured brain natriuretic peptide (BNP) expression in fetal hearts and left ventricles of adult offspring in the control (C) and PR groups because BNP is a marker of left ventricular dysfunction during volume overload or cardiac fibrosis (Nishikimi et al. Cardiovasc Res. 2006). Methods and results: Pregnant CD1 mice were placed on C (18% casein) or PR (9% casein) diet. Fetal hearts were collected on day 12 of gestation (C, n =11, PR, n =10) and the left ventricles (LV) of adult offspring at 6 months (C, n =17, PR, n =17). Fetal heart BNP mRNA expression relative to unit total RNA as measured by real-time PCR was similar in C and PR (C, 0.858F0.104 vs. PR, 0.761F0.096, p =NS). However, BNP expression in adult LV was greater in the PR than C (C, 7.043F0.68 vs. PR, 11.012F1.54, p =0.04). Conclusion: These results indicate that protein restriction in pregnancy induces cellular changes (indicated by cyclin G1 changes) in the fetal heart which places it under stress in adulthood (elevated BNP production). Because BNP can suppress ventricular remodelling, we are presently investigating cardiac structural changes to assess whether these alterations are adaptive or maladaptive.Objectives: Multiple pregnancy affects size at birth and growth pattern from as early as 8 weeks gestation (Iffy et al., 1983. Am. J. Obstet. Gynecol. 146, 970—972). Male embryos grow at a greater rate than females (Pedersen, 1980. Br. Med. J. 281, 1253). We hypothesised that moderate maternal undernutrion in early gestation will have a greater effect on male offspring growth, particularly if combined with the increased constraint of being a twin. Methods: Welsh Mountain ewes received 100% (C, n =41) or 50% nutrient requirements (U, n =47) from 1 to 31 days gestation (dGA), and 100% thereafter. Ewes were weighed weekly and blood samples were collected at 1, 30, and 65 dGA for cortisol analysis (Immulite analyser, DPC). Results: At day 31, U ewes had gained less weight than C ewes and had a lower plasma cortisol concentration ( p b0.05). During 1—31 dGA, twin bearing ewes gained less weight than singleton bearing ewes. At birth, twins were smaller than singleton lambs ( p b0.05). Weight gained between birth and 12 weeks old and weight at 12 weeks old were greater in U males compared to C males, an effect that was predominantly in twins ( p b0.01). Data were analysed by ANOVA. Conclusion: The increased constraint of being a twin and a male embryo in a nutrient-restricted intrauterine environment induces a phenotype more likely to gain weight in a good postnatal environment. Supported by the British Heart Foundation.