Developmental Plasticity Sensitive Periods and Risk of Obesity

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1 Developmental Plasticity Sensitive Periods and Risk of Obesity Karen A Lillycrop Institute of Developmental Sciences Centre for Biological Sciences University of Southampton

2 Genotype E P I G E N O M E Environment Environment Environment Chronic disease susceptibility (CVD, obesity, type II diabetes, cancer) 2

3 Early life environment can alter the epigenome Establishment of imprinting Erasure of parental methylation Cell lineage specific methylation Gametogenesis conception birth weaning Growth maturation aging Epigenome Prenatal Neonatal Pubertal Nutrition/ Body composition Maternal stress /childhood adversity Endocrine disruptors /pollution

4 What is the evidence that early life environment can induce epigenetic and phenotypic changes? High fat High fat Protein restriction Biochemical Journal , Matthew J. Warner and Susan E. Ozanne

5 Maternal protein restriction induces the hypomethylation of the PPARα Promoter in the liver of the offspring Maternal protein restriction Hypertension Dyslipideamia Insulin resistance Hepatic PPAR methylation PN34 DNA methylation compared to control (%) * Control PR Maternal dietary group mrna concentration compared to control (%) PPAR expression PN34 * Control PR Maternal dietary group hyroxybutyrate concentration ( mol/l) Β oxidation PN34 con PR maternal dietary group Lillycrop et al., 2005,

6 Maternal protein restriction induces a change in PPAR α methylation in fetal liver % Methylation ( mean ±SD) Con PR * ** Con PR Con * * * * PR E18 PN34 PN84

Maternal high fat feeding alters the methylation of the FADS2 gene Female Liver 20:4n-6 % Total Fatty acids 40 30 20 10 7% Butter 21% Butter 7% Fish Oil 21% Fish oil 0 7% Butter 21% Butter 7% Fish

7 Maternal high fat feeding alters the methylation of the FADS2 gene Female Liver 20:4n-6 % Total Fatty acids % Butter 21% Butter 7% Fish Oil 21% Fish oil 0 7% Butter 21% Butter 7% Fish Oil 21% Fish oil Female Hepatic FADS2 expression CpG -394 Log DD Ct (mean SD) % Butter 21% Butter 7% Fish Oil 21% Fish oil % methylation 7% 80Butter 21% Butter 7% 60Fish Oil 21% Fish oil % Butter 21% Butter CpG % Fish Oil 21% Fish oil Expression (normalised Ct) 3 r 2 = 0.56, P< % Butter 21% Butter 2 7% Fish Oil 21% Fish oil CpG methylation 7 7

8 Over feeding in early postnatal life leads to obesity and the hypermethylation of the POMC promoter Rat pups raised in small litters (SL) POMC methylation in the hypothalamus (PN21) Rapid fat accumulation Hyperglyceamia Obesity NL SL Plagemann et al., 2009

Nutrition in early life can induce long term changes in DNA

sperm IL-13R, PPARa GR, PPAR, PEPCK HNF4,ATR1 GR, PPARa FADS2

High Fat Protein restriction Protein restriction Global

9 Nutrition in early life can induce long term changes in DNA methylation conception birth weaning Growth maturation aging sperm IL-13R, PPARa GR, PPAR, PEPCK HNF4,ATR1 GR, PPARa FADS2 Epigenome POMC Long term changes in gene expression & metabolism High Fat Protein restriction Protein restriction Global restriction High fat Over feeding Paternal diet Maternal diet Altered disease risk

The Dutch Hunger Winter A period of severe food shortage in the Netherlands in 1944.

Individuals born to women exposed to famine during pregnancy have an increased risk of CVD, T2D and obesity in later life

10 The Dutch Hunger Winter A period of severe food shortage in the Netherlands in Energy intakes dropped from 1800 to between 400 and 800 kcal per day (equivalent g pasta). Individuals born to women exposed to famine during pregnancy have an increased risk of CVD, T2D and obesity in later life Alterations in DNA methylation has been shown in individuals from mothers who Were exposed to famine during pregnancy compared to their non exposed siblings IL10 GNASAS IGF2 Lep ABCA1 MEG3 Tobi et al., 2009

11 Developmental plasticity Same genome, different phenotype Early embryo Normal Adapted Future Actual Postnatal Environment e.g. High Calorie Diet Developmental Plastic Stage prenatal environment High Disease Risk MISMATCH Environmental Cue e.g. Poor Nutrition Reduce energy demands Increase capacity for fat storage Less investment in muscle mass Response Alternative Developmental Path Future Actual Postnatal Environment e.g. Poor Nutrition Waddington 1942 Low Disease Risk MATCH 11

12 conception birth weaning Growth maturation aging GR, PPAR, PEPCK P16, p21,hnf4,atr1 GR, PPARa FADS2 p16 Epigenome POMC FADS2 Long term changes in gene expression & metabolism Protein restriction Global restriction High fat Over feeding Folic acid Detect changes in methylation Altered disease risk Predict metabolic capacity & future disease risk

13 Epigenetic marks as biomarkers? In humans limited tissue availability? Available tissues: Umbilical cord Cord blood Placenta buccal cells Blood

14 Methylation at the retinoid X receptor α (RXRA) promoter at birth vs child s fat mass PAH children age 9 yrs SWS children age 6 yrs r= 0.32 P=0.009 n=64 r= P=0.002 n= Child's fat mass age and sex adjusted (kg) Child's fat mass age and sex adjusted (kg) Umbilical cord RXRA methylation Umbilical cord RXRA methylation Godfrey et al., 2011 Values are means + SEM

Genome wide methylation array Methyl DNA capture Genomic DNA from SWS subjects sonicated and

Array 60 mer probes spaced across promoter regions of 17,000 best characterised transcripts covering

the Bayesian algorithm BATMAN Differentially methylated Regions (DMRs 100 nt) identified (Fishers

15 Genome wide methylation array Methyl DNA capture Genomic DNA from SWS subjects sonicated and precipitated using His-tagged MBD2b to enrich for methylated sequences Hybridised to Human Promoter Array 60 mer probes spaced across promoter regions of 17,000 best characterised transcripts covering -8kb to +2kb downstream of TSS DATA analysis Methylation levels of 100nt regions were estimated using the Bayesian algorithm BATMAN Differentially methylated Regions (DMRs 100 nt) identified (Fishers Exact test) associated with % fat mass age 6 yrs 93 DMRs identified, associated with changes in % fat mass age 6 years

16 Among the identified DMRs, the top pathway enriched was DNA replication & repair Confidential This pathway includes a DMR linked with cyclin-dependent kinase inhibitor 2A (CDKN2A) UCN IDH1 Igm CD40 MapK GATA6 AKAP8 SAA1 Akt Jnk ENDRB HLA-C CDKN2A CCND1 P13K complex capsase Hintone H3 APP GAPDH PCPB2 E2f cyclin A SP1 NFKb complex RNA pol II 26s proteosome SIAH1 OAS1 PCNA NGFR Alpha tubulin FEN1 VMP1 GTPBP4 ID1

17 CDKN2A Encodes for 2 cell cycle inhibitors p14 ARF and P16 INK4a, which play roles in cell proliferation, differentiation and senescence

18 Lower umbilical cord CDKN2A methylation is associated with higher child s % fat mass (SWS children) At 6 years CpG1 CpG3 CpG4 CpG6 P=0.003 n=208 P=0.003 n-204 P=0.005 n=231 P=0.015 n=230 At 4 years P=0.007 n=221 P=0.007 n=215 P=0.007 n=247 P=0.04 n=244

Lower CDKN2A methylation at birth associated with greater ponderal index at age 18 months in 161 GUSTO infants, reflecting higher adiposity P=0.

19 Lower CDKN2A methylation at birth associated with greater ponderal index at age 18 months in 161 GUSTO infants, reflecting higher adiposity P=0.015 Perinatal CDKN2A methylation in an independent cohort from Singapore with adiposity data at age 18 months Lillycrop & Godfrey et al, unpublished

20 Epigenetic biomarkers Temporal stability of DNA methylation Recent data suggests that DNA methyation can be dynamically regulated

Methylation stability over time The Early Bird Study Longitudinal

Annual measurements include physical activity, body composition and insulin

co-activator (PGC))-1α Energy homeostasis Muscle fibertype switching

21 Methylation stability over time The Early Bird Study Longitudinal non-intervention study of healthy children between ages 5 to 16. Annual measurements include physical activity, body composition and insulin sensitivity and annual whole blood samples taken Sirt1/PGC-1a (PPAR gamma co-activator (PGC))-1α Energy homeostasis Muscle fibertype switching Mitochondrial biogenesis Sirt1 (energy sensor) PGC1α HNF4a Adipogenesis Glucose metabolism Insulin secretion

22 Methylation stability over time M e th y la tio n (% ) M e th y la tio n (% ) Sirt1 8 0 PGC1a HNF4a C p G C p G A g e (y e a r s ) A g e (y e a r s ) Methylation not affected by the proportion of neutrophils or lymphocytes (all P > 0.1; neutrophils r = to 0.34, lymphocytes r=0.001 to 0.23).

Methylation of CpG loci in PGC-1α was associated with adiposity at 14 years Significant association between the methylation of 4 of the 7 CpG s at 5-7

23 Methylation of CpG loci in PGC-1α was associated with adiposity at 14 years Significant association between the methylation of 4 of the 7 CpG s at 5-7 years and % body fat from 9-14 years (P<0.05). For each 10% difference in methylation at 5-7 years, % body fat differed by 6.3% to 12.5% (P<0.03) at age 14.

24 Does differential methylation of the CpG loci within PGC1a have functional significance? Methylation increases binding of the pro-adipogenic HOXB9/PBX1 heterodimer.

Early life environment is critical for future health

Maternal/paternal Nutrition Maternal behavior

Early life environment is an important determinant of

Early life environment can alter the epigenome and

significant contribution to later phenotype and

25 Early life environment is critical for future health In vitro Culture? Maternal/paternal Nutrition Maternal behavior Nutrition 1.Early life environment is an important determinant of later obesity risk 2.Early life environment can alter the epigenome and these epigenetic changes induced in early life make a significant contribution to later phenotype and disease susceptibility. 3.Detection of such altered epigenetic marks in early life may allow the identification of individuals at increased risk of metabolic diease in later life.

Pathways to obesity Are there modifiable epigenetic marks? Epigenetic signatures? CDKN2A? mis-match pathway? obesity? Epigenetic signatures? PGC1a/Sirt1 Are there modifiable epigenetic marks?

age 4 Maternal drivers? Ill-prepared for pregnancy. Minimal changes in diet and health behaviours Maternal drivers?

26 Pathways to obesity Are there modifiable epigenetic marks? Epigenetic signatures? CDKN2A? mis-match pathway? obesity? Epigenetic signatures? PGC1a/Sirt1 Are there modifiable epigenetic marks? Rapid catch up growth Poor educational attainment Poor diet Take less exercise Obese Greater fat mass, less lean mass and lower IQ at age 4 Maternal drivers? Ill-prepared for pregnancy. Minimal changes in diet and health behaviours Maternal drivers? Undernourished Dietary restriction IUGR Over nutrition Maternal high energy diet Maternal obesity GDM

Southampton Developmental Epigenetics group Graham

Nicki Irvine Elisoe Cook Leanie Grenfell Becki

Jordan Price Rob Murray Emma Garratt Sam Hoile

Singapore Joanna Holbrook Walter Strunkel

27 Southampton Developmental Epigenetics group Graham Burdge Keith Godfrey Mark Hanson Teejal Bhatt Nicki Irvine Elisoe Cook Leanie Grenfell Becki Clarke Paula Costello Mark Burton Danya Agfa Jordan Price Rob Murray Emma Garratt Sam Hoile Charlie Simmons New Zealand Peter Gluckman Singapore Joanna Holbrook Walter Strunkel Australia RaeChi Huang Leiden Bas Zwaan Plymouth Terry Wilkin Jo Hosking Jon Pinkey

Epigenetic processes are fundamental to development because they permit a

Early Life Nutrition and Epigenetic Markers Mark Hanson, PhD Epigenetic processes are fundamental to development because they permit a range of phenotypes to be formed from a genotype. Across many phyla