“ EPIGENÉTICA DE LA NUTRICIÓN Y LA OBESIDAD” 11 y 12 de Noviembre de 2011 X CONGRESO DE LA SOCIEDAD DE ENDOCRINOLOGÍA, NUTRICIÓN Y DIABETES DE LA COMUNIDAD DE MADRID Departamento de Nutrición, Ciencias de la alimentación, Fisiología y Toxicología Universidad de Navarra. Pamplona
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EPIGENÉTICA DE LA NUTRICIÓN Y LA OBESIDAD” · “ epigenÉtica de la nutriciÓn y la obesidad” 11 y 12 de noviembre de 2011 x congreso de la sociedad de endocrinologÍa, nutriciÓn
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“ EPIGENÉTICA DE LA NUTRICIÓN Y
LA OBESIDAD”
11 y 12 de Noviembre de 2011
X CONGRESO DE LA SOCIEDAD DE ENDOCRINOLOGÍA, NUTRICIÓN Y DIABETES DE LA
COMUNIDAD DE MADRID
Departamento de Nutrición, Ciencias de la alimentación, Fisiología y Toxicología Universidad de Navarra. Pamplona
DNA Methylation and Histone modification. (From Molecular Development - Epigenetics by Dr Mark Hill.)
Epigenetic modifications
http://www.youtube.com/watch?v=eYrQ0EhVCYA&NR=1
- DNA methylationAddition or removal of a methyl group (CH3), predominantly where cytosine is followed by a guanine (CpGs).
-Post-translational modifications on N-terminal tails of histones, phosphorylation, sumoylation, ubiquitination, acetylation, and methylationThese modifications alter chromatin structure to influence gene expression. In general, tightly folded chromatin (heterochromatin) tends to be shut down, or not expressed, while more open Chromatin (euchromatin) is functional, or expressed.
-Other mechanims
Gene expression maybe modified by miRNA, transposons, etc
Epigenetic Phenomena
Me
DNA Methylation
Histone Modifications DNA Packaging around Nucleosomes
Me
Me
Chromatin Packaging Alterations
Me
Me
Me
MeMe
Me
Me
Me
Me Me
Me MeAc Ac Ac
Ac
Epigenetic modifications
The carbon atom at the 5' position of cytosines within a CpG site can be methylated by DNA methyltransferases (DNMTs).
Methylated CpGs are recognized and bound by specific proteins including histone deacetylases (HDACs).Histone deacetylation alters the nucleosomal structure and decreases the chromatin transcriptional activity.
Subsequent methylation of histone tails by histone methyltransferases (HMTs) enhance the formation of transcriptionally incompetent heterochromatin. Lodygin D et al. Cell Research (2005) 15, 237–246.
Relationship between DNA methylation and chromatin structure
- Erasure of methylation imprints is almost exclusively of two stages:• primordial germ cells• blastocyst development
Periods of life in which DNA methylation processes take place
- Maternal care, ageing, dietary compounds,toxins,inflammation,regulate DNA methylation.
- Transgenerational Transmission
Individuality and epigenetics in obesity.Campion J, Milagro FI and Martinez JA.Obesity Rev 2009; 10:383-92
Many genes related to Nutrition are regulated by DNA methylation
A diet low in methionine, folate and choline induces steatosis in mice and decreases DNA methylation.
Number of unmethylated CpG sites
Pogribny IP et al. J Hepatol. 2009; 51:176-86.Diet modifies DNA methylation
DIET MODIFIES DNA METHYLATION
I. INTRODUCCIÓN
Ng S-F et al. Nature 2010; 467: 963-6.
HFD leads to adiposity, glucose intolerance and insulin resistance in fathers.
a, Body-weight trajectoriesb, Blood glucose during glucose tolerance test
Fathers (fed control or HFD diet) Female offspring (fed control diet)
b b
Chronic high-fat diet in fathers programs β-cell dysfunction in female rat offspring
In Vivo Methylation Patterns of the Leptin Promoter in Human and MouseStöger R.Epigenetics 1:4, 155-162, 2006
BACKGROUND
Proporcion en nutrientes de las dietas
0
10
20
30
40
50
60
70
80
Proteínas Lípidos (%) Hidratos de carbono (%)
Prop
orci
on (%
)
Control
Cafeteria
Proteins Lipids Carbohydrates
x2
n=5
n=6
Control
Cafetería
MATERIAL AND METHODS
77 days
oooo
J Physiol Biochem 2009; 65: 1-8
Retroperitoneal adipose tissue
gDNA Extraction
Sodium Bisulfite treatment(converts cytosine to uracyl)
SequencingMethylation-amplification PCR
U
Adipocyte Isolation(colagenase digestion)
Caf C
EXPERIMENTAL DESIGN
Control Diet High fat Diet0.0
0.5
1.0
1.5
2.0
2.5 *
Lept
in m
RNA
expr
essi
on(R
etro
perit
onea
l W
AT)
Control Diet High fat Diet0.0
2.5
5.0
7.5
10.0 **Le
ptin
(ng
/ml)
Control Diet High fat Diet0
5
10
15
20
25 **
Retro
perit
onea
lW
hite
adip
ose t
issu
e (g)
Control Diet High fat Diet0
2
4
6**
HOM
A
Control Diet High fat Diet0
100
200
300
400
500**
Fina
l Bo
dy W
eigh
t (g
)
Control Diet High fat Diet0
50
100
150
200
250
300
350 **
Food
Int
ake (
Kca
l/day
)
Control Diet High fat Diet0
10
20
30
40
50
60
Tota
l Met
hyla
tion
(%)
RESULTS
Transcription
CpGs
-613 -597-536
-549
0
-664
0 +2000-2000 +8000 +10000
-700 -600 -500 -400
-529 -498 -419 -398-447-443
-477 -395
MethylatedUnmethylated
Control Diet High fat Diet0.00
0.25
0.50
0.75 *
% -4
43 C
pG M
ethy
latio
n
RESULTS
Leptin gene
CpGs
% Methylation
0 25 50 75 1000.0
2.5
5.0
7.5
10.0
12.5 Control DietHighFat Diet
r=-0.899p=0.015
r=-0.400p=0.505
-443 CpG Methylation (%)
Lept
in (n
g/m
l)
0 25 50 75 100300
400
500
600 Control DietHighFat Diet
r=-0.841p=0.036
r=-0.400p=0.505
Total Methylation (%)
Fina
l Bo
dy W
eigh
t (g
)
Association between circulating leptin and methylation of the -443 CpG (A), and between the total methylation of the distal island and rat final body weights (B).
High-fat diet-induced obesity in rodents is able to enhance the methylation pattern of the leptin promoter in adipocytes.Those subjects more methylated in the HF diet, showed lower leptin levels and lower body weight.
DIET MODIFIES DNA METHYLATION
J Physiol Biochem 2009; 65: 1-8
8 weeks of Low Calorie Diet
(n=6)
(n=21)
(n=27)
Subcutaneous Adipose Tissue
(SAT)
SAT
Blood
Blood
Low vs. High responders before diet
Epigenetic biomarkers in weight loss
Cordero P et al. J Physiol Biochem 2011
LEPTIN
TNFα
0
10
20
30
40
50
60
70
Leptina TNF-alfa
Respuesta
No respuesta
*
TBASELINE methylation %
LEPTIN TNFa
Responders
No Responders
Ladder
sample 1 sample 2
met
unmet unmet
met
MSP
Epigenetic biomarkers in weight loss
Cordero P et al. J Physiol Biochem 2011
MetabolicCompounds
“Histone-Code”
Histone-modifying enzymes
mRNA levels
Protein levels
Activity as enzymatic substrate
Diet and nutrients
MetabolicStatus Diseases
Repressed genes Activated genesOCCH3
OCCH3
OCCH3OCCH 3OCCH3
OCCH3
OCCH3OCCH3Ub
Ac
Ac P
P UbUb
Transcription
Ac
Ub
Sum
Sum
Me-C
Biotin
Influence of metabolic compounds and diseases on the activity of histone-modifying enzymes and gene transcription regulation
Main histone modifications, enzymes involved and donors
Methyl deficient diet is lipogenic and induces liver steatosis and alters the histone methylation pattern.Pogribny IP et al. J Hepatol. 2009 Jul;51(1):176-86.
Methyl deficient diet induces liver steatosis in mice
H3K9Me3
H4K20Me3
Diet and histone methylation
Photograph by Todd Bauders
Ac
Ac
Ac Ac
Ac
Slgt-1 Promoter
DIET MODIFIES HISTONE ACETYLATION
Intraceccal administration of sulforaphane inhibits HDAC activity and histone acetylation in colonocytes and inhibits tumor cell proliferation.Myzak MC et al. FASEB J. 2006 .
Inhibits colon cancer proliferation
Dietary inhibitors of histone deacetylases (HDACs)
Green tea’s teophylline increases deacetylase activity and supresses the expression of pro-inflammatory genes.Ito K et al. Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8921-6.
Dietary inhibitors of histone deacetylases (HDACs)
Resveratrol increases NAD-dependent HDAC activity
Yeung F, et al. Embo J. 23:2369-2380, 2004
Dietary activators of histone deacetylases (HDACs)
Caloric restriction increases SIRT1 expression (a protein deacetylase) in a variety of rat tissues as well as longevity
Cohen HY, et al. Science 305:390-392, 2004.
Dietary activators of histone deacetylases (HDACs)
Methylation and Histone modifications involved in the repression (left) or activation (right) of gene transcription
Repressed genes Activated genes
OCCH3
OCCH3
OCCH3OCCH3OCCH3
OCCH3
OCCH3OCCH3Ub
Ac
Ac P
P UbUb
Transcription
Ac
Ub
OCCH3
Ub
Ac
P
Sum
Methylated residues
Ubiquitylated residues
biotinylatedresidues
Phosphorylatedresidues
Sumoylatedresidues
Sum
Sum
Me-C Methylated cytosine
Me-C
The Histone Code
Biotin
Biotin
Acetylatedresidues
CpG Island Analysis in the promoters of different genesRole in obesity Gene symbol
adipogenesis PPARGC1A
adipogenesis NR0B2
adipogenesis FGF2
adipogenesis PPARG
adipogenesis PTEN
adipogenesis and cell cycle CDKN1A
adipogenesis and inflammation LEP
adipogenesis ESR1
adipogenesis NR3C1
inflammation TNF
inflammation PLA2G4A
inflammation SOD3
inflammation SOCS1/3
inflammation and apoptosis CASP8
energy metabolism COX7A1
fat metabolism LPL
fat metabolism FABP4
insulin signalling CAV1
insulin signalling PIK3CG
insulin resistance and adiposity HSD11B2
insulin resistance IGFBP3
BSP
Pyrosequencing
Capillary Electrophoresis
U
Technology
EpiTYPER Sequenom
Microrarray
+ Sequencing
MSPMethyLight
Methyl Chip-on-Chip
Technology
Table 1. Available techniques for research in epigenetics.
DNA methylation Histone modifications and non-histone proteins Enzymes involved miRNA
Global LUMA, IRE, HPLCHPLC, Western Blot, PAGE,
HPCE, RP-HPLC and hydrophilic Interaction LC
Western blot, enzymatic activity, RT-
PCR
Locus-specificBSP, MM, Pirosequencing, MS-Snupe, HMR, COBRA,
MSPChip Assay Chip Assay RT-PCR
Genome wide analysis Human Methylation 27 BeadChips, Medip
Chip on chip Chip on seq
Chip on chip, Microarrays, Proteomics Microarrays
Technology
HS diet
12
11
C
HF
< 6% METHYLATION
6 - 25% METHYLATION
25 - 40% METHYLATION
40 - 80% METHYLATION
> 80% METHYLATION
Diet modifies DNA methylation
Lomba A et al. Mol Genet Metab. 2010; 101: 273-8
Table 1. Differentially methylated regions in several obesogenic genes by Illumina microarray and their corespondent analysis by Sequenom Epityper approach.MICROARRAY SEQUENOM
Symbol Chrom. Chromosomic CpG position
Illumina ID PRIMERS CpGs present in the region
CpG dinucleotide equivalent to
AQP9 15 56217683 cg11098259 LEFT aggaagagagTGAAAATTTTTTTTGGATTAGGGTT RIGHT cagtaatacgactcactatagggagaaggctAA
TCCTCACTTTCACAACCAAATAA
7 CpG 1
56217974 cg11577097 CpG 7
ATP10A 15 23577342 cg11015241 LEFT aggaagagagAGGTTGGTTTTTTTATTTAGGTTGG RIGHT cagtaatacgactcactatagggagaaggctCT
CCCAAATTCAAATAATTCTCCTA
22 CpG 3 and CpG4
23577440 cg17260954 CpG 5, CpG10, CpG16
CD44 11 35117468 cg18652941 LEFT aggaagagagGGATATTATGGATAAGTTTTGGTGG RIGHT cagtaatacgactcactatagggagaaggctCC
TTTCTAAAAAACCCATTACCAAC
31 CpG 2 and CpG3
35117805 cg04125208 CpG 26
IFNG 12 66839844 cg26227465 LEFT aggaagagagTGTGTTGTATTTTTTTTGGTTGTTG RIGHT cagtaatacgactcactatagggagaaggctAA
AAAACTTCCTCACCAAATTATTC
5 CpG 1
MEG3 14 100362433 cg05711886 LEFT aggaagagagATGGGTTTTGTTTTTTTGGATATGT RIGHT cagtaatacgactcactatagggagaaggctTA
AACTAAAATCCCTACCACCCAAC
6 CpG 2
NTF3 12 5473803 cg04740359 LEFT aggaagagagTTTTTTTAGAATGTTTAGAGGGGAG RIGHT cagtaatacgactcactatagggagaaggctAA
AAACCTCAACTTTAAACAAAATACTCT
6 CpG 6
POR 7 75421357 cg20748065 LEFT aggaagagagGGGGTAAGGTTTAGTATTTAGGTGG RIGHT cagtaatacgactcactatagggagaaggctTC
TAACAAAAAAACAAAACCCAAAA
11 CpG 8
TNFRS9 1 7922901 cg08840010 LEFT aggaagagagTATAAGAGGTTGAATGATTTTGTTGTG RIGHT cagtaatacgactcactatagggagaaggctAA
AAAATACACCCTCAAACTTTAACAA
4 CpG 3
WT1 11 32406026 cg04096767 LEFT aggaagagagGGGAGATTAGTTTTAATTTTTTTTAAG RIGHT cagtaatacgactcactatagggagaaggctCT
AAATCTCCCTCCATCCCAAATAC
34 CpG 9 and CpG10
32406214 cg12006284 CpG 21
Some promoter methylable regions on putative obesogenes
effect residue and methylation affected experimental model citeincrease H3K4me1 insulin via ROS, and high glucose, choline defficiency Kabra09 Brassachio Davison 09
H3K4me2 Methyl defficency in mice, Hypoxia, Hexavalent Chromium, aging, and glucose Dobosy 08 Jonhson 08 Xia 2009 Sun 2009 Schnekenburger 2007El-meyayen 2009Miao 07 burke 2009
H3K9me1Protein restriction, nickel ion exposure, insulin via ROS with hyperglycemia, deprivation of glucose, gestational choline supply, hypoxia, chromium, high glucose
Inductors of the different histone modifications, enzymes implicated and amino acidic residues affected
.Campion J, Milagro FI and Martinez JA (2010)
1
Maternal alterations
Caloric restriction Increase of genomic methylation of ras DNA
Reduction of rat uterine blood flow Alteration of the methylation status of p53 in the kidney of the offspring
Dietary protein restriction of pregnant rats Hypomethylation of hepatic gene expression (glucocorticoid receptor and PPARalpha) in the offspring
Methyl donors Restriction of cobalamine, folate, methionine DNA methylation in the preovulatory oocyte and the preimplantation embryo Folate deficiency Impact on DNA methylation (i.e.in rat liver) and colon cancer susceptibility
Folic acid, vitamin B12, choline, and betaine Prevention of transgenerational amplification of body weight in A(vy) mice
Microminerals Differerent micronutrient intake Epidemiologic data relating methylation in p16(INK4a) with low consume of folate, vitamin A, vitamin B1, potassium and iron.Selenium defficiency Modification of methyl metabolism in different tissues
Arsenite defficiency Hypomethylation of Caco-2 cells not treated with arsenite
Vegetarian diet Vegetarian vs omnivore diet 3-fold increase in the expression of the MnSOD gene and decreased CpG methylation in its promoter regionFatty acids Butyrate supplementation Demethylation of RARbeta2 in cancer cellsOther compounds Soy genistein Reversal of hypermethylation of several genes and
Tea polyphenols supplementation Inhibition of DNA methyltransferase in cancer cells
Diallyl disulfide Increase of histone acetylation in human colon tumor cell lines
Sulforaphane Inhibition of histone deacetylases
Royal jelly intake Modifications in reproductive and behavioural status of honeybees
Toxins/Drugs Bisphenol A DNA hypomethylation and body weight gain in rats
Epigenetic modifications due to different nutritional conditions
EPIOBESOGENES?. A review,,,,,,,,,,,,? Volunteers for a Methylepigenome?
PROGRAMACIÓN PERINATAL Y ENF. CRÓNICAS
NUTRITION, PERINATAL PROGRAMING AND DISEASE
Genetics Genomics Epigenetics
Rather than there being an ‘optimal’ human diet, there are a range of adequate diets which depend upon individual biological and cultural variation.
EPIGENETIC MARKS GENETIC BACKGROUND
PHYSICAL ACTIVITY
LIKES AND DISLIKESALLERGIES AND INTOLERANCES
FAMILY HISTORY
PREVIOUS DISEASESCULTURE
Personalized diet
PERSONALIZED NUTRITION
Nutrition and Health
San Sebastián/Donostia 2009
But genetics/epigenetcs is only the tip of the iceberg
THANK YOU !
DNA Methylation
ProfilingSNP Profiling
Personalized nutriepigenomics
The children born small or large for gestational age are more likely to be obese.Both birth weight extremes are hyper-insulinemic, insulin-resistant states.If the mother suffers from gestational diabetes, the risk is higher.There are differences between monozygotic twins.
How could diet and lifestyle alter the epigenetic (methylation) pattern ?
Wonders:
1- How the dietary-induced epigenetic marks could be inherited and influencethe obesity susceptibility and metabolic alterations of the offspring ?.
2- How the diet could influence the epigenetic pattern (DNA methylation) of gene promoters and epigenetics could be concerned with obesity ?.
3- How the epigenetic marks related with inflammation could be used as biomarkers of disease risk or weight loss/weight gain susceptibility ?.
Nutriepigenomics
Supplementation of maternal diet with genistein and other compounds induced alterations in DNA methylation that were reflected in offspring coat color changes.Dolinoy DC et al. Environ Health Perspect. 2006; 114: 567-72
Nutriepigenetics
Methyl donor supplementation prevents transgenerational amplification of obesity.
Avy/a mice fed a diet supplemented with extra folic acid, vitamin B12, betaine and choline, that induces DNA hypermethylation.
Body weight is relatively constant in the supplemented group, but increases transgenerationally in the unsupplemented group (P=0.000006).Waterland RA et al. Int J Obes 2008;32:1373-9.
Nutriepigenomics
Supplementation of maternal diet with choline reduces Igf2 promoter methylation and inhibits Dnmt1 methyltransferase. It ameliorates memory !
Kopacheva VP et al. J Biol Chem. 2007; 282; 31777-88.
Choline supplementation increases SAM
Choline defficiency increases Igf2 methylation
Choline defficiency increases Igf2 and Dnmt1 expressions
Diet and histone metylation
I. INTRODUCCIÓN
Ng S-F et al. Nature 2010; 467: 963-6.
HFD leads to adiposity, glucose intolerance and insulin resistance in fathers.
a, Body-weight trajectoriesb, Blood glucose during glucose tolerance test
Fathers (fed control or HFD diet) Female offspring (fed control diet)
b b
Chronic high-fat diet in fathers programs β-cell dysfunction in female rat offspring
Prenatal nutrition induces differential changes to the methylation of individual CpGs in juvenile rats which persist in adults.
Control
Protein-restricted
Transcription factors
PPAR
-alphaBACKGROUND
In this case, feeding pregnant rats a protein-restricted diet persistently alters the methylation of specific cytosines in the hepatic PPAR alpha promoter of the offspring Lillycrop KA et al. Br J Nutr. 2008; 100: 278-82
Maternal care (licking, grooming, and nursing methods) alters cytosinemethylation of glucocorticoid receptor promoter in their pups. DNA methylation and histone acetylation at the hippocampus and the long-term behavior of their offspring. Weaver IC et al. Nature Neurosci 2004;7: 847-54.
BACKGROUND
Progressive increases in DNA methylation have been found with aging..
Mean bisulfite pyrosequencing percent methylation of FZD9_E458 and 5 downstream CpGs in adult bloods Christensen BC et al. PLoS Genet. 2009; 5: e1000602.
BACKGROUND
Wonders:
1- How the dietary-induced epigenetic marks could be inherited and influencethe obesity susceptibility and metabolic alterations of the offspring ?.
2- How the diet could influence the epigenetic pattern (DNA methylation) of gene promoters and epigenetics could be concerned with obesity ?.
3- How the epigenetic marks related with inflammation could be used as biomarkers of disease risk or weight loss/weight gain susceptibility ?.
Nutriepigenomics
May high energy or high-fat diets affect gene promoter methylation,
affecting thus obese “phenotype”?
The methylation pattern of leptin promoter is modified by high fat diet-induced obesity in rats.Milagro FI, Campión J, García-Díaz DF, Goyenechea E, Paternain L, Martínez JA. J Physiol Biochem 2009; 65: 1-8.
HYPOTHESIS
In Vivo Methylation Patterns of the Leptin Promoter in Human and MouseStöger R.Epigenetics 1:4, 155-162, 2006
BACKGROUND
Proporcion en nutrientes de las dietas
0
10
20
30
40
50
60
70
80
Proteínas Lípidos (%) Hidratos de carbono (%)
Prop
orci
on (%
)
Control
Cafeteria
Proteins Lipids Carbohydrates
x2
n=5
n=6
Control
Cafetería
MATERIAL AND METHODS
77 days
oooo
J Physiol Biochem 2009; 65: 1-8
Retroperitoneal adipose tissue
gDNA Extraction
Sodium Bisulfite treatment(converts cytosine to uracyl)
SequencingMethylation-amplification PCR
U
Adipocyte Isolation(colagenase digestion)
Caf C
EXPERIMENTAL DESIGN
Control Diet High fat Diet0.0
0.5
1.0
1.5
2.0
2.5 *
Lept
in m
RNA
expr
essi
on(R
etro
perit
onea
l W
AT)
Control Diet High fat Diet0.0
2.5
5.0
7.5
10.0 **Le
ptin
(ng
/ml)
Control Diet High fat Diet0
5
10
15
20
25 **
Retro
perit
onea
lW
hite
adip
ose t
issu
e (g)
Control Diet High fat Diet0
2
4
6**
HOM
A
Control Diet High fat Diet0
100
200
300
400
500**
Fina
l Bo
dy W
eigh
t (g
)
Control Diet High fat Diet0
50
100
150
200
250
300
350 **
Food
Int
ake (
Kca
l/day
)
Control Diet High fat Diet0
10
20
30
40
50
60
Tota
l Met
hyla
tion
(%)
RESULTS
Transcription
CpGs
-613 -597-536
-549
0
-664
0 +2000-2000 +8000 +10000
-700 -600 -500 -400
-529 -498 -419 -398-447-443
-477 -395
MethylatedUnmethylated
Control Diet High fat Diet0.00
0.25
0.50
0.75 *
% -4
43 C
pG M
ethy
latio
n
RESULTS
Leptin gene
CpGs
% Methylation
0 25 50 75 1000.0
2.5
5.0
7.5
10.0
12.5 Control DietHighFat Diet
r=-0.899p=0.015
r=-0.400p=0.505
-443 CpG Methylation (%)
Lept
in (n
g/m
l)
0 25 50 75 100300
400
500
600 Control DietHighFat Diet
r=-0.841p=0.036
r=-0.400p=0.505
Total Methylation (%)
Fina
l Bo
dy W
eigh
t (g
)
Association between circulating leptin and methylation of the -443 CpG (A), and between the total methylation of the distal island and rat final body weights (B).
High-fat diet-induced obesity in rodents is able to enhance the methylation pattern of the leptin promoter in adipocytes.Those subjects more methylated in the HF diet, showed lower leptin levels and lower body weight.
DIET MODIFIES DNA METHYLATION
J Physiol Biochem 2009; 65: 1-8
Wonders:
1- How the dietary-induced epigenetic marks could be inherited and influencethe obesity susceptibility and metabolic alterations of the offspring ?.
2- How the diet could influence the epigenetic pattern (DNA methylation) of gene promoters and epigenetics could be concerned with obesity ?.
3- How the epigenetic marks related with inflammation could be used as biomarkers of disease risk or weight loss/weight gain susceptibility ?.
Nutriepigenomics
Campión J, Milagro FI and Martinez JA.TNF-alpha promoter methylation as a predictive biomarker for weight-loss response.
Obesity 2009; 17: 1293-7.
Does epigenetically-mediated inflammatory regulation participate in body weight control?
Could the epigenetic control of inflammation-related gene promoters be implicated in the susceptibility to lose body weight by a hypocaloric diet?
Is inflammation-related epigenetic status a good marker of weight loss?
hypocaloric diet/exercise
Microarray and validation
PBMCsWhite cells
Epigenetic biomarkers in weight loss
Bouchard L et al. Am J Clin Nutr. 2010; 91: 309-20
Epigenetic biomarkers and weight loss
Epigenomic and transcriptomic responses of 14 overweight and obese postmenopausal women to a caloric restriction intervention.
Epigenetic biomarkers in weight loss
Bouchard L et al. Am J Clin Nutr. 2010; 91: 309-20
Epigenetic biomarkers and weight loss
Subcutaneous adipose tissue
Epigenetic biomarkers in weight loss ( Bouchard et al.)
Sullivan KE et al.Epigenetic regulation of tumor necrosis factor alpha. Mol Cell Biol 2007, 27: 5147-5160.
TNF-a promoter methylation is tissue specific
24 obese subjects(BMI: 30.5 ± 1.5 kg/m2)
12 men 12 female
Serum: TNF-aBlood, PBMC: DNA
8-week hypocaloric diet
Weigh loss ≥ 5% of initial body
Responders Non-responders
Serum: TNF-a
EXPERIMENTAL DESIGN
TNF-α gene
% Methylation
Campion et al Obesity 2009
Weight Loss
Baseline TNF-α
Final TNF-α
Subjects less methylated lose more weight
Subjects with morebaseline TNF have the promotermore methylated
Subjects with the promoter more methylated decreaseless the TNF levels
RESULTS
Campion et al Obesity 2009
8 weeks of hypocaloric diet
Before
After
IlluminaMicroarray
Milagro FI et al. FASEB J (2011)
Epigenetic biomarkers in weight loss
Microarray Changes by the diet
625 CpG hypermrth
945 CpG hypometh
Before vs. After diet
(20 % variation p < 0.05)
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
APOA5 PTEN GNAS H19 IL26
Inicial
Final
***
***
***
*** ***
(% metilación)
APOA5 PTEN GNAS H19 IL26
Methylation changes induced by an energy restricted diet
Milagro FI et al. FASEB J (2011).
A B
C
* *
*
D
*
Methylation patterns after an energy-restriction intervention
Table 3. DNA methylation levels of different CpG sites from several obesogenic genes by Illumina microarray and Sequenom Epityper analysis. Results are compared upon the effects of the diet (before vs. after) and upon the techniques (microarray vs. epityper).
Basal (n=12) After intervention (n=10) Microarray vs Epityper
$ p<0.05 for comparisons between dietary groups (before vs. after) by paired t-test, results from microarray# p<0.05 for comparisons between dietary groups (before vs. after) by paired t-test, results from epityper* p<0.05 for comparisons between techniques (Microarray vs. Epityper) by Spearman correlation test
(1) before intervention, (2) after intervention, or (3) both before and after internvention together.
Methylation levels of CpG sites by two different approaches
Rather than there being an ‘optimal’ human diet, there are a range of adequate diets which depend upon individual biological and cultural variation.
EPIGENETIC MARKS GENETIC BACKGROUND
PHYSICAL ACTIVITY
LIKES AND DISLIKESALLERGIES AND INTOLERANCES
FAMILY HISTORY
PREVIOUS DISEASESCULTURE
Personalized diet
PERSONALIZED NUTRITION
Nutrition and Health
San Sebastián/Donostia 2009
But genetics/epigenetcs is only the tip of the iceberg
Conclussion?
THANK YOU !
November 17th-20th 2010
www.isnn2010navarra.com
Bisulfite Sequencing PCR (BSP)
Sodium Bisulfite treatment(converts cytosine into uracyl)
U
PCR products are cloned and transformed in bacteria
PCR with specific BSP primers
Sequencing with accurate primers
Methods in DNA methylation
MSP / MethyLight
Methylation Specific PCR (MSP) is a bisulfite conversion-based PCR technique.
MethyLight method is based on MSP, but using quantitative real-time PCR
Capillary Electrophoresis
Pyrosequencing
Methyl Chip-on-Chip for Methyl-CpGs Binding Proteins (MBDs)
ChIP-on-chip is used to investigate interactions between proteins and DNA in vivo.It combines chromatin immunoprecipitation (ChIP) with microarray technology (chip).it allows the identification of binding sites of DNA-binding proteins on a genome-wide basis.
Methods in Protein-DNA Interactions
Epigenetics & Developmental Origins of Health and Disease
Molecular Nutrition today
Nutrients
Epigenetics & Developmental Origins of Health and Disease
Developmental Origins of the Metabolic SyndromeMcMillen C et al. Physiol Rev 2005; 85 : 571–633.
Metabolic programming in the pathogenesisof insulin resistanceDevaskar SU et al. Rev Endocr Metab Disord (2007) 8:105–113
Developmental Origins of Health and Disease (DOHAD)
The developmental origins of adult disease (Barker) hypothesisDe Boo H et al. Australian and New Zealand Journal of Obstetrics and Gynaecology 2006; 46: 4–14
Developmental Origins of Health and Disease (DOHAD)
DNA METHYLATION
I. INTRODUCCIÓN
↑ ↓
DNA METHYLATION INHIBITS GENE EXPRESSION
↑ ↓
↓ ↑
DNA METHYLATION INHIBITS GENE EXPRESSION
I. INTRODUCCIÓN
↑ ↓
DNA METHYLATION INHIBITS GENE EXPRESSION
Chronic high-fat diet in fathers programs β-cell dysfunction in female rat offspring
Ng S-F et al. Nature 2010; 467: 963-6.
HFD leads to adiposity, glucose intolerance and insulin resistance in fathers.
a, Body-weight trajectoriesb, Blood glucose during glucose tolerance test
Hyperm ethylat ion is associat ed with non-small cell lung cancer
(82)
Evidence for dietary regulation of microRNA expression in cancer cells.Davis CD, Ross SA.Nutr Rev. 2008 Aug;66(8):477-82.
MicroRNAs induced during adipogenesis that accelerate fat cell development are downregulated in obesity.Xie H, Lim B, Lodish HF.Diabetes. 2009 May;58(5):1050-7.
Methylation Specific PCR (MSP) is a bisulfite conversion-based PCR technique for the study of DNA CpG methylation.
The MethyLight method is based on MSP, but using quantitative real-time PCR
Methods in DNA methylation
BSP PCR Pyrosequencing Capillary Electrophoresis
Sequencing by synthesis +DNA polymerase incorporates dNTPs +releases pyrophosphate (PPi) ATP
Methods in DNA methylation
BSP PCR Pyrosequencing Capillary Electrophoresis
Chain-termination methods +thin-layer electrophoresis +fluorescent detection of dNTPs
Methods in DNA methylation
Up to now, the development of fast technologies analyzing DNA sequencing and mRNA expression have allowed to describe:
-Polymorphisms (SNPs)-Gene expression levels
There is a lot of information concerning polymorphisms and gene expression (in different tissues) in relation to:
-differential response to diets,-susceptibility to meabolic diseases,-intake of different nutrients.
However, it lacks the integration of this information.
Polyunsaturated fatty acids modulate the effect of TCF7L2 (transcription factor 7-like 2 gene) gene variants (SNPs) on postprandial lipemia.Warodomwichit D et al. J Nutr. 2009;139:439-46.
SNPs
Microarrays PCR-based methods
Fluorescence in situ hybridization (FISH) DNA Sequencing
Methods in nutrigenetics (CNVs)
While much nutrigenomic research has focused on SNPs, the human genome also has structural variation resulting from:- nucleotide insertions or deletions,- chromosomal segment rearrangements and variation,- increases or decreases in the number of genes (i.e. copy number variants)
Individuals whose ancestors were exposed to either high-starch or low-starch environments differed in the number of amylase genes:Perry GH et al. Nat Genet. 2007; 39(10): 1256–60
CNVs
Nutrigenomics (miRNA)
microRNAs (miRNA) are single-stranded RNA molecules of 21-23 nucleotides, which regulate gene expression.
miRNA is complementary to a part of one or more messenger RNAs (mRNAs). It promotes cleavage of the RNA and inhibits gene expression.
MicroRNAs induced during adipogenesis that accelerate fat cell development are downregulated in obesity.Xie H, Lim B, Lodish HF.Diabetes. 2009 May;58(5):1050-7.
Nutrigenomics (miRNA)
Comparison by RT-PCR of miRNA regulation during 3T3-L1 differentiation (mature adipocytes vs. enriched preadipocytes )
Are there other reasons to explain obesity pandemics ?
Marti A et al. Int J Obesity (2004) 28, S29–S36.
Intake Expenditure
Genetics
Resistance to high-fat diet in the female progeny of obese mice fed a control diet during the periconceptual, gestation and lactation periods.Gallou-Kabani C et al. Am J Physiol Endocrinol Metab (2006).
When mice are cloned, they have normal birth weights but often develop adult-onset obesity.Tamashiro KL et al. Nat Med. 2002; 8:262-7.
3 – Are diet-induced epigenetic marks inherited by the offspring,affecting thus obesity pandemics?
Nutriepigenetics can furnish long-term or static biomarkers for individual disposition towards diet and nutritional imprinting.
Epigenetics does not look at DNA sequence but at post-translational modifications of:- DNA-binding proteins (e.g., histones and chromatin) - DNA itself (methylation)
- Other mechanisms (iRNA, transposons,…)
which influence DNA accessibility and, thereby, transcription.
These effects could even be transmitted from one generation to another.
Supplementation of maternal diet with genistein and other compounds induced alterations in DNA methylation that were reflected in offspring coat color changes.Dolinoy DC et al. Environ Health Perspect. 2006; 114: 567-72
Decreases in cell number. Organ growth fetal growthDecreased vasculgenesis, angiogenesis
Premature differentiation of functional capacity
Restricted
substrate
supply during
embryogenesis
organogenesis
and fetal life
Increased
nutrient supply
in neonatal,
postnatal or
adult life
McMillen, et al. 2005
Resistance to high-fat diet in the female progeny of obese mice fed a control diet during the periconceptual, gestation and lactation periods.Gallou-Kabani C et al. Am J Physiol Endocrinol Metab (2006).
When mice are cloned, they have normal birth weights but often develop adult-onset obesity.Tamashiro KL et al. Nat Med. 2002; 8:262-7.
1 – Are diet-induced epigenetic marks inherited by the offspring,affecting thus obesity or diabetes pandemics?
Up to now, the development of fast technologies analyzing DNA sequencing and mRNA expression have allowed to describe:
-Polymorphisms (SNPs)-Gene expression levels
There is a lot of information concerning polymorphisms and gene expression (in different tissues) in relation to:
-differential response to diets,-susceptibility to metabolic diseases,-Intake/requirements of different nutrients.
However, it lacks the integration of this information.
Genetic Methods in Nutrition Research
ADVANCES IN EXPERIMENTAL TECHNOLOGIES for analyzinggenomes, proteins, metabolites, and transcripts are layingthe foundation for developing recommendations for personalizednutrition and optimizing medical treatments for each individual.
Genomics started with the sequencing of genome. Then, expanded to DNA (SNPs, structure) and RNA studies (transcriptomics).Genomics is extending to protein and metabolite studies (proteomics/metabolomics). Systems biology aims to study biology as a whole, integrating the previous information.