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Nutritional epigenomics
of the metabolic syndrome
Periconceptual, fetal, neonatal,
lifelong and transgenerational
Pr. Claudine Junien
- Inserm U781, Hôpital Necker-Enfants Malades, Paris France
Developmental, environmental origin of the MetS
Indulgent lifestyle
Energy imbalance
Oxidative stress
Aging …
Previous
generations
experiences
behavior,
nutrition
Epigenetics
CH3
CH3CH3
Genotype
Oscillatory,
circadian, seasonal
rhythms perturbation
Metabolic,
neuronal
malprogramming
Mitochondrial
dysfunction
Chromosome/
DNA damage
Epigenetic programming dynamics
Environmental transient / permanent impacts
IG
Transposons
Genes
Gametes
Methylation
Embryoadult
cells,
tissues
Somatic
Zygote
tissues
Genes,
transposons
Aging
Gastrula
Blastocyst
ExtraEmbryonic
tissues
Primordial
germ
cellsgametes
Methylation
Zygote
Gametes
Imprinted
genes
Primordial Gonadal
ridge
germ
cells
(Gallou-Kabani & Junien Diabetes 2005)
I
CIRCADIAN-LIFELONG
epigenetic deteriorations
Pr. Claudine Junien
- Inserm U781, Hôpital Necker-Enfants Malades, Paris France
Circadian nutritional epiphenotype ?
The epigenetic
connection
Oscillatory, circadian,
seasonal rhythms
Sleep-wake
Feeding-fasting
Thermogenesis
• Circadian
rhythms
in
H3
acetylation and RNA pol II binding
of the core clock
• Clock coHAT p300
(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)
with EZH2 polycomb,
•Rythmic gene expression : ± 10%
or > genes :
Temporal coactivator recruitment
and HAT-dependent chromatin
remodeling on the promoter of
clock controlled genes
(Curtis et al 2004; Etchegaray et al
2003, 2006)
Gene-specific aberrant methylation
Age-related diseases
Normal colon
Atherogenesis:
IFN
PDGFA
MMP2-7-9
TIMP
ICAM
ERa-b
EC-SOD
HSD11B2
P53…
(Issa et al. PNAS 1996)
(Hiltunen & Yla-Herttuala
ATVB 2003)
Genome-wide methylation
Age- and diet-related diseases
Human
arteries
Apoe-/mice
arteries
Apoe-/mice
WT
4-weeks
old
6-months
old
(Hiltunen & Yla-Herttuala ATVB 2003)
(Lund et al. JBC 2004)
Genetic basis for epigenetic instability
Susceptibility to environment/ diets ?
CIMP : CpG island methylator phenotype
MTHFR
DNMT ?
Etc…
II : DOAD
Developmental
Origin
of Adult Diseases
Pr. Claudine Junien
- Inserm U781, Hôpital Necker-Enfants Malades, Paris France
DOAD :
Diet and/or specific dietary component
Diet
Protein restriction
Carbohydrate-rich
Lipid-rich
Amino-acids
Thr, Met, gly
Tau etc..
Sugar
Glucose, fructose
Fatty acids
SAT, MUFA, PUFA
TFA
LP/C
C
HFD/LFD/C
suckling
LP/C
HHC
HFD/LFD/C
weaning
HFD/C
HHC/C
HFD/LFD/C
Lifespan
Hypertension
Glucose Metabolism
Liver methyl.
Pancreas devel
Membrane FA.
Hyperinsulinism
Obesity
Preference (CH/F)
Hyperinsulinism
Hypertension
Obesity
Preference (CH/F)
gestation
Outcome
(Armitage et al, J. Physiol 2004)
1 - Can we identify epigenetic alterations
responsible for
nutritional
malprogramming ?
2 - Are they reversible ?
How : diet? drugs? lifestyle? …
Sex-specific adaptive resistance to a high fat diet
Crossing and diet scheme
 
F0 Mating
F1HF
F1 Gestation-Lactation
weaning
83%
Rˇsistante
Sensible
n=87
n=106
Adult
HFD
Mating
F2
17%
F2HF
Gestation
57%
1er trim.
2e trim.
Lactation
Weaning
HFD
Adult
p=
0.001
n=35
(Gallou-Kabani et al 2006)
HFD
n=47
43%
A « satiety »
phenotype
Plausible candidates for adaptation ?
Imprinted genes ?
Coevolution: Placenta and Imprinting (mammals)
Monoallelic Expression
Maternal allele
silenced
0 - 10 %
Paternal allele
expressed
90 - 100 %
Fetal and placental growth
Brain development - behaviors
Postnatal nutritional adaptation
Co-adaptation mother-infant (evolution)
Epigenetic lability by nutrients
Non-expression
Biallelic expression
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)
Satiety phenotype
1 - Custom microarrays
Dgat 1 Gata 3
60
Imprinted
genes
« Epigenetics energy
homeostasis »
500 genes
Decorin
Esx1
Acads
Gata 1
Decorin
Esx1
Igf 2 Riken cDNA Pparg
Igf 2 Riken cDNA
Grb10
Grb10
Nr1h3
Slc2a5
Kcnq 1 Slc2a5
Placenta
Nnat
Nr1h3
Ube3a
Liver
(Vigé et al CGR 2005)
Nnat
Satiety phenotype
2 - Candidate gene approach Q-RT-PCR
Females
0,6
stomach,
muscle,
adipose tissue,
hypothalamus,
liver
Peg3
0,4
0,3
Peg1 & Peg 3:
0,2
Paternally expressed
0,1
0
FN
F1C
F1HF
F1HF
F2HF sen
F2HF res
F2-S
F2-R
Imprinted genes
increased in DIO
60
Peg1:
50
Peg1
Unitˇ arbitraire
28 genes
Unitˇ arbitraire
0,5
Adipocyte size marker
40
30
20
10
0
FN
F1C
F1HF
F1HF
F2HF sen
F2HF res
F2-S
F2-R
(Moraes et al 2003; Takahashi et al 2004; Curley et al 2004)
Satiety phenotype
DNA methylation
Candidate genes :
Bisulphite-Pyrosequencing
- Liver : Scd1, Snrpn = no difference..so far
- Adipose tissue : Lep, Peg1, Peg3 = no
difference
70
% de méthylation
3 - Epigenetic signatures?
Femelles Peg1_MSP6 Tissu Adipeux
80
60
F1N
F1HF
F2HF Res
F2HF Sen
50
40
30
20
10
0
Position 1
Position 2
Position 3
P = 0.02
P = 0.03
Hypomethylation
0,45
0,4
0,35
Genome-wide:
Luminometric Methylation Assay (LUMA)
Satiety phenotype, liver :
Hypomethylation F1HF -> adaptation F2
0,3
0,25
Hypermethylation
0,2
0,15
F1N F1HF F2HFR F2HFS
Histones alterations
Candidate genes:
Chromatin ImmunoPrecipitation(ChIP)
Genome-wide:
ChIP
(Karimi et al, Epigenetics, 2006, Umlauf et al, Nat Genet, 2006)
Can we identify placental markers
for early events of malprogramming,
tracing back the in utero
nutritional and metabolic course?
DBA/2 X
C57B/6
C57B/6 X
DBA/2
MetS : Placental markers of nutritional and
metabolic epigenetic malprogramming
E 0.5
Control vs
≠ diets
E 15.5
Maternally expressed
Slc22a3
Paternally expressed
Rtl1 (Peg11)
Epigenetic signatures
III
Transgenerational effects
Pr. Claudine Junien
- Inserm U781, Hôpital Necker-Enfants Malades, Paris France
Modes of transmission
Paternal
Maternal
Germ cells
Developmental
Germ cells
Gametes
programming
Gametes
Male transmission on 4 generations
Endocrine disruptors
& fertility:
Apoptosis
Sperm
- number
- mobility
Methylation :
25
sequences
(Anway et al Science 2005)
Maternal effect
Gestation-postnatal/lactation
First generation
High-carbohydrate diet during suckling
Hyperinsulinism
(Srinivasan et al Diabetes 2003)
Second generatiion
Control diet (HC mother)
T2D, mortality : only paternal grandparents !
XX XY
GP
Y
X
(Kaati et al 2002;
Pembrey et al, 2005)
GM
(Kaati et al 2002)
Acknowledgements
Bioinformatics -statistics
JP Jais (Hôp.Necker SBIM)
U383-U781
Beta oxydation fatty acids
C Gallou-Kabani, A Vigé,
F. Djouadi, J. Bastin
E Boudadi, H Pilet, MS Gross,
(Inserm U393, Paris)
Desaturation index ,FFA
A Belaid, C. Junien
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)
Financing
INRA, ATC- INSERM, PRNH
T. Ekstrom (Karolinska, Suède
INSERM, AFD, AFERO
I.B. Delessert, Lab Fournier, Nestlé
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)
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)
Epigenetic patterns
Involvement of an imprinted gene ?
Promotor ?
Differentially methylated Region (DMR)
Proportional to the adipocyte size ?
Adaptation to caloric intake
heritable ?
-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?
Plausible candidates for resistance to HFD ?
Spatiotemporal windows ?
Markers ? Placenta ? WBC?
-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?