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Nutritional epigenomics of the metabolic syndrome

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1 Nutritional epigenomics of the metabolic syndrome
Periconceptual, fetal, neonatal, lifelong and transgenerational Pr. Claudine Junien - Inserm U781, Hôpital Necker-Enfants Malades, Paris France Using appropriate dietary protocols during gestation and lactation, by transcriptome analysis (with a custom « epigenetic-energy homeostasis” oligochips including all known imprinted genes, genes involved in epigenetic processes & genes involved in metabolism and neurotransmission) and/or Q-RT-PCR, and epigenomic approaches we seek: a) – to characterize the critical spatiotemporal windows of murine fetal/neonatal programming; b) – to identify dietary signatures on genes subject to transcriptional regulation including potentially involved master genes or gene networks & imprinted genes and, c) - to identify the “epigenetic signatures” underlying these transcriptional changes on candidate genes and on the whole genome in rodent placenta and adult tissues. The identification of markers in these rodent models should lead to the identification in humans of diagnostic and prognostic markers useful for appropriate dietary recommendations or interventions before and during pregnancy and lactation and in infancy. To this aim we coordinate a « human placentae network » to collect and study human placentae representing, for different clinical and scientific purposes, a spectrum of conditions associated with altered programming leading to components of MetS. Using epigenetic approaches rather than genetics our ultimate goal is to raise the issue, until now never considered, of the "locking" or "lability" of epigenetic modifications induced by diet/drug and/or transmitted by the parents in order to determine the potential reversibility of these changes using appropriate dietary, therapeutical or surgical protocols.

2 Developmental, environmental origin of the MetS
Indulgent lifestyle Energy imbalance Oxidative stress Aging … Epigenetics CH3 Previous generations experiences behavior, nutrition Genotype Oscillatory, circadian, seasonal rhythms perturbation Metabolic, neuronal malprogramming Mitochondrial dysfunction Chromosome/DNA damage In the last ten years, a series of studies — notably those by Barker, Hales and co-workers, who first coined the term "fetal programming" leading to a “thrifty phenotype” — have demonstrated that common disorders like obesity, CVD, diabetes, hypertension, asthma and even schizo take root in early nutrition, during gestation and lactation hence the new term “developmental programming”. It is now widely accepted that individuals with metabolic syndrome (MetS) may not only show a lifelong imbalance between energy intake and energy expenditure but also have suffered improper «epigenetic programming» during their early development due to placental insufficiency, maternal inadequate nutrition and metabolic disturbances as well as « transgenerational effects », due to experiences including behavior and nutrition of previous generations. Epigenetic alterations are linked with several other processes that are known to deteriorate during progression to MetS, under the influence of oxidative stress, aging and the folate status These include the oscillatory, circadian, and reasonal rythms with the rythmic expression of histone acetyl transferase: metabolic syndrome is associated with the KO of Clock, with the early neurotrophic processes including epigenetic modifications during differentitaion leading for example to the development of arcuate nucleus neurons by leptin in early the neonatal period.

3 ? Epigenetic programming dynamics
Environmental transient / permanent impacts Maternal care Carcinogenes M16 medium Diet-induced obesity FCS medium Litter size Develop- -ment. Amino acid medium Crop TGE Agouti Folates Transposons IG Genes Synth diets Somatic tissues Zygote Embryo- adult cells, tissues Gametes Methylation Aging Genes, transposons Gastrula Extra- Embryonic tissues Blastocyst ? Zygote Primordial germ cells Gonadal ridge Gametes Methylation Primordial germ cells-gametes Imprinted genes Adult Peri- conception Fertlization Implantation Birth Lactation Weaning SGP Puberty Figure 2. Impact of nutrients or diet intervention on DNA methylation reprogramming during early development and throughout life. After fertilization, the paternal and maternal genomes in the zygote undergo rapid demethylation at coding sequences (genes) and at repetitive sequences (transposable elements) (full line). In theory epigenetic marks are erased. With the exception of a brief period of global demethylation in the early mammalian embryo, transposons are normally silenced by promoter CpG methylation. However, transposons that escape this epigenetic silencing can interfere with the expression of neighboring genes in several ways. after implantationActive de novo methylation takes place, to various extents, depending on the part of the embryo concerned. After implantation, the bulk of the genome becomes hypermethylated in the embryonic ectoderm and mesoderm, through active de novo methylation, whereas the genome of extra-embryonic cells, such as the primary endoderm and trophoblast, remain hypomethylated. There is a sequence of de novo methylation that dictates the structure and function of each somatic tissue, through a finely tuned pattern involving the switching on and off of gene expression. During aging, a global hypomethylation is observed with hyper or hypomethylation of CpG in the promoter of several genes thus altering their level of expression.and promoting the occurrence of age related diseases In Germ cells, the parental methylation imprints in imprinted genes escape this demethylation process and de novo methylation (dotted line). Imprinted genes imprints are eliminated before primordial germ cells (PCG) reach the gonadal ridge and are appropriately reinstalled during male and female gametogenesis, being completed during the slow growth period (SGP) before puberty (dotted line for male, pecked line for female). In the male, the acquisition of DNA methylation patterns begins before birth in prospermatogonia and is completed for many sequences after birth, prior to the end of the pachytene stage of meiosis. In contrast to the male, in female germ cells, most gametic DNA methylation only begins to be acquired postnatally, during the oocyte growth phase, following the pachytene phase of meiosis (reviewed in ). The SGP is associated with the emergence of the first viable pools of spermatocytes and initiation of the programming of methylation imprints. At every stage during this cascade of epigenetic fluctuations (during both fetal development and the aging process), the nutritional balance must be optimal. There is compelling evidence that DNA methylation varies between tissues, individuals and disease conditions in humans and various animals and with aging, with both hyper- and hypomethylation observed. The impacts of nutrients/diets are indicated by arrows, they may concern imprinted genes (striped), transposons (black), genes (white). Arrows indicate the window during which the impact was observed. Several reports have shown that epigenetic programming is tightly time and space regulated during fetal development and lactation. Therefore, there are critical windows for specific nutrients supply / placenta, demand of the fetus. (Gallou-Kabani & Junien Diabetes 2005)

4 I CIRCADIAN-LIFELONG epigenetic deteriorations
Pr. Claudine Junien - Inserm U781, Hôpital Necker-Enfants Malades, Paris France The first type of alterations are those occuring everyday and accumulating overtime as age , diet, and disease-related deteriorations Curtis AM JBC 2004 Histone acetyltransferase-dependent chromatin remodeling and the vascular clock. Rhythmic gene expression is central to the circadian control of physiology in mammals. Transcriptional activation of Per and Cry genes by heterodimeric bHLH-PAS proteins is a key event in the feedback loop that drives rhythmicity; however, the mechanism is not clearly understood. Here we show the transcriptional coactivators and histone acetyltransferases, p300/CBP, PCAF, and ACTR associate with the bHLH-PAS proteins, CLOCK and NPAS2, to regulate positively clock gene expression. Furthermore, Cry2 mediated repression of NPAS2:BMAL1 is overcome by overexpression of p300 in transactivation assays. Accordingly, p300 exhibits a circadian time-dependent association with NPAS2 in the vasculature, which precedes peak expression of target genes. In addition, a rhythm in core histone H3 acetylation on the mPer1 promoter in vivo correlates with the cyclical expression of their mRNAs. Temporal coactivator recruitment and HAT-dependent chromatin remodeling on the promoter of clock controlled genes in the vasculature permits the mammalian clock to orchestrate circadian gene expression. Etchegaray JP, Lee C, Wade PA, Reppert SM. Rhythmic histone acetylation underlies transcription in the mammalian circadian clock.Nature 2003 In the mouse circadian clock, a transcriptional feedback loop is at the centre of the clockwork mechanism. Clock and Bmal1 are essential transcription factors that drive the expression of three period genes (Per1-3) and two cryptochrome genes (Cry1 and Cry2). The Cry proteins feedback to inhibit Clock/Bmal1-mediated transcription by a mechanism that does not alter Clock/Bmal1 binding to DNA. Transcriptional regulation of the core clock mechanism in mouse liver is accompanied by rhythms in H3 histone acetylation, and that H3 acetylation is a potential target of the inhibitory action of Cry. The promoter regions of the Per1, Per2 and Cry1 genes exhibit circadian rhythms in H3 acetylation and RNA polymerase II binding that are synchronous with the corresponding steady-state messenger RNA rhythms. The histone acetyltransferase p300 precipitates together with Clock in vivo in a time-dependent manner. Moreover, the Cry proteins inhibit a p300-induced increase in Clock/Bmal1-mediated transcription. The delayed timing of the Cry1 mRNA rhythm, relative to the Per rhythms, is due to the coordinated activities of Rev-Erbalpha and Clock/Bmal1, and defines a new mechanism for circadian phase control. Brain and muscle Arnt-like protein-1 (BMAL1), a component of the molecular clock, regulates adipogenesis. Shimba S, PNAS 2005

5 Circadian nutritional epiphenotype ? Oscillatory, circadian,
seasonal rhythms Circadian rhythms in H3 acetylation and RNA pol II binding of the core clock Clock co- with EZH2 polycomb, HAT p300 Rythmic gene expression : ± 10% or > genes : Temporal coactivator recruitment and HAT-dependent chromatin remodeling on the promoter of clock controlled genes The epigenetic connection (Curtis et al 2004; Etchegaray et al 2003, 2006) Sleep-wake Feeding-fasting Thermogenesis (Staels B Nat Med 2006) (Turek et al 2005; Rudic et al 2004; Oishi et al 2005; Shimba et al 2005; Inoue et al 2005; Zvonic et al 2006; Kreier F 2003) Oscillatory, circadian, seasonal rhythms In both the SCN neurons and peripheral cells, the circadian clockwork is constructed from interconnected transcriptional and post-translational feedback loops in gene expression that function in a cell-autonomous fashion. The activity of transcription factor BMLA1 (MOP3) and CLOCK are rhythmycally counterbalanced by period (PER) and cryptochrome (CRY) proteins to govern time of day dependent gene expression. In addition the circadian clock dictates the rhythmic production of output regulators, other transcription factors such as DBP, Hlf and Tef. These TF in turn, regulate downstream target genes involved in different biochemical pathways including glucose, cholesterol and triglyceride metabolism and including the sleep-wake cycle, thermogenesis, and feeding Pathways downstream of the clock genes include the nuclear receptor PPARA. The expression of hepatic PPARA shows circadian rhythmicity directly modulated by the Clock and BMLA1 proteins and modulated by glucocorticoids Recent studies show that deletion of the Clock and BMLA1 genes results not only in circadian disturbances but also in metabolic abnormalities of lipid and glucose homeostasis – a phenotype resembling the MetS. Mutations in these genes influence the development of glucose intolerance and insulin resistance in response to a high fat diet. Thus although built to fluctuate at or near a 24h cycle, the clock can be entrained by light, activity or food. The epigenetic connection. The master circadian oscillator in SCN uses epigenetic mechanisms, including histone acetylation and phosphorylation, to generate circadian patterns of gene expression and to modulate gene expression in response to phase-resetting stimuli. As shown by Etchegaray et al (2003) and by Curtis AM JBC 2004, the transcriptional coactivators and histone acetyltransferases, p300/CBP, PCAF, and ACTR associate with the bHLH-PAS proteins, CLOCK and NPAS2, to regulate positively clock gene expression. Thus transcriptional regulation of the core clock mechanism in mouse liver is accompanied by rhythms in H3 acetylation, The histone acetyltransferase p300 precipitates together with Clock in vivo in a time-dependent manner. Reversibility ? Thus factors such as methionine or trichostatin A which can modulate histone modification and DNA methylation are good candidate to explore their effects on desynchronisation.

6 Gene-specific aberrant methylation
Age-related diseases Normal colon IFN PDGFA MMP2-7-9 TIMP ICAM ERa-b EC-SOD HSD11B2 P53… (Hiltunen & Yla-Herttuala ATVB 2003) Atherogenesis: (Issa et al. PNAS 1996) Aberrant methylation starts in normal tissues Issa 1996 We have previously linked aging, carcinogenesis, and de novo methylation within the promoter of the estrogen receptor (ER) gene in human colon. We now examine the dynamics of this process for the imprinted gene for insulin-like growth factor II (IGF2). In young individuals, the P2-4 promoters of IGF2 are methylated exclusively on the silenced maternal allele. During aging, this promoter methylation becomes more extensive and involves the originally unmethylated allele. Most adult human tumors, including colon, breast, lung, and leukemias, exhibit increased methylation at the P2-4 IGF2 promoters, suggesting further spreading during the neoplastic process. In tumors, this methylation is associated with diminished or absent IGF2 expression from the methylated P3 promoter but maintained expression from P1, an upstream promoter that is not contained within the IGF2 CpG island. Our results demonstrate a remarkable evolution of methylation patterns in the imprinted promoter of the IGF2 gene during aging and carcinogenesis, and provide further evidence for a potential link between aberrant methylation and diseases of aging.

7 Genome-wide methylation Age- and diet-related diseases
(Lund et al. JBC 2004) 4-weeks old 6-months WT Apoe-/- mice (Hiltunen & Yla-Herttuala ATVB 2003) Apoe-/- mice arteries Human -Validity of epigenetic mechanisms as causative agents in the development of nutritionally linked chronic disease? -Can human cell-based models be used effectively to study epigenetic programming in vitro? -What kind of environmental variables initiate the emergence of an epigenetic phenotype? -What are the molecular methods that can most efficiently identify epigenetic changes? Fatty streaks/advanced lesions

8 Genetic basis for epigenetic instability
Susceptibility to environment/ diets ? CIMP : CpG island methylator phenotype MTHFR DNMT ? Etc…

9 - Inserm U781, Hôpital Necker-Enfants Malades, Paris France
II : DOAD Developmental Origin of Adult Diseases Pr. Claudine Junien - Inserm U781, Hôpital Necker-Enfants Malades, Paris France

10 DOAD : Diet and/or specific dietary component
Protein restriction Carbohydrate-rich Lipid-rich Lifespan Hypertension Glucose Metabolism Liver methyl. Pancreas devel Membrane FA. LP/C HFD/C Amino-acids Thr, Met, gly Tau etc.. Sugar Glucose, fructose Hyperinsulinism Obesity Preference (CH/F) HHC HHC/C C Hyperinsulinism Hypertension Obesity Preference (CH/F) HFD/LFD/C Fatty acids SAT, MUFA, PUFA TFA gestation suckling weaning Outcome (Armitage et al, J. Physiol 2004) Recent experiments have shown that the features of MetS in the adult offspring of fat-fed rats may be acquired antenatally and during suckling. Moreover, exposure during pregnancy confers adaptive protection against endothelial dysfunction — but not against hypertension — due to maternal fat-feeding during suckling. A smaller number of studies have dealt with the consequences of a high-carbohydrate or fat-rich diet, conditions corresponding more closely to the current epidemic of MetS. However, it remains unclear whether MetS can be reliably induced by the interventions made, due to differences between protocols, diets (e.g. type of fatty acids), sex and time periods examined . A recent review by Armitage examined animal studies in which the fetal and postnatal environment had been manipulated by changing maternal dietary intake or modifying uterine artery flow. Most studies examined the consequences of protein restriction during gestation, conditions not fully matching the features of the current epidemic of MetS. However, these data only partially reflect the features of MetS because pregnant mothers were not overeweight and therefore did not display the metabolic disturbances of MetS that could also interfere with fetal/postnatal programming. Malnourished fetuses adopt several strategies to optimize their chances of survival during the neonatal period, but these strategies assume that the same type of nutritional conditions will prevail. A selective distribution of nutrients ensures that brain growth is given priority over the growth of other organs such as the liver, muscle and pancreas. The adaptations adopted during fetal programming may prove to be detrimental if food becomes more abundant . Thus, any change in conditions may have deleterious consequences. A significant and increasing proportion of women (14-27%) are overweight when pregnant. Whereas the long-term effects of gestational diabetes are well documented, the consequences of MetS in the mother, together with an unbalanced diet and metabolic disturbances during the periconceptual period, gestation and lactation, for fetal programming, and for the various critical windows of development, and during aging are poorly documented and remain to be explored .

11 nutritional 1 - Can we identify epigenetic alterations responsible for
malprogramming ? 2 - Are they reversible ? How : diet? drugs? lifestyle? … In contrast with M. Szyf approach with maternal care

12 Sex-specific adaptive resistance to a high fat diet
  Crossing and diet scheme p = F1HF 83% 17% 57% 43% F2HF F0 n=106 n=35 n=47 n=87 HFD Mating F1 Gestation-Lactation weaning Adult Mating F2 Gestation Lactation Weaning A « satiety » phenotype Adult (Gallou-Kabani et al 2006) Given the increasing proportion of women who are overweight/obese and overfed, and the limited success of weight loss strategies we investigated whether reasonable dietary changes during pregnancy/lactation in a background of maternal obesity may interfere with fetal :neonatal programming of MetS. C57Bl/6 mice were placed on a low-fat (10%) or high-fat (60 %) diet. Over five months on the high-fat diet both male and female F1 mice developed features of MetS, notably obesity, hyperinsulinemia, and glucose intolerance. High-fat feeding led to normal/decreased serum triglycerides and hypercholesterolemia comprised of both HDL and LDL particles. Interestingly on the next generation, despite a hyperfat diet ad libitum after weaning, half of the F2 female mice offspring, but not F2 male offspring, of obese F1 mothers fed a normal diet during pregnancy/lactation displayed a “satiety phenotype” and remained lean. Thus, adaptation of diet during pregnancy and/or lactation may help to slow the alarming transgenerational increase in obesity. This corresponds to the inverse of the “predictive adaptive response” hypothesis, suggesting that an appropriate dietary fatty acid profile and intake during pregnancy and/or lactation helps the offspring to cope with deleterious maternal milieu conditions, slowing the alarming transgenerational increase in obesity.

13 Plausible candidates for adaptation ? Buffering or « rheostat » System
Imprinted genes ? Maternal allele silenced % Paternal allele expressed % Monoallelic Expression Biallelic expression Non-expression Coevolution: Placenta and Imprinting (mammals) Fetal and placental growth Brain development - behaviors Postnatal nutritional adaptation Co-adaptation mother-infant (evolution) Epigenetic lability by nutrients Non erasing of epigenetic marks (except gametes) Altered imprinting syndromes and obesity, T2D Buffering or « rheostat » System (Pembrey M. 1996, Junien C. 2000, Beaudet A. 2002, Pembrey M. 2002) Labilité épigénétique des Gènes Soumis à Empreinte (GSE) modulables par l’alimentation Rôle des GSE dans la croissance fœtale et placentaire Rôle des GSE dans le développement du cerveau Déséquilibres de l’empreinte et phénotypes d’obésité et diabète Coévolution placenta GSE

14 « Epigenetics - energy homeostasis »
Satiety phenotype 1 - Custom microarrays Slc2a5 Dgat 1 Gata 3 Acads Esx1 Decorin Igf 2 Riken cDNA Gata 1 Nr1h3 Nnat Grb10 Kcnq 1 Ube3a Pparg Decorin Esx1 Nr1h3 Nnat Grb10 Igf 2 Riken cDNA 60 Imprinted genes « Epigenetics - energy homeostasis » 500 genes Placenta Liver (Vigé et al CGR 2005)

15 Satiety phenotype 28 genes Females Peg3 Peg1
2 - Candidate gene approach Q-RT-PCR Females 28 genes stomach, muscle, adipose tissue, hypothalamus, liver Peg3 Peg1 & Peg 3: Paternally expressed Imprinted genes increased in DIO Peg1: Adipocyte size marker F2-S F1C F1HF F2-R Peg1 F2-S F1C F1HF F2-R (Moraes et al 2003; Takahashi et al 2004; Curley et al 2004)

16 3 - Epigenetic signatures?
Satiety phenotype 3 - Epigenetic signatures? DNA methylation Candidate genes : Bisulphite-Pyrosequencing - Liver : Scd1, Snrpn = no difference..so far - Adipose tissue : Lep, Peg1, Peg3 = no difference Genome-wide: Luminometric Methylation Assay (LUMA) Satiety phenotype, liver : Hypomethylation F1HF -> adaptation F2 0,15 0,2 0,25 0,3 0,35 0,4 0,45 P = 0.03 P = 0.02 F1N F2HFR F1HF F2HFS Hypomethylation Hypermethylation Histones alterations Candidate genes: Chromatin ImmunoPrecipitation(ChIP) Genome-wide: ChIP (Karimi et al, Epigenetics, 2006, Umlauf et al, Nat Genet, 2006)

17 Can we identify placental markers for early events of malprogramming,
tracing back the in utero nutritional and metabolic course? In the last ten years, a series of studies — notably those by Barker, Hales and co-workers, who first coined the term "fetal programming" leading to a “thrifty phenotype” — have demonstrated that common disorders like obesity, CVD, diabetes, hypertension, asthma and even schizo take root in early nutrition, during gestation and lactation. Because of these early and permanent alterations caused by unbalanced diet in an otherwise “normal” or at risk individual, it is clear that patients with monogenic inborn errors of metabolism may also suffer the same types of alterations with the specific diets that circumvent their metabolic defect.

18 Epigenetic signatures
MetS : Placental markers of nutritional and metabolic epigenetic malprogramming Control vs ≠ diets DBA/2 C57B/6 X E 0.5 E 15.5 C57B/6 X Maternally expressed Slc22a3 Paternally expressed Rtl1 (Peg11) Epigenetic signatures

19 III Transgenerational effects
Pr. Claudine Junien - Inserm U781, Hôpital Necker-Enfants Malades, Paris France Using appropriate dietary protocols during gestation and lactation, by transcriptome analysis (with a custom « epigenetic-energy homeostasis” oligochips including all known imprinted genes, genes involved in epigenetic processes & genes involved in metabolism and neurotransmission) and/or Q-RT-PCR, and epigenomic approaches we seek: a) – to characterize the critical spatiotemporal windows of murine fetal/neonatal programming; b) – to identify dietary signatures on genes subject to transcriptional regulation including potentially involved master genes or gene networks & imprinted genes and, c) - to identify the “epigenetic signatures” underlying these transcriptional changes on candidate genes and on the whole genome in rodent placenta and adult tissues. The identification of markers in these rodent models should lead to the identification in humans of diagnostic and prognostic markers useful for appropriate dietary recommendations or interventions before and during pregnancy and lactation and in infancy. To this aim we coordinate a « human placentae network » to collect and study human placentae representing, for different clinical and scientific purposes, a spectrum of conditions associated with altered programming leading to components of MetS. Using epigenetic approaches rather than genetics our ultimate goal is to raise the issue, until now never considered, of the "locking" or "lability" of epigenetic modifications induced by diet/drug and/or transmitted by the parents in order to determine the potential reversibility of these changes using appropriate dietary, therapeutical or surgical protocols.

20 Developmental programming
Modes of transmission Germ cells Gametes Paternal Maternal Germ cells Gametes Developmental programming -How do you determine the modes of transmission of some epigenetic phenomena? -Validity of epigenetic mechanisms as causative agents in the development of nutritionally linked chronic disease? -What kind of environmental variables initiate the emergence of an epigenetic phenotype? -Is there a genetic basis to epigenetic inheritance? Are certain genotypes more prone to epigenetic programming? -How do you identify epigenetic biomarkers?

21 Male transmission on 4 generations
Endocrine disruptors & fertility: Apoptosis Sperm - number - mobility Methylation : 25 sequences (Anway et al Science 2005) Transgenerational effects of environmental toxins require either a chromosomal or epigenetic alteration in the germline. Anway et al recently showed that transient exposure of a gestating female rat during the period of gonadal sex determination to the endocrine disruptors vinclozolin (an antiandrogenic compound) or methoxychlor (an anti estrogenic compound) induced an adult phenotype in the F1 generation of decreased spermatogenic capacity (cell number and viability) and increased incidence of male infertility. These effects were transferred through the male germline to nearly all males of all subsequent generations examined (F1 to F4). The effects on reproduction correlate with altered DNA methylation patterns in the germline. The ability of an environmental factor (for example, endocrine disruptor) to reprogram the germline and to promote a transgenerational disease state has significant implications for evolutionary biology and disease etiology. About 25 different PCR products were identified that had altered DNA methylation patterns after the endocrine disruptor treatment. Two of the DNA fragments were sequenced and mapped on 8q32 and 6q32 (lysophospholipase and cytokine inducible SH2 containing protein). Whether the genes identified are causal factors or simply markers (i.e., downstream) of the transgenerational epigenetic phenotype remains to be determined. Methylation-sensitive restriction ezyme PCR analysis of the methylation state of clone 17 (CISP) gene in epididymal sperm from F2 and F3 generations from control and vinclozolin-treated animals. The epigenetic alterations observed involve both hypermethylation and hypomethylation events Methylation- of clone 17 (CISP-gene) in epididymal sperm from F2 and F3 generations

22 Gestation-postnatal/lactation
Maternal effect Gestation-postnatal/lactation  First generation High-carbohydrate diet during suckling  Second generatiion Control diet (HC mother) Hyperinsulinism (Srinivasan et al Diabetes 2003) As shown by Srinivasan and colleagues, feeding rats a high CH diet during suckling rapidly leads to hyperinsulinism. Moreover, when these female rats become pregnant hyperinsulinism also rapidly appears in their offspring even though they are fed a normal diet, sucling milk from their mothers. This example clearly shows that the early postnatal period is an important period for programming and that marks acquired during this period can influence the outcome of the next generation. Recent experiments by Khan et al have shown that the features of MetS in the adult offspring of fat-fed rats may be acquired antenatally and during suckling. Moreover, exposure during pregnancy confers adaptive protection against endothelial dysfunction — but not against hypertension — due to maternal fat-feeding during suckling.

23 T2D, mortality : only paternal grandparents !
XX XY X Y GP 8 - Héritage Transgénérationnel Conséquences des bonnes/mauvaises récoltes pendant la période prébubertaire Des données ont été collectées à partir de trois cohortes nées en 1890, 1905 et 1920 dans le nord de la Suède jusqu’à leur décès ou jusqu’en L’accès à la nourriture des grands-parents ou des parents au cours de leur SGP a été déterminé en se réferrant aux données historiques sur les récoltes et les prix des aliments, aux archives des réunions des communautés et les faits historiques généraux. Si les aliments n’étaient pas vraiment disponibles pendant la SGP du père, la mortalité par maladie cardiovasculaire était réduite. La mortalité par diabète augmentait si le grand-père paternel était exposé à une surabondance de nourriture pendant sa SGP (OR 4,1, p=0,01). Un mécanisme lié à la nutrition semble avoir influencé le risque CVD et le risque de diabète à travers la lignée paternelle . GM (Kaati et al 2002; Pembrey et al, 2005) (Kaati et al 2002)

24 E Boudadi, H Pilet, MS Gross,
Acknowledgements Bioinformatics -statistics JP Jais (Hôp.Necker SBIM) Beta oxydation fatty acids F. Djouadi, J. Bastin (Inserm U393, Paris) Desaturation index ,FFA P.Gambert (Inserm, Dijon) Lipid fraction analysis J. Fruchart (Inserm, Lille) LDL, HDL, TG, C. Boileau, J.P. Rabès (Biochimie Hôp.A. Paré, Boulogne) Absorptiometry P. Letteron, B. Fromenty (Hôp.Bichat CERFI Paris) Microarray fabrication L.Talini, M.C. Pottier (Genescore, ESPCI,Paris) Energy metabolism (Ind Calorimetry) P. Even, D. Tomé, C. Larue (INA-PG, Paris) Methylation by pyrosequençing/LUMA I Gut, J. Tost (CNG, Evry) T. Ekstrom (Karolinska, Suède Network ATC-Nutrition – PRNH Inserm Inra Coord C. Junien C Junien (Inserm Paris) J. P. Jaïs (SBIM, Paris), H. Vidal (Inserm, Lyon) D. Langin (Inserm, Toulouse) K. Clément (Inserm, Paris) J.D.Zucker (Paris XIII) U383-U781 C Gallou-Kabani, A Vigé, E Boudadi, H Pilet, MS Gross, A Belaid, C. Junien Placenta network Coord C. Junien (Paris) F. Andreelli (Paris) C. Levy-Marchal (Paris) MA Charles (Villejuif) A Vambergue (Lille) I Fajardy (Lille) D Vieau (Villeneuve d’Asq) B. Reusens (Louvain) G.Moore (Londres) R. Frydman (Paris) Y. Dumez (Paris) D. Vaiman (paris) J Tost (Evry) Financing INRA, ATC- INSERM, PRNH INSERM, AFD, AFERO I.B. Delessert, Lab Fournier, Nestlé

25 Involvement of an imprinted gene ?
Epigenetic patterns Involvement of an imprinted gene ? Promotor ? Proportional to the adipocyte size ? Differentially methylated Region (DMR) Adaptation to caloric intake heritable ? Analyse des modifications épigénétiques Une fois l’expression différentielle validée, les modifications épigénétiques seront étudiés par 2 approches différentes : a) Profils de méthylation, après traitement par le bisulfite, par la technique du pyroséquençage, en collaboration avec la plate-forme du CNG à Evry (Ivo Gut) b) Les modifications des histones sont analysées par chromatin immunoprecipitation (ChIP) en utilisant des anticorps spécifiques de la méthylation et de l’acétylation des histones. c) Enfin il est prévu, à terme, de faire « designer » (par la sociéte Genescore) des microarrays dédiés comportant les ilôts CpG des gènes cibles identifiés (directs comme l’ER ou indirects comme les cibles de l’ER) pour étudier par ChIP-on-chip à la fois l’expression des gènes, la méthylation de l’ADN et les modifications des histones. Il existe en effet pour l’homme, mais pas pour la souris, d’autres types de microarrays permettant d’analyser simultanément l’expression des gènes et leurs modifications épigénétiques. Les ECISTs (Expressed CpG island sequence tag microarray) (Shi H 2002) permettent à la fois l’analyse de l’expression des gènes et celle de la méthylation de l’ADN. Plus récemment l’utilisation de microarrays comportant des milliers d’ilôts CpG (CGI couplée à l’immunoprécipition de la chromatine par différents anticorps, a permis d’étudier la commutation de l’acétylation à la méthylation de la lysine en position 9 de l’histone H3 (H3K9) grâce à des anticorps, anti-H3-K9 méthylé, et anti-H3-K9 acétylé. Cette approche a permis de sélectionner des clones montrant un niveau élevé du rapport méthylation sur acétylation de H3K9 (K9 Me/Ac). La commutation de l’acétylation à la méthylation contribue à l’extinction de gènes dans les cellules montrant une extinction associée à la méthylation de l’ADN (Kondo Y 2004).

26 -Validity of epigenetic mechanisms as causative agents in the development of nutritionally linked chronic disease? -How are additional models developed, when and how do we study them? -What will be the effective methodologies in terms of culture models and molecular techniques for determining epigenetic marks? -How do we explore the nutritional factors and their effects on C1 metabolism? -Can human cell-based models be used effectively to study epigenetic programming in vitro? -What kind of environmental variables initiate the emergence of an epigenetic phenotype? -Is there a genetic basis to epigenetic inheritance? Are certain genotypes more prone to epigenetic programming? -What kind of epigenetic modifications could be physiologically advantageous? -How do you identify epigenetic biomarkers? -What are some simple model systems? Phenotype? -How do you determine the modes of transmission of some epigenetic phenomena? -What are the molecular methods that can most efficiently identify epigenetic changes? In utero vs. postnatal impacts? In the last ten years, a series of studies — notably those by Barker, Hales and co-workers, who first coined the term "fetal programming" leading to a “thrifty phenotype” — have demonstrated that common disorders like obesity, CVD, diabetes, hypertension, asthma and even schizo take root in early nutrition, during gestation and lactation. Because of these early and permanent alterations caused by unbalanced diet in an otherwise “normal” or at risk individual, it is clear that patients with monogenic inborn errors of metabolism may also suffer the same types of alterations with the specific diets that circumvent their metabolic defect.

27 Plausible candidates for resistance to HFD ?
Spatiotemporal windows ? Markers ? Placenta ? WBC? In the last ten years, a series of studies — notably those by Barker, Hales and co-workers, who first coined the term "fetal programming" leading to a “thrifty phenotype” — have demonstrated that common disorders like obesity, CVD, diabetes, hypertension, asthma and even schizo take root in early nutrition, during gestation and lactation. Because of these early and permanent alterations caused by unbalanced diet in an otherwise “normal” or at risk individual, it is clear that patients with monogenic inborn errors of metabolism may also suffer the same types of alterations with the specific diets that circumvent their metabolic defect.

28 -Is there a genetic basis to epigenetic inheritance
-Is there a genetic basis to epigenetic inheritance? Are certain genotypes more prone to epigenetic programming? -How do you determine the modes of transmission of some epigenetic phenomena? -What kind of environmental variables initiate the emergence of an epigenetic phenotype? In the last ten years, a series of studies — notably those by Barker, Hales and co-workers, who first coined the term "fetal programming" leading to a “thrifty phenotype” — have demonstrated that common disorders like obesity, CVD, diabetes, hypertension, asthma and even schizo take root in early nutrition, during gestation and lactation. Because of these early and permanent alterations caused by unbalanced diet in an otherwise “normal” or at risk individual, it is clear that patients with monogenic inborn errors of metabolism may also suffer the same types of alterations with the specific diets that circumvent their metabolic defect.


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