Best Practice & Research Clinical Endocrinology & Metabolism

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1 Best Practice & Research Clinical Endocrinology & Metabolism 26 (2012) Contents lists available at SciVerse ScienceDirect Best Practice & Research Clinical Endocrinology & Metabolism journal homepage: 8 Epigenetic mechanisms linking early nutrition to long term health Karen A. Lillycrop, Reader in Epigenetics a, *, Graham C. Burdge, Reader in Human Nutrition b a Centre for Biological Sciences, Institute of Developmental Sciences, Faculty of Natural and Environmental Sciences, University of Southampton, Southampton SO16 6YD, UK b Academic Unit of Human Health and Development, Faculty of Medicine University of Southampton, Southampton SO16 6YD, UK Keywords: epigenetics nutrition transcription early life Traditionally it has been widely accepted that our genes together with adult lifestyle factors determine our risk of developing noncommunicable diseases such as type 2 diabetes mellitus, cardiovascular disease and obesity in later life. However, there is now substantial evidence that the pre and early postnatal environment plays a key role in determining our susceptible to such diseases in later life. Moreover the mechanism by which the environment can alter long term disease risk may involve epigenetic processes. Epigenetic processes play a central role in regulating tissue specific gene expression and hence alterations in these processes can induce long-term changes in gene expression and metabolism which persist throughout the lifecourse. This review will focus on how nutritional cues in early life can alter the epigenome, producing different phenotypes and altered disease susceptibilities. Ó 2012 Elsevier Ltd. All rights reserved. Abbreviations: A vy, Agouti viable yellow; CVD, cardiovascular disease; CpG, cytosine and guanine nucleotides linked by phosphate; DMR, differentially methylated region; Dnmt, DNA methyl transferase; GR, glucocorticoid receptor; HDAC, histone deacetylase; HNF4a, Hepatocyte nuclear factor 4a; HFD, high-fat diet; HMT, histone methyl transferase; HOTAir, HOX transcript antisense RNA; 11bHSDII, 11b-hydroxysteroid dehydrogenase type II; IAP, intracisternal-a particle; IGF2, insulin like growth factor 2; Il13 ra2, interleukin 13 receptor a3; IUGR, intrauterine growth restricted; k, lysine; MeCP, methyl CpG binding protein; mirna, microrna; ncrna, non-coding RNA; NCD, non-communicable disease; PAR, predictive adaptive response; PEPCK, phosphoenolpyruvate carboxykinase; Pdx1, pancreatic and duodenal homeobox 1; POMC, Pro-opiomelanocortin; PR, protein restricted; PPAR, peroxisomal proliferator-activated receptor; RXRa, retinoid X receptora; Xist, X-inactive specific transcript. * Corresponding author. Tel.: þ44 (0) ; Fax: þ44 (0) address: kal@soton.ac.uk (K.A. Lillycrop) X/$ see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi: /j.beem

2 668 K.A. Lillycrop, G.C. Burdge / Best Practice & Research Clinical Endocrinology & Metabolism 26 (2012) Introduction Non-communicable disease (NCD) such as diabetes mellitus, cardiovascular disease (CVD) and obesity, account for 60% of all deaths globally. The incidence of NCDs has risen sharply over the past two decades. This increase in NCDs is not restricted to industrialised nations but is becoming particularly prevalent in developing nations as those countries undergo socioeconomic improvement. 1 Although it is widely established that genotype in combination with adult lifestyle factors are critical determinants of NCD risk, there is increasing recognition that fixed genomic variations only account for a small proportion of the variation in NCD risk 2 and that the rise in incidence of NCDs has occurred too rapidly to be explained solely by such factors. There is now substantial evidence that the fetal and early postnatal environment strongly influences the risk of developing NCD and that epigenetic processes play a critical role in the mechanism by which early life environment influences future disease risk. This review will focus on the evidence that early life nutrition can induce the altered epigenetic regulation of genes leading to persistent changes in metabolism and physiology, and as a consequence altered disease susceptibility. Early life environment and future disease risk The association between the quality of the early life environment and future risk of adult disease was first described by Forsdahl in 1977, who found that infant mortality rates were positively associated with an increased risk of CVD in middle age. 3 Subsequent studies in the UK by David Barker and colleagues found an inverse relationship between birth weight and increased CVD mortality. 4 Numerous retrospective epidemiological studies have since confirmed the association between low birth weight and later risk of CVD and shown that low birth weight is also associated with an increased risk of obesity, hyperytension and type 2 diabetes mellitus. 5 However, in all of these studies, birth weight is thought to represent a very crude indicator of the intrauterine environment, which may have been compromised through a variety of maternal, environmental or placental factors. 6 The effect in particular of early life nutrition on subsequent disease susceptibility has however been most clearly shown in studies of the Dutch Hunger Winter, a famine which occurred in the Netherlands during the winter of These studies have shown that individuals whose mothers were exposed to famine periconceptually and in the first trimester of pregnancy did not have reduced birth weights compared to unexposed individuals, but did as adults exhibit an increased risk of obesity and CVD, whereas individuals whose mothers were exposed in the later stages of gestation had reduced birth weights and showed increased incidence of insulin resistance and hypertension. 7 However it is not just under-nutrition which has long term phenotypic effects, over-nutrition in early life is also associated with an increased susceptibility to metabolic disease which may account for the U-shaped or J-shaped relationships observed in a number of studies between birth weight and risk of obesity or insulin resistance in later life. 8,9 Animal models of nutritional programming These findings from the human epidemiological studies have been replicated in a variety of animal models which have generally used either rats or mice fed either an isocaloric low protein diet, global dietary restriction or a high-fat diet during pregnancy and/or lactation. Interestingly, offspring born to dams fed these different diets exhibit to varying extents characteristics of humans with cardiometabolic disease including obesity, insulin resistance, hypertension and raised serum cholesterol levels. For instance feeding rats a protein restricted diet (PR) during pregnancy has been reported to result in impaired glucose homeostasis, 10 vascular dysfunction, 11 impaired immunity, 12 increased susceptibility to oxidative stress, 13 increased fat deposition and altered feeding behavior. 14,15 The induction during early life of persistent changes to the physiology of the offspring by alterations in maternal diet implies the induction of long term changes to gene expression which in turn results in the altered activities of metabolic pathways and homeostatic control processes. 16,17 For example, feeding a PR diet to pregnant rats increased glucocorticoid receptor (GR) expression and reduced expression of 11b-hydroxysteroid dehydrogenase type II (11bHSD)-2, the enzyme that inactivates

3 K.A. Lillycrop, G.C. Burdge / Best Practice & Research Clinical Endocrinology & Metabolism 26 (2012) corticosteroids, in liver, lung, kidney and brain in the offspring. 18 In the liver, increased GR activity upregulates phosphoenolpyruvate carboxykinase (PEPCK) expression and activity leading to an increased capacity for gluconeogenesis. 10 Long-term changes in gene expression have also been reported in adult offspring of dams fed a global under-nutrition diet during pregnancy. Gluckman et al. (2007) have shown that expression of PPARa and GR are both down-regulated in adult offspring born to dams fed a global nutrient-restricted diet of 30% ad libitum during pregnancy. 19 With the recent rise in rates of NCDs throughout the world, and concerns over western style diets, a number of new animal models of over-nutrition during pregnancy have also been developed. Feeding an obesogenic diet to female rats from before mating and through lactation has been shown to lead to maternal obesity as well as hyperphagia, increased adiposity, decreased muscle mass, reduced locomotive activity and accelerated puberty in the offspring. 20 There is also increasing evidence that the period of susceptibility extends into postnatal life as studies of rats in cross-fostering experiments show that high-fat feeding in the suckling period leads to an increase in adiposity, hyperleptinaemia, and hypertension in the adult offspring fed a normal diet after weaning. 21,22 Animal studies have also shown the potential importance of paternal diet in future NCD risk. Carone et al., 2010 showed that offspring of males, who were fed a low protein diet from weaning until sexual maturity, exhibited elevated hepatic expression of many genes involved in lipid and cholesterol biosynthesis, and decreased levels of cholesterol esters. 23 A number of mirnas were also persistently altered in the offspring (mirna 21, let-7, mir-199 and mir98, mir210), many of which are associated with cellular proliferation. Ng et al. (2010) have also shown that a paternal high-fat diet (HFD) led to increased body weight, adiposity, impaired glucose tolerance and insulin sensitivity in the female offspring. 24 Paternal HFD altered the expression of 642 pancreatic islet genes in adult female offspring; these genes included those involved in cation and ATP binding, cytoskeleton and intracellular transport. Developmental plasticity The induction of different phenotypes by perturbations in early life nutrition has been suggested by Gluckman and Hanson to reflect a predictive adaptive response (PAR) whereby the organism acting through the process of developmental plasticity can adjust its developmental programme in response to environmental cues to aid fitness/survival in later life. 25 For instance, poor maternal nutrition may signal to the fetus that nutrients are scarce; the fetus may then adapt its metabolism to conserve energy demands, increase its propensity to store fat and accelerate puberty. If the prediction is correct then the metabolism of the organism will be matched to the environment and that individual will be of low disease risk. In support of this, there is evidence in both rat and pig models of maternal overnutrition during pregnancy followed by continued high-fat feeding in postnatal life, that this does not lead to deleterious effects. 26,27 However, if the prediction is incorrect, the organism s metabolism will be mismatched to its environment and that individual would have an increased risk of metabolic disease. This would explain why human populations undergoing socioeconomic change or migration from rural to urban areas show increased risk of chronic disease. There is now increasing evidence that the mechanism by which an organism can produce different phenotypes from a single genome in response to the environment is through the altered epigenetic regulation of genes. Epigenetics Epigenetic processes are integral in determining when and where specific genes are expressed. Alterations in the epigenetic regulation of genes can lead therefore to profound changes in phenotype. 28,29 The major epigenetic processes are DNA methylation, histone modification and non-coding RNAs. DNA methylation Methylation at the 5 position of cytosine in DNA within a CpG dinucleotide (the p denotes the intervening phosphate group) is a common modification in mammalian genomes and constitutes a stable epigenetic mark that is transmitted through DNA replication and cell division. 30 CpG

4 670 K.A. Lillycrop, G.C. Burdge / Best Practice & Research Clinical Endocrinology & Metabolism 26 (2012) dinucleotides are not randomly distributed throughout the genome but are clustered at the 5 0 ends of genes in regions known as CpG islands. Hypermethylation of these CpG islands is associated with transcriptional repression, while hypomethylation of CpG islands is associated with transcriptional activation. 31,32 DNA methylation is important for asymmetrical silencing of imprinted genes, 33 X chromosome inactivation 34,35 and for cell specification and tissue specific gene expression. 30 Methylation of CpGs is largely established during embryogenesis or in early postnatal life. Following fertilisation, maternal and paternal genomes undergo extensive demethylation followed by global methylation de novo just prior to blastocyst implantation 36,37 during which 70% of CpGs are methylated, mainly in repressive heterochromatin regions and in repetitive sequences such as retrotransposable elements. 38 Lineage-specific methylation of tissue specific genes occurs throughout prenatal development and early postnatal life and determines the developmental fates of differentiating cells. The methylation of CpG dinucleotides de novo is catalyzed by the DNA methyl transferases (Dnmt) 3a and 3b, and is maintained through mitosis by gene-specific methylation of hemi-methylated DNA by Dnmt1. 39 DNA methylation however once established is essentially maintained throughout life, although gradual hypomethylation does occur during aging and such age-related alterations in methylation have been linked with cancer. 40 Histone modification The DNA in our cells is packaged as chromatin. The basic unit of chromatin is a nucleosome which comprises 147bp of DNA wrapped around a core of histone proteins (two copies of histone H2A, H2B, H3 and H4). Histone proteins contain 2 domains - a globular domain and an N terminal tail domain. The N terminal tails of the histones are subject to modifications including acetylation, methylation, ubiquitination, sumoylation, and phosphorylation. 41 Histone modification leads to the recruitment of effector proteins which in turn bring about specific cellular processes. The establishment of these marks on the histone tails is often referred to as the histone code. Histone acetylation is exclusively associated with active chromatin states, while the methylation of lysines (K) can either be an active or repressive mark depending on the specific lysine involved. 41 Many families of histone-modifying enzymes have been identified, the so called writers of the code include the histone acetyl transferases, and methyl transferases, while the erasers include the deacetylases, and demethylases. 42,43 Crosstalk between DNA methylation and histone modification clearly occurs. Methylated DNA is bound by Methyl CpG binding protein-2 (MeCP2) which can recruit both histone deacetylases (HDACs), which remove acetyl groups from the histones, a signal of transcriptionally active chromatin and histone methyl transferases (HMTs) such as Suv39H 44 which methylates lysine 9 on H3, resulting in a closed chromatin structure and transcriptional silencing. Recent studies have however shown that Dnmt1 is recruited by a number of histone-modifying enzymes such as HDAC1 and HDAC2, and the histone methyl transferases SUV39 and EZH2, 45,46 suggesting that chromatin structure may also determine DNA methylation status and that there is a reciprocal relationship between these two processes. Non-coding RNAs Non-coding RNAs (ncrnas) have also been implicated in the epigenetic regulation of gene expression. Non-coding RNAs can either act in cis or in trans The cis-acting ncrnas are the long/macroncrnas (up to 100,000 nt), which include Xist and HOTAir, Xist plays a pivotal role in X chromosome inactivation where it coats the inactive X chromosome and directs histone H3K27 and DNA methylation leading to gene silencing. The trans acting ncrnas include the micrornas, these mostly target the 3 0 untranslated region of mrnas for degradation. 47 However more recent studies have shown that the human mirnas can also induce chromatin remodelling 48,49 ), suggesting that DNA methylation, histone modification and mirnas may work in concert to regulate gene expression. Early life nutrition and altered epigenetic regulation DNA methylation is the most stable epigenetic mark. However there is growing evidence that the epigenome is susceptible to a number of environmental factors during the prenatal, and early postnatal

5 K.A. Lillycrop, G.C. Burdge / Best Practice & Research Clinical Endocrinology & Metabolism 26 (2012) periods. Moreover the changes once induced by environmental factors then appear to be maintained throughout life leading to long term changes in phenotype. One of the best examples of how diet can alter the phenotype is seen in studies on the honeybee. Female larvae fed different diets develop into either sterile worker bees or fertile queen bees, even though they are genetically identical. 50 However, the proportion of larvae developing into queen bees compared to sterile worker bees was significantly increased when the expression of DNA methyl transferase 3 (Dnmt3) was silenced, suggesting that DNA methylation plays a key role in nutritional programming of phenotype. 50 Alterations in DNA methylation have also been linked to nutritional programming of the phenotype in rodents. Differences in the intake of methyl donors and the cofactors for 1-carbon metabolism during pregnancy altered DNA methylation and induce differences in the coat colour of the offspring. The murine A vy mutation results from the insertion of an intracisternal-a particle (IAP) retrotransposon upstream of the agouti gene, which regulates the production of yellow pigmented fur. Supplementation of pregnant mice with betaine, choline, folic acid and vitamin B 12 led to increased methylation of the agouti gene and shifted the distribution of coat colour of the offspring from yellow (agouti) to brown (pseudo-agouti). 51 There is also evidence that in models of nutritional programming where perturbations in maternal diet are associated with an altered metabolic phenotype, that changes in the epigenetic control of a number of key regulators of lipid and glucose metabolism occur. Feeding pregnant rats a PR diet induced hypomethylation of the GR and PPARa promoters in the livers of juvenile and adult offspring which was associated with increased mrna expression of these genes. 16,52 This was the first evidence that moderate changes in macronutrient intake during pregnancy can alter the epigenome. Increased expression was associated with an increase in acetylation of histones H3 and H4 and methylation of histone H3 at lysine K4 53 Sequencing analysis of the PPARa promoter showed that four specific CpGs were hypomethylated, and that two CpGs located within transcription factor response elements predicted the level of the transcript. 54 Thus the effects of the maternal PR diet on the offspring are targeted to specific CpGs. In contrast to the effect of the maternal PR diet, adult female offspring of dams which experienced 70% reduction in total nutrient intake during pregnancy showed hypermethylation and decreased expression of the GR and PPARa promoters in the liver. 19 Thus, the effects of maternal nutrition on the epigenome of the offspring depend upon the nature of the maternal nutrient challenge. Similarly alterations in paternal diet have also been associated with altered DNA methylation in the offspring. Reduced representation bisultifte sequence analysis on DNA from the liver of offspring whose fathers were fed a low protein diet prior to mating, revealed widespread modest changes in DNA methylation (10 20%) between the control and paternal low protein fed offspring, including a substantial increase in methylation at an intergenic CpG island 50 kb upstream of the PPARa gene. 55 Ng et al. (2010) have also shown that the interleukin 13 receptor alpha 2 (Il13ra2) promoter was hypomethylated in female offspring after high-fat feeding of the fathers. 56 Mechanisms for nutritional induced changes in the epigenome The mechanism by which nutrition in early life induces changes in the epigenome is unknown. DNA methylation was thought to be a very stable modification and that demethylation only occurred passively through a failure to maintain DNA methylation patterns through cell division. However active demethylation has been observed 39,57,58 and a number of DNA demethylases have been proposed. 59,60,61,62 Thus, environmental exposures which alter the activity of the Dnmts and/or demethylases provide a possible mechanism by which a change in methylation could occur and interestingly Dnmt1 expression has been shown to be down-regulated in the liver of PR offspring (Fig. 1). 53 However with the knowledge that DNA methylation and histone modification are intimately linked, a number of groups have explored the role of histone changes in phenotype induction and the relationship between DNA methylation and histone modification. Park et al., have shown that in a model of IUGR a persistent decrease in Pdx1 expression is mediated through a series of epigenetic changes, 63 neonatal histone modifications at the Pdx1 promoter was followed in adulthood by methylation of the CpG island in the promoter of Pdx1 and permanent gene silencing. Moreover the analysis of the

6 672 K.A. Lillycrop, G.C. Burdge / Best Practice & Research Clinical Endocrinology & Metabolism 26 (2012) Nutrition in early life (prenatal, neonatal and puberty) induces long term changes in gene expression and phenotype (Protein restriction, Global restriction. Over nutrition, micronutrient intake) K9-me K9-me DNA demethylases? HATs K9-ac K9-ac K9-ac K9-ac Me Me Me Inactive gene HDACs Dnmts HMTs Active gene mirnas mrna Fig. 1. Effect of early life nutrition on the epigenome. Nutrition in early life induces long term changes in gene expression and phenotype though the altered epigenetic regulation of genes. Nutritional status affects the balance between gene methylation and demethylation. Histone deacetylases (HDACs), histone methyl transferases (HMTs) and DNA methyl transferases (Dnmts), promote histone deacetylation, the methylation of K9 on histone H3 and DNA methylation resulting in a closed chromatin configuration and gene silencing. mirnas expression can also be affected leading to decreased mrna stability or repression of translational. In contrast, histone acetyl transferases (HATs) promote the acetylation of lysine residues within the histone tails including acetylation of H3K9 resulting in an open chromatin configuration and gene transcription. DNA demethylases have been postulated to exist and may also participate in promoting gene transcription but no clear demethylase enzyme has yet been described. HNF4a locus in control and maternal low protein fed offspring showed that while only small changes in DNA methylation at the proximal promoter and enhancer of HNF4a were observed, large changes in the repressive histone mark H3K27 were seen at the HNF4a locus. 64,65 Interestingly initial studies have implicated altered H3K27 methylation in the induction of an altered phenotype through the paternal line. Carone et al., found that in the sperm of control and low protein fed males DNA methylation patterns, including the enhancer region of PPARa which was subsequently demethylated in the juvenile offspring, were very similar, they did observed a consistent decrease in H3K27me3 in the sperm of the low protein fed males compared to control males. 55 Together then these findings suggest that histone modifications especially histone methylation may play a key role in the response of the epigenome to the environment and potentially in the transmission of the signal from parent to offspring. Recent advances in next generation sequencing technologies will enable further clarifications of the environmentally susceptible loci both at the level of DNA and histone methylation and the relationship between the two. Evidence that early nutrition can alter the epigenome in humans There is also now growing evidence that early life nutrition can induce persistent changes in DNA methylation not only in animals but also in humans. Heijmans et al. (2008) has reported hypomethylation of the imprinted IGF2 gene in genomic DNA isolated form whole blood from individuals who were exposed to famine in utero during the Dutch Hunger Winter compared to unexposed samesex siblings. 66 Tobi et al also found that insulin like growth factor was hypomethylated in individuals whose mothers were periconceptually exposed to famine, while interleukin-10, leptin, ATP binding cassette A1, guanine nucleotide binding protein were hypermethylated. 67 Steegers-Theunissen et al.

7 K.A. Lillycrop, G.C. Burdge / Best Practice & Research Clinical Endocrinology & Metabolism 26 (2012) have also shown that differential methylation of5 CpGs in the IGF2 DMR in children whose mothers did or did not take 400 mg of folic acid per day in the periconceptional period. 68 The change in DNA methylation in these human studies are far smaller than those reported in the animal studies, this may reflect the fact that in the animal studies, both the environmental stimulus and the postnatal environment are highly controlled. If a similar mechanism does operate in humans as in animals and early life environment can induce epigenetic alterations in the fetus which then persist throughout the lifecourse, characterisation of such altered epigenetic marks in early life may allow the identification of those individuals at increased risk of developing NCD in later life. Godfrey et al., has recently reported in two independent cohorts, the methylation status of a single CpG site in the promoter region of the transcription factor RXRA was strongly related to childhood adiposity in both boys and girls 69 ; RXRA promoter methylation explained over a fifth of the variance in childhood fat mass, suggesting that epigenetic alterations may contribute a far greater proportion of an individual s risk to NCD than has previously thought. Interestingly these measurements were made in umbilical cord, suggesting that methylation levels in cord and perhaps in other readily available tissue such as blood or buccal may provide useful proxy markers of methylation in more metabolically relevant tissues. Although tissue specific differences in gene methylation do clearly exist, if the environmental constraint occurred early on in development then this may affect all germ layers and an imprint of this altered epigenetic mark may be detectable in all tissues, while exposures occurring later on in gestation may only induce tissue specific methylation effects. It will certainly be important to further our understanding of how these altered marks are established, and their potential as biomarkers of future disease risk, to examine DNA methylation in tissues from the different germ layers in response to the timing and degree of environmental constraint. Interventions to prevent/reverse epigenetic and phenotypic changes A number of studies have shown that interventions to prevent and/or reverse the effects of early life environment on the epigenome and phenotype are possible. Treatment with leptin of neonatal rats born to dams exposed to 70% global reduction in food intake during pregnancy between postnatal days 3 and 13 reversed the induced phenotype and hypermethylation and increased expression of the PPARa and GR promoters in their livers. 19 Induction of an altered phenotype in the offspring of rats fed PR diet during pregnancy can be prevented by supplementation of the PR diet with glycine or folic acid Hypomethylation of the hepatic GR and PPARa promoters was also prevented by increasing the addition of folic acid content of the maternal PR diet 73 suggesting that impaired 1-carbon metabolism plays a central role in the induction of the altered epigenetic regulation of GR and PPARa and in the induced of an altered phenotype by maternal protein restriction. Interestingly folic acid supplementation of the diet of juvenile-pubertal rats born to dams fed a PR diet did not simply reverse the altered epigenotype or phenotype but induced a different pattern of epigenetic change 74 and further alterations in phenotype. Thus although the juvenile-pubertal period may represent a period of plasticity where it may be possible to reverse the adverse effect of prenatal nutrition, folic acid itself appears to have a deleterious effect down regulating fatty acid b oxidation and increasing weight gain. Summary There is now a considerable body of evidence to suggest that our genotype is not the sole determinant of disease risk but that variations in the quality of the early life environment affects future disease risk through the altered epigenetic regulation of genes. The demonstration of a role for altered epigenetic regulation of genes in the developmental induction of NCD together with the identification of potential epigenetic biomarkers of future disease risk suggest the possibility that individuals at increased risk could be identified at an early stage of the lifecourse and their long term risk of NCD modified either through nutritional or lifestyle interventions. However further understanding of the mechanism by which nutrition can modify the epigenome, the periods of epigenetic susceptibility, the nutritional factors that induce epigenetic changes and the stability of the induced changes are all critical for both the robust identification of individuals at risk and for the development of novel intervention strategies, to reverse this current epidemic of NCD.

8 674 K.A. Lillycrop, G.C. Burdge / Best Practice & Research Clinical Endocrinology & Metabolism 26 (2012) Practise points It is critical that there is a greater awareness of the importance of early life environment as a key determinant of future disease risk. Public information on what constitutes a healthy early life environment is needed. The use of predictive epigenetic biomarkers of NCD risk requires further studies to determine tissue specific differences in susceptible loci in response to environmental exposures. The effect of the postnatal environment and stability of the epigenetic marks also needs to be defined. Research agenda Further research using animal models is required to understand the mechanism and interplay between DNA methylation, histone and mirnas. The periods of susceptibility of the epigenome needs to be further defined. The use of next generation sequencing platforms to identify loci susceptible to environmental change is required. References 1. Ramachandran A & Snehalatha C. Rising burden of obesity in Asia. Journal of Obesity 2010: Manolio TA, Collins FS, Cox NJ et al. Finding the missing heritability of complex diseases. Nature 2009 Oct 8; 461(7265): Forsdahl A. Are poor living conditions in childhood and adolescence an important risk factor for arteriosclerotic heart disease? British Journal of Preventive & Social Medicine 1977 Jun; 31(2): Barker DJ, Winter PD, Osmond C et al. Weight in infancy and death from ischaemic heart disease. Lancet 1989 Sep 9; 2(8663): *5. Godfrey KM & Barker DJ. Fetal programming and adult health. Public Health Nutrition 2001 Apr; 4(2B): *6. Hanson MA & Gluckman PD. Developmental processes and the induction of cardiovascular function: conceptual aspects. Journal of Physiology 2005 May 15; 565(Pt 1): Painter RC, Roseboom TJ & Bleker OP. Prenatal exposure to the Dutch famine and disease in later life: an overview. Reprod Toxicol 2005 Sep; 20(3): Curhan GC, Willett WC, Rimm EB et al. Birth weight and adult hypertension, diabetes mellitus, and obesity in US men. Circulation 1996 Dec 15; 94(12): McCance DR, Pettitt DJ, Hanson RL et al. Birth weight and non-insulin dependent diabetes: thrifty genotype, thrifty phenotype, or surviving small baby genotype? British Medical Journal 1994 Apr 9; 308(6934): Burns SP, Desai M, Cohen RD et al. Gluconeogenesis, glucose handling, and structural changes in livers of the adult offspring of rats partially deprived of protein during pregnancy and lactation. Journal of Clinical Investigation 1997 Oct 1; 100(7): Torrens C, Brawley L, Anthony FW et al. Folate supplementation during pregnancy improves offspring cardiovascular dysfunction induced by protein restriction. Hypertension 2006 May; 47(5): Calder PC & Yaqoob P. The level of protein and type of fat in the diet of pregnant rats both affect lymphocyte function in the offspring. Nutrition Research 2000; 20(7): Langley-Evans SC & Sculley DV. Programming of hepatic antioxidant capacity and oxidative injury in the ageing rat. Mechanisms of Ageing and Development 2005 Jun; 126(6 7): Bellinger L, Lilley C & Langley-Evans SC. Prenatal exposure to a maternal low-protein diet programmes a preference for high-fat foods in the young adult rat. British Journal of Nutrition 2004 Sep; 92(3): Bellinger L, Sculley DV & Langley-Evans SC. Exposure to undernutrition in fetal life determines fat distribution, locomotor activity and food intake in ageing rats. International Journal of Obesity (London) 2006 May; 30(5): Lillycrop KA, Phillips ES, Jackson AA et al. 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. Proceedings of the Nutrition Society 2006; 65: 65A. 17. Maloney CA, Gosby AK, Phuyal JL et al. Site-specific changes in the expression of fat-partitioning genes in weanling rats exposed to a low-protein diet in utero. Obesity Research 2003 Mar; 11(3): Whorwood CB, Firth KM, Budge H et al. Maternal undernutrition during early to midgestation programs tissue-specific alterations in the expression of the glucocorticoid receptor, 11beta-hydroxysteroid dehydrogenase isoforms, and type 1 angiotensin II receptor in neonatal sheep. Endocrinology 2001 Jul; 142(7): Gluckman PD, Lillycrop KA, Vickers MH et al. Metabolic plasticity during mammalian development is directionally dependent on early nutritional status. The Proceedings of the National Academy of Sciences of the United States of America 2007 Jul 31; 104(31):

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