J.L. Slater-Jefferies

Epigenetic Gene Promoter Methylation at Birth Is Associated With Child’s Later Adiposity

OBJECTIVE Fixed genomic variation explains only a small proportion of the risk of adiposity. In animal models, maternal diet alters offspring body composition, accompanied by epigenetic changes in metabolic control genes. Little is known about whether such processes operate in humans. RESEARCH DESIGN AND METHODS Using Sequenom MassARRAY we measured the methylation status of 68 CpGs 5′ from five candidate genes in umbilical cord tissue DNA from healthy neonates. Methylation varied greatly at particular CpGs: for 31 CpGs with median methylation ≥5% and a 5–95% range ≥10%, we related methylation status to maternal pregnancy diet and to child’s adiposity at age 9 years. Replication was sought in a second independent cohort. RESULTS In cohort 1, retinoid X receptor-α (RXRA) chr9:136355885+ and endothelial nitric oxide synthase (eNOS) chr7:150315553+ methylation had independent associations with sex-adjusted childhood fat mass (exponentiated regression coefficient [β] 17% per SD change in methylation [95% CI 4–31], P = 0.009, n = 64, and β = 20% [9–32], P < 0.001, n = 66, respectively) and %fat mass (β = 10% [1–19], P = 0.023, n = 64 and β =12% [4–20], P = 0.002, n = 66, respectively). Regression analyses including sex and neonatal epigenetic marks explained >25% of the variance in childhood adiposity. Higher methylation of RXRA chr9:136355885+, but not of eNOS chr7:150315553+, was associated with lower maternal carbohydrate intake in early pregnancy, previously linked with higher neonatal adiposity in this population. In cohort 2, cord eNOS chr7:150315553+ methylation showed no association with adiposity, but RXRA chr9:136355885+ methylation showed similar associations with fat mass and %fat mass (β = 6% [2–10] and β = 4% [1–7], respectively, both P = 0.002, n = 239). CONCLUSIONS Our findings suggest a substantial component of metabolic disease risk has a prenatal developmental basis. Perinatal epigenetic analysis may have utility in identifying individual vulnerability to later obesity and metabolic disease.

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.

Effect of sex and dietary fat intake on the fatty acid composition of phospholipids and triacylglycerol in rat heart

Variations in the fatty acid composition of lipids in the heart alter its function and susceptibility to ischaemic injury. We investigated the effect of sex and dietary fat intake on the fatty acid composition of phospholipids and triacylglycerol in rat heart. Rats were fed either 40 or 100 g/kg fat (9:1 lard:soybean oil) from weaning until day 105. There were significant interactive effects of sex and fat intake on the proportions of fatty acids in heart phospholipids, dependent on phospholipid classes. 20:4n-6, but not 22:6n-3, was higher in phospholipids in females than males fed a low, but not a high, fat diet. There was no effect of sex on the composition of triacylglycerol. These findings suggest that sex is an important factor in determining the incorporation of dietary fatty acids into cardiac lipids. This may have implications for sex differences in susceptibility to heart disease.

RRR-alpha-tocopherol supplementation has pro- and anti-inflammatory and pro- and antioxidant effects in peripheral blood mononuclear cells in healthy middle-aged men depending upon dosage and genotype for xenobiotic metabolising enzymes

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.

Influence of superoxide dismutase polymorphism and overweight status on clinical outcome and oxidant production and stress in healthy middle-aged men and rheumatoid patients receiving high-dose RRR-alpha-tocopherol supplementation

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.

Feeding a protein-restricted diet during pregnancy in the rat induces changes to DNA methylation in the liver of the F-1 and F-2 offspring after weaning.

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.

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.