“ 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
Conclussion?
CH
OLi
pid s
Prot
e in
sAl
coho
l
BM
RTe
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e nei
sAd
ctiv
ida d
Fi
sic a
INTAKE NEEDS
Nutrient Balance
Efferent SignalsAfferent Signals
CNSNeuropeptides
Genétics / habits / neuroendocrine status
mins VITs R
estr
uctu
re
Nutritional Balance
Regulatory Elements Estructural Functions
Proteíns
Energy
Nutrients
NUTRIENT FUNCTIONS
Fatty AcidsRetinoidsVitamin DGlucoseEnergy
Transcriptión mRNA DNA ProteínsTranslation
Amino ácidsIronSelenium
Postranslation
MineralsVitamins
GENE EXPRESSION NUTRITIONAL CONTROL
Cascade Kinases
Mechanisms : - Receptors (membrane/nucleus)Nutrients
Nutrients
�
Nutrients &Metabolites
- Metabolism intermediate
FT
- Transcription Factors (FT): affinity & concentrations
RE
GENE EXPRESSION CONTROL: MECHANISMS
DNA RNA PROTEINS
Transcription Translation
Gene Expression= f(DNA × Environmental factors)
GENOTYPE → PHENOTYPE
Nutrition and Genetic interactions
Possible interactions of dietary intake with genetic variability to affect disease risk.
Jenab MZ et al. Hum Genet (2009) 125: 507–525
Personalized Medicine and Nutrition
Genetic make up can determine unique nutritional requirements and responses to different foods.
Based on:
- The sequencing of the human genome,
- subsequent analyses of human genetic variation,
-studies that associate gene variants with disease markers
-Impact of nutrition/nutrients on gene expression
Nutriepigenetics
Epigenetic processes modulate gene activity and expression without changes in the DNA nucleotide sequence
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
b
Female offspring demonstrate impaired glucose tolerance.
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
aK, acetyl lysine; meR, methyl arginine; meK, methyl lysine ; uK-ubiquinated lysine; pS, phosphorylated serine; bK, biotinylated lysine; AR, ADP-ribosylation
H3
H3
H4
H4
H2A
H2A
H2B
H2B
aK
aK
aK
aK
aK
aK
aK
aK
aKaK
meK
meKmeK
meKmeR
meK
meR
pS
pSuK
uK
uK
AR
bK
bKAR
NAD+
Acetyl CoA
HATs
HDACs
Serine kinaseATP
Serine phosphatase
KMTs
KDMsSAM
biotinidaseholocarboxylase synthetase
Biotinebiotinidase
H3
H3
H4
H4
H2A
H2A
H2B
H2B
aK
aK
aK
aK
aK
aK
aK
aK
aKaK
meK
meKmeK
meKmeR
meK
meR
pS
pSuK
uK
uK
AR
bK
bKAR
NAD+
Acetyl CoA
HATs
HDACs
Serine kinaseATP
Serine phosphatase
KMTs
KDMsSAM
biotinidaseholocarboxylase synthetase
Biotinebiotinidase
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
H3K4me3 Hexavalent Chromium, Arsenite nickel, LPS, aging, hypoxia, glucose, ischemia, benzoapirene Sun 2009 Zhou 2009 Ara 08 El-meyayen 2009 Schnekenburger 2007
Xia 2009 Johnson 08 burke 2009 Zager 2009
H3K9me1Protein restriction, nickel ion exposure, insulin via ROS with hyperglycemia, deprivation of glucose, gestational choline supply, hypoxia, chromium, high glucose
Lyllicrop Kabra09 Murayama 2008Xia 2009 Sun 2009
Chen 2006 Davison 09 Chen 2006 Miao 07H3K9me3 food restriction, methyl deficient model, hexavalent chromium Sharif 07 Pogribny 06 Pogribny 09
Sun 2009H3K27me3 food restriction, hypoxia, gestational choline supply Sharif 07 Johnson 08 Davison 09
H3K36me3 hypoxia Xia 2009H3K79me2 hypoxia Johnson 08H4R3me2 hypoxia Johnson 08
reduction H3K4me1 insulin via ROS with hyperglycemia Kabra09H3K4me2 High glucose, cAMP glucose induced, diabetic state Miao 07 burke 2009 Miao 07
H3K4me3 food restriction, chromium, benzoapirene, methyl donors, LPS Sharif 07 Schnekenburger 2007Ara 08
H3K9me1 insulin via ROS Kabra09H3K9me2 Protein restriction, choline/methionine restriction, BIX-01294, high glucose Lyllicrop Dobogy 08 Miao 07
Dobosy 08 Kubicek 2007
H3K9me3 db/db mice, adenosinealdehide Villeneuve 08 Heit 2009
H3K27me3 DZNEP, adenosinealdehide, sinefungin, hypoxia, hexavalent chromium, methyl defficient diet
miranda 09 Sun 2009Johnson 08 Pogribny 09
H3R17 Insulin Hall 07H3R2me2 Hexavalent Chromium Sun 2009H4K20me3 food restriction, adenosinealdehide, methyl deficient diet, cAMP glucose induced Sharif 07 Heit 2009 burke 2009
Pogribny 06 Pogribny 09H3K20me3 DZNEP miranda 09
Examples of experimental changes in the level of histone methylations (lysines, K and arginines, R) by different metabolic conditions.
IsothiocianatesAlcoholButyrateNADCaffeinaeResveratrolCalóric RestrictionHigh-salt dietsLipoic acid, vit. E
InflammationInfections (virus…)HypoxiaOxidative StressSmokingPhysical activity
HDACs and SIRTInhibitors
DIETARY FACTORS
METABOLISM
DRUGS
BiotinNicotinamide (niacin)Anacárdic AcidGarcinolCurcuminTeofilinCupperChromiumNickel
EstrógensHyperglycemia
HAT inhibitotsAcetyl CoA inhibitors
DIETARY FACTORS
METABOLISM
DRUGS
aK
aK
aKaK
HISTONE ACETYLATION
INH
IBITIO
NA
CTI
VATI
ON
Dietary and metabolic factors involved in histone acetylation and deacetylation
Phosphorylation ADP-Ribosylation
Sumoylation Ubiquitination Deimination Biotinylation Proline isomerization
Enzymes Serine kinasesSerine phosphatases
poly-ADPribose polymerasemono-ADP-ribosyltransferaseADP-ribosyl cyclasesSirtuin SirT4
Sumo-protein ligases
Ubiquitin ligase Peptidylarginine deiminases (PAD 1-6)
BiotinidaseHolocarboxylase synthetase
CyclophilinsFK506-binding proteinsPin1
Amino acids
Serine, threonine, tyrosine
Arginine, glutamic acid, phosphoserine
Lysine Arginine Lysine Proline
Inducers DNA breaksBenzeneBisphenol AUVAOxidative stressHypoxiaButyrate (inhibitor)
Oxidative stressGenotoxicsDNA breaksTryptophanNAD+Nicotinamide (niacin)
Ginkgolic acidAnacardic acidKerriamycin B
PYR-41 InflammationOxidative stress
BiotinCell proliferationOxidative stress
Juglone
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
b
Female offspring demonstrate impaired glucose tolerance.
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
Baseline High responders Low responders p (n=6) (n=6) Units Mean SEM Mean SEM Body Weight Kg 96.7 ± 2.7 93.8 ± 4.2 n.s. Body Mass Index (BMI) Kg/m2 31.0 ± 0.7 30.2 ± 0.6 n.s. Fat Mass kg 26.7 ± 1.3 26.6 ± 2.0 n.s. Waist Girth cm 102 ± 3 100 ± 2 n.s. Systolic Blood Pressure mm Hg 127 ± 5 131 ± 2 n.s. Diastolic Blood Pressure mm Hg 67 ± 3 77 ± 6 n.s. Total Cholesterol mg/dL 196 ± 21 237 ± 22 n.s. HDL-Cholesterol mg/dL 47 ± 3 46 ± 2 n.s. LDL-Cholesterol mg/dL 127 ± 19 168 ± 17 n.s. Triglycerides mg/dL 108 ± 17 117 ± 20 n.s. Glucose mg/dL 92 ± 5 95 ± 3 n.s. Insulin μU/mL 17.9 ± 5.6 11.2 ± 1.8 n.s. HOMA 4.1 ± 1.4 2.6 ± 0.4 n.s. Leptin ng/mL 13.2 ± 2.9 13.8 ± 2.1 n.s. Adiponectin μg/mL 8.6 ± 1.6 7.5 ± 0.7 n.s. Ghrelin pg/mL 982 ± 120 941 ± 55 n.s. PAI-1 ng/mL 172 ± 16 139 ± 19 n.s. IL-6 pg/mL 1.3 ± 0.1 3.1 ± 1.0 n.s. TNF-α pg/mL 0.9 ± 0.2 1.5 ± 0.3 n.s. Plasma Malondialdehyde μM 2.1 ± 0.4 2.1 ± 0.3 n.s.
Epigenetic biomarkers in weight loss
Milagro FI et al. FASEB J (2011).
602 CpG hypermethylation
432 CpG hypomethylation
Low vs. High responders before 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
1
IL10 AR GNAS SIRT1 CASP8
Respuesta
No respuesta
***
***
***
***
***
(% methylation)
IL10 AR GNAS SIRT1 CASP8
Milagro FI et al. ( FASEB J , 2011)
Epigenetic biomarkers in weight loss
Milagro FI et al. FASEB J (2011).
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
Illumina Epityper Microarray Epityper Microarray Epityper
1-be
fore
2-A
fter
3-be
fore
+a
fter
Symbol ID site Mean ± SEM Mean ± SEM Mean ± SEM Mean ± SEM Spearman
AQP9 cg11098259 CpG 1 45.4% ± 6.3% 48.4% ± 2.9% 26.1% ± 3.1% $ 32.7% ± 4.3% # *AQP9 cg11577097 CpG 7 28.9% ± 3.0% 59.5% ± 6.1% 37.6% ± 2.3% $ 60.1% ± 6.0% *ATP10A cg11015241 CpG 3-4 78.1% ± 8.1% 73.6% ± 4.6% 89.9% ± 1.6% 80.3% ± 4.4% *ATP10A cg17260954 CpG 10 73.5% ± 6.0% 27.2% ± 0.6% 76.2% ± 3.2% 26.7% ± 0.7% *CD44 cg18652941 CpG 2-3 8.5% ± 4.4% 0.7% ± 0.2% 5.6% ± 0.9% 0.4% ± 0.2%CD44 cg04125208 CpG 26 11.3% ± 6.2% 8.1% ± 2.3% 6.2% ± 0.6% 9.1% ± 2.3% * *IFNG cg26227465 CpG 1 69.0% ± 2.6% 0.8% ± 0.2% 73.6% ± 2.7% 1.0% ± 0.4%MEG3 cg05711886 CpG 2 47.0% ± 6.6% 47.0% ± 0.3% 46.7% ± 5.2% 45.2% ± 1.0%NTF3 cg04740359 CpG 6 53.4% ± 8.2% 62.6% ± 6.6% 72.1% ± 1.8% $ 73.6% ± 6.7%POR cg20748065 CpG 8 51.4% ± 6.3% 55.2% ± 2.2% 33.5% ± 2.7% $ 51.4% ± 3.3% *TNFRSF9 cg08840010 CpG 3 53.8% ± 5.3% 4.5% ± 0.7% 36.1% ± 2.2% $ 8.0% ± 2.3%WT1 cg04096767 CpG 9-10 32.0% ± 6.6% 38.0% ± 1.9% 30.6% ± 2.4% 38.7% ± 2.6% * *WT1 cg12006284 CpG 21 23.4% ± 4.1% 21.7% ± 1.8% 24.2% ± 2.4% 27.1% ± 2.3% # * *
$ 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
Milagro FI et al. FASEB J (2011).
* *
High Responders
Low Responders
Before diet
(% methylation)
10
9
0
0
43
14
3
2
8
11
15
7
18
10
3
1
2
2
5 26 292-3 4-28 6-7 8 9-10 11-12 13 14 15 16 17 18 19-20-21 22 23 24-25 27 4-28 30 31
0
0
2
2
3
6
19
16
0
0
0
0
2
2
8
7
14
6
3
0
32
24
23
16
CD44
Fat mass change (%)
BMI change
Final leptin (ng/ml)
Waist girth change (cm)
CpG 9-10 r2(p)
0.255 (p=0.001)
0.364 (p=0.001)
0.142 (p=0.069)
0.246 (p=0.01)
CpG 14 r2(p)
0.297 (p=0.006)
0.304 (p=0.005)
0.179 (p=0.044)
0.181 (p=0.038)
CpG 30 r2(p)
0.194 (p=0.027)
0.299 (p=0.005)
0.050 (p=0.107)
0.120 (p=0.090)
p = 0.001 p = 0.003p = 0.005
*
High Responders
Low Responders
Before diet
(% methylation)
25
28
16
15
32
31
19
22
90
94
15
24
100
100
66
72
3-4 5-10-16 6 9-191-2 14 15 185-10-16 5-10-16 19
*Fat mass change (%)
BMI change
Final leptin (ng/ml)
Waist girth change (cm)
CpG 3-4 r2(p)
0.040 (p=0.353)
0.016 (p=0.540)
0.336 (p=0.003)
0.119 (p=0.087)
CpG 5-10-16 r2(p)
0.078 (p=0.147)
0.172 (p=0.038)
0.010 (p=0.635)
0.036 (p=0.363)
CpG 18 r2(p)
0.375 (p=0.001)
0.434 (p<0.001)
0.377 (p=0.001)
0.382 (p=0.001)
p < 0.001 p = 0.001
ATP10A
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
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
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
b
Female offspring demonstrate impaired glucose tolerance.
a, Blood glucose during glucose tolerance test b, Plasma insulin during glucose tolerance test
Fathers (fed control or HFD diet) Female offspring (fed control diet)
b b
Table 1. Some examples of dynamical changes in DNA methylation pattern and histone modifications due to different nutritional conditions.
Nutritional condition Model Dietary / Nutritional condition Dose Treatment DurationMagnitude of epigenetic and/or metabolic effect Reference
Maternal alterations Rat Reduction of uterine blood flow during pregnancy Ligation of uterine arteries 3 days <43% in p53 methylation (-179 bp)
(25)
Rat Dietary protein restriction during pregnancy 90 protein vs 180 g/kg Pregnancy ≈<10% in PPARalpha and GR methylation
(26)
Methyl donors Sheep Restriction of cobalamine, folate and methionine Sulphur <50% Cobalt <95% Pregnancy80% hypomethylated, 20% hypermethylated
(29)
Rat Folate deficiency in postweaning period 0 mg folic acid/kg vs 2 or 8 Postweaning period> 34-48% in DNA methylation
(30)
Rat Folate supplementation in elderly period 18 µmol folate/ kg 8 / 20 weeks> 30% in DNA methylation
(38)
Mouse Dietary methyl supplementation with folic acid, vitamin B12, choline and betaine
5 g/kg Choline, 5 g/kg Betaine, 5 g/kg Folic acid, 0.5 mg/kg Vitamin B12
Pregnancy Lower body weight (34)
Microminerals Human Different micronutrient intake Consume less methyl donors Long duration ns (43)
Rat Selenium deficiency 0.2 mg selenium / kg Weanling rats< 20% in DNA methylation
(44)
Caco-2 cells Arsenite deficiency 0, 1 or 2 µmol arsenite / L 7 days< 20% in DNA methylation
(45)
Vegetarian diet Human Vegetarian vs omnivore diet ns Years< 5-20% in MnSOD methylation
(46)
Fatty acids HCT116 / HT-29 cells Butyrate supplementation 4 mM 24 hours< 70% in RARbeta2 methylation (48)
Other compounds KYSE 510 / 150 cells Soy genistein supplementation 5, 10, or 20 µmol/L 6 days --- (50)
OSCC cells Tea polyphenols (epigallocatechin-3-gallate) suppl. 20 or 50 µ M 6 days --- (51)
Rat colonocytes Diallyl disulfide supplementation 200 µ M 3-6 hours --- (52)
Mouse Sulforaphane oral adminitration 10 µ mol 6 hours < 50-65% in histone deacetylase activity
(57)
Honeybee Royal jelly intake Fed with royal jelly ns < 10-15% in dynactin p62 methylation
(58)
Toxins/Drugs Rat Bisphenol A during pregnancy and lactation 50 mg/kg diet Perinatal< 15% in CabpIAP methylation, and body weight gain
(32)
--- Shown in figure but not quantified. ns - Not showed or determined; unapplicable
Table 2. Examples of several human genes described recently as regulated by epigenetic mechanisms and involved in obesity (EpiObesigenes)
Role in obesity Gene symbol Name Epigenetic evidence References
adipogenesis PPARGC1Aperoxisome proliferat or act ivat ed receptor gamm a coact ivator-1 alpha
im port an t in human islet insulin secret ion (59)
adipogenesis NR0B2 nuclear recep tor sm all heterodimer part ner
t um or suppresor methylat ion-relat ed (60)
adipogenesis FGF2 Fibroblast Growth Factor-2 hom ocyst eine disrup t s ECs t h rough alt ered promoter DNA methylat ion
(61)
adipogenesis PPARG peroxisome proliferat or-act ivat ed receptor gamm a
Changes in DNA m et hylat ion during cellular aging and atherosclerosis
(62)
adipogenesis PTEN phosphat ase and tensin homolog
epigenet ic role in colorect al cancer and gliomas (63)
adipogenesis RARA ret inoic acid receptor, alpha P romoter hypermethylat ion is associat ed with prost at e and m amm ary cancer
(64)
adipogenesis and cell cycle
CDKN1A cyclin-dependent k inase inhibit or 1A (p21, Cip1)
aberran t prom oter methylat ions related t o cancer (65)
adipogenesis and in flamm at ion
LEP Lept in post zygot ic developm ent , adipocyt e m at urat ion and cellular aging
(62, 66-68)
adipogenesis ESR1 Est rogen recep tor alpha prognost ic value of ER hypermethylat ion (69)
adipogenesis NR3C1 Glucocort icoid recep tor M ethylat ion st at us is sensit ive t o prenat al mat ernal mood
(70)
inflamm at ion and insulin resist ance TNF
t umor necrosis factor (T NF superfamily , m ember 2)
Epigenet ic silencing during endot oxin t o lerance and myeloid differen t iat ion (71)
inflamm at ion PLA2G4A plasm a secretory phospholipase A(2) t ype IIA
malignant p rost ate cells (72)
inflamm at ion SOD3 Ext racellular superoxide dismut ase
developm ent o f foam cells (73)
inflamm at ion SOCS1 Suppressor o f cy tok ine signaling 1
Severit y of liver fibrosis and hepatocarcionma (74)
inflamm at ion SOCS3 Suppressor o f cy tok ine signaling 3
ro le in cellular growt h and m igrat ion and melanomas
(75)
inflamm at ion and apoptosis
CASP8 caspase 8 hyperm et hylat ion in neuroblastom as and medulloblastom as
(76)
energy metabolism COX7A1 cytochrome c ox idase subunit VIIa po lypept ide 1 (m uscle)
Age in fluences DNA m ethylat ion (77)
fat m et abolism LPL lipopro t ein lipase Changes in DNA m et hylat ion during cellular aging (62)
fat m et abolism FABP4 fat t y acid binding pro t ein 4 , adipocyt e
Changes in DNA m et hylat ion during cellular aging (62)
insulin signalling CAV1 Caveolin 1 Aberrant methylat ion is associated with hepatocellular carcinoma
(78)
insulin signalling PIK3CG phosphoinosit ide-3-k inase, catalyt ic, gamma pp
CpG hypermethylat ion is associated wit h progression of colorect al cancer
(79)
insulin resist ance SSTR2 somatost at in recep tor 2 t issue-specific relat ed (80)
insulin resist ance and adiposit y HSD11B2
11 bet a-hydroxyst ero id dehydrogenase 2
in v ivo Epigenet ic repression and relat ion t o hypert ension (81)
insulin resist ance IGFBP3 insulin -like growth factor binding prot ein 3
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.
Nutrigenomics (miRNA)
A supply of new methyl groups can be provided from foodCholine comes from lecithin, eggs, lettuce, peanuts and liver.
Methionine comes from chicken, beef, fish (defficiency in legumes)
Betaine is in sugar beet.
We can make methyl groups from precursor chemicals Folic acid comes from legumes, fruits and grains.
To transport the methyl groups within the body, and attach them safely to DNAZinc:
Vitamin B12 or cobalamin
High levels of homocysteine have been associated with atherosclerosis and an increased risk of heart attacks and stroke.
However Folic acid, Vitamin B12, Choline increase methylation!
Homocysteine
Betaine + ZnBHMT
HistoneBetaine + Zn
BHMT
Histone
Methyl donors and the one-carbon (methionine) cycle
BSP PCR Pyrosequencing Capillary Electrophoresis
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
BSPMSP
MethyLight Pyrosequencing Capillary Electrophoresis
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?
Individuality and epigenetics in obesity.Campion J, Milagro FI and Martinez JA.Obesity Rev 2009; 10:383-92
Many genes related to obesity are regulated by DNA methylation
Individuality and epigenetics in obesity.Campion J, Milagro FI and Martinez JA.Obesity Rev 2009; 10:383-92
i.e., 10 promoters specially susceptible to epigenetic regulation according to bioinformatics Methprimer sequence analysis (epiobesigenes).
1 – Do hypercaloric diets induce DNA methylation ?
Perturbation
NutritionCaloric restriction Glycemic index
aboriginal/cafeteria Protein restriction
Taurineglycine
Micronutrient restrictionIronCalcium
Environment Ambient temperature
HyperthermiaCold
Hypoxia Exogenous agents
Cigarette smokeExperimental/therapeutic drugs
GlucocorticoidsEstrogensStilboestrolStreptozotocin
Utero-placenta circulation Embolization Uterine artery ligation Restriction of placental growthPre-implantationEnvironmental/conditions Local nutrient concentrations
SerumAmino acids Leucine McMillen, et al. 2005
Organ system
Organ size/cellular phenotye Heart
Cardiomyocytes Coronary arteries Vascular
Smooth muscleEndothelium
Brain Liver
Zonation Adrenals Pancreas
Isletsbcells
KidneyNephron number
Lung Fat
Adipocyte numberAdipocyte size
MuscleType I and IIFibers
Bone
Vasculogenesis/Angiogenesis
Set points
Pathology
Cardiovascular disease Coronary artery disease Stroke
HypertensionDiabetes Type I/Type II diabetes Impaired glucose Tolerance Insulin resistanceDyslipidemiaObesity Fat distributionOsteoporosisHypercortisolismGH/IGF programmingTumors Childhood leukemia Breast cancerRespiratory DiseasePsychiatric disorders Schizophrenia
Maternal
Placental
Fetal
Neonatal
Postnatal
EpigeneticMechanisms
Are there other reasons to explain obesity pandemics ?
Hales & Barker. 2001Type 2 Diabetes Metabolic Syndrome
Hales & Barker. 2001Type 2 Diabetes Metabolic Syndrome
Are there other reasons to explain obesity pandemics ?
Hales & Barker. 2001Type 2 Diabetes Metabolic Syndrome
Methylation is a biochemical process that starts with methionine.Anything that depletes SAM (aging, alcohol) lowers methylation.
DNA methyltransferases
DNA methylation
Betaine + ZnBHMT
Cytosine
One-carbon cycle
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.
DNA Methylation and Histone modification. (From Molecular Development - Epigenetics by Dr Mark Hill.)
Epigenetic modifications
Langley, et al. 1994
Response of rats exposed to 18 or 6% casein in utero to an intravenous glucose load, at 9 weeks of age.
HYPERGLICEMIA PROGRAMMING AS AFFECTED BY PERINATAL FEEDING
Fernández-Twinn, et al. 2005
INSULINEMIA AS AFECTED BY PERINATAL FEEDING
Perinatal Nutrient Restriction
Pancreas Liver Skeletal Muscle Adipocyte
b cell massand secretory
capacity
Glucose uptake
Gluconeogenesis
Lipid oxidation
Insulin sensitivity
Insulin inhibition of lipolysis
Decreased pancreatic functional capacity glucose intolerance
insulin resistance
TYPE 2 DIABETES McMillen, et al. 2005
PERINATAL PROGRAMING
PerturbationNutrition Caloric restriction Glycemic index
aboriginal/cafeteria Protein restriction
Taurine, glycine Micronutrient restriction
Iron, CalciumEnvironment Ambient temperature
HyperthermiaCold
Hypoxia Exogenous agents
SmokingExperimental/therapeutic drugs
GlucocorticoidsEstrogensStreptozotocin
Utero-placenta circulation Embolization Uterine artery ligation Restriction of placental growthPre-implantationEnvironmental/conditions Local nutrient concentrations
SerumAmino acids Leucine McMillen, et al. 2005
Organ system Organ size/cellular phenotye Heart
Cardiomyocytes Coronary arteries Vascular
Smooth muscleEndothelium
Brain Liver
Zonation Adrenals Pancreas
Isletsbcells
KidneyNephron number
Lung Fat
Adipocyte numberAdipocyte size
MuscleType I and IIFibers
Bone
Vasculogenesis/Angiogenesis
PathologyCardiovascular disease Coronary artery disease Stroke
HypertensionDiabetes Type I/Type II diabetes Impaired glucose Tolerance Insulin resistanceDyslipidemiaObesity Fat distributionOsteoporosisHypercortisolismGH/IGF programmingTumors Childhood leukemia Breast cancerRespiratory DiseasePsychiatric disorders Schizophrenia
Maternal
Placental
Fetal
Neonatal
Postnatal
EpigeneticMechanisms
PROGRAMACIÓN PERINATAL Y ENF. CRÓNICAS
PERINATAL PROGRAMING AND DISEASES
Dietary and metabolic factors involved in histone acetylation and deacetylation
IsothiocyanatesDiallyl sulfideGenisteinAlcoholsButyrateNicotinamide(niacin)CaffeineResveratrolCalorie restrictionHigh-salt dietLipoic acid, vitamin E?
InflammationImmune activityInfections (virus…)HypoxiaOxidative stressUV lightSmokingPhysical exerciseGlucose
HDACs and SIRT inhibitors
DIETARY FACTORS
METABOLISM
DRUGS
BiotinAnacardicacidGarcinolCurcumin
NAD+ (for sirtuins)
HATs inhibitorsAcetyl-CoA analogues
DIETARY FACTORS
METABOLISM
DRUGS
aK
aK
aKaK
HISTONE ACETYLATIONIN
HIB
IT ION
ACTI
VATI
ON
IsothiocyanatesDiallyl sulfideGenisteinAlcoholsButyrateNicotinamide(niacin)CaffeineResveratrolCalorie restrictionHigh-salt dietLipoic acid, vitamin E?
InflammationImmune activityInfections (virus…)HypoxiaOxidative stressUV lightSmokingPhysical exerciseGlucose
HDACs and SIRT inhibitors
DIETARY FACTORS
METABOLISM
DRUGS
BiotinAnacardicacidGarcinolCurcumin
NAD+ (for sirtuins)
HATs inhibitorsAcetyl-CoA analogues
DIETARY FACTORS
METABOLISM
DRUGS
aK
aK
aKaK
HISTONE ACETYLATIONIN
HIB
IT ION
ACTI
VATI
ON
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
Nutriepigenetics
Insulin Resistance Syndrome; Syndrome X; Dysmetabolic Syndrome; Multiple Metabolic Syndrome
1923-1930: Kylin / Marañón describe the possible association of Hyperglycemia, hypertension and gout
RISK
Evolution of Metabolic Syndrome
Insulin Resistance Syndrome; Syndrome X; Dysmetabolic Syndrome; Multiple Metabolic Syndrome
1988: Reaven describes “Syndrome X” – hypertension, hyperglycemia, glucose intolerance, elevated triglycerides, and low HDL cholesterol
Evolution of Metabolic Syndrome
ReavenG. Diabetes. 1988;37:1565-1607.
InsulinResistance
InsulinInsulinResistanceResistance
GlucoseIntoleranceGlucoseGlucose
IntoleranceIntolerance HyperinsulinemiaHyperinsulinemiaHyperinsulinemia á TGáá TGTG â HDL-Câ HDL-C HypertensionHypertension
CORONARY HEART DISEASECORONARY HEART DISEASECORONARY HEART DISEASE
Evolution of Metabolic SyndromeBody SizeBody Size
BMIBMI Central AdiposityCentral Adiposity
Body SizeBody Size BMIBMI
Central AdiposityCentral Adiposity
GlucoseGlucoseMetabolismMetabolismGlucoseGlucose
MetabolismMetabolismUric AcidUric Acid
MetabolismMetabolismUric AcidUric Acid
MetabolismMetabolism DyslipidemiaDyslipidemiaDyslipidemiaDyslipidemia HemodynamicHemodynamic Novel RiskFactors
Novel RiskFactors
CORONARY HEART DISEASECORONARY HEART DISEASECORONARY HEART DISEASECORONARY HEART DISEASE
Insulin ResistanceInsulin ResistanceInsulin ResistanceInsulin Resistance
HyperinsulinemiaHyperinsulinemiaHyperinsulinemiaHyperinsulinemia++
l TGTGl PP lipemiaPP lipemial HDL-CHDL-Cl PHLAPHLAlSmall, dense LDLSmall, dense LDL
l± Glucose± Glucoseintoleranceintolerance
l Uric acidUric acidl Urinary uricUrinary uric
acid clearanceacid clearance
l SNS activitySNS activityl Na retentionNa retentionlHypertensionHypertension
l CRPCRPl PAI-1PAI-1l FibrinogenFibrinogen
Genetics and epigenetics do not explain everything.
Epigenetics & Developmental Origins of Health and Disease
Epigenetics literally means “in addition to changes in genetic sequence”.
Mothers that, during gestation and
lactation, suffered malnutrition, gave birth
children with more probability to suffer
obesity, cardiovascular events and type
2diabetes in adulthood.Roseboom TJ, et al, 2003
The Netherlands, winter 1944
Developmental Origins of Health and Disease (DOHAD)
Developmental origins of Health and Disease
Hales & Barker. 2001
Type 2 Diabetes Metabolic Syndrome
Developmental origins of Health and Disease
Hales & Barker. 2001
•BMI
•BMI
•WEIGHT
• WEIGHT• HEIGHT
• HEIGHT
THE THRIFTY PHENOTYPE
Hales & Barker. 2001
Developmental origins of Health and Disease
Perinatal Nutrient Restriction
Pancreas Liver Skeletal Muscle Adipocyte
beta cell massand secretory
capacity
Glucose uptake
Gluconeogenesis
Lipid oxidation
Insulin sensitivity
Insulin inhibition of lipolysis
Decreased pancreatic functional capacity glucose intolerance insulin
resistance
TYPE 2 DIABETES McMillen, et al. 2005
PerturbationNutrition Caloric restriction Glycemic index
aboriginal/cafeteria Protein restriction
Taurine, glycine Micronutrient restriction
Iron, CalciumEnvironment Ambient temperature
HyperthermiaCold
Hypoxia Exogenous agents
SmokingExperimental/therapeutic drugs
GlucocorticoidsEstrogensStreptozotocin
Utero-placenta circulation Embolization Uterine artery ligation Restriction of placental growthPre-implantationEnvironmental/conditions Local nutrient concentrations
SerumAmino acids Leucine McMillen, et al. 2005
Organ system Organ size/cellular phenotye Heart
Cardiomyocytes Coronary arteries Vascular
Smooth muscleEndothelium
Brain Liver
Zonation Adrenals Pancreas
Isletsbcells
KidneyNephron number
Lung Fat
Adipocyte numberAdipocyte size
MuscleType I and IIFibers
Bone
Vasculogenesis/Angiogenesis
PathologyCardiovascular disease Coronary artery disease Stroke
HypertensionDiabetes Type I/Type II diabetes Impaired glucose Tolerance Insulin resistanceDyslipidemiaObesity Fat distributionOsteoporosisHypercortisolismGH/IGF programmingTumors Childhood leukemia Breast cancerRespiratory DiseasePsychiatric disorders Schizophrenia
Maternal
Placental
Fetal
Neonatal
Postnatal
EpigeneticMechanisms
PROGRAMACIÓN PERINATAL Y ENF. CRÓNICAS
PERINATAL PROGRAMING AND DISEASES
PROGRAMACIÓN PERINATAL Y ENF. CRÓNICAS
PERINATAL PROGRAMING AND DISEASES
PROGRAMACIÓN PERINATAL Y ENF. CRÓNICAS
PERINATAL PROGRAMING AND DISEASESMaterial and/or fetal neuroendocrine adaptations
Decreased anabolic hormones (Insulin, IGFs, thyroidhormones, GH/PRL)Increased stress hormones (Glucocorticoids, catecholamines)
Programmed Set points in Physiological Systems
Cardiovascular, renal, metabolic, HPA, etc
Altered neuroendocrine regulation of energy balancehomoestoasis, growth etc
Insulin synthetic/secretory capacity
Hepatic glucose
production
Skeletal muscle mass
Cardiac and renal functional capacityAltered vascular reactivity
HPA activityAltered set points for GH/IGF, appetite regulatory axes
Increased insulin sensitivity-muscle, adipoctyeIncreased appetiteCatch up growth
Visceral obesity, increased circulating FFAsHepatic glucose intolerance
Fat accomodationInsulin resistance-hepatocyte, muscleDecreased insulin systhetic/secretory capacity
Type 2 Diabetes Obesity CV Disease
Decreased GFRIncreased intravascular volumeIncreased vascular reactivityHypertensionCardiac Hypertrophy
Substrate Restriction
Decreased O2,glucose, amino acids etcAmino acid balance: homocysteinte/glycine
Changes in Organ Development and Growth
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?
Human CpG-Island 15K ArraySubcutaneous biopsies
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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.
Nutrients
Systems
biology and
bioinformatics
Homeostasis
ProteinsPersonalized
NutritionProteomics
DNA
Genomics
Metabolomics
and functional
genomics
Proteomics
mRNA
Dietary bioactive compounds
Physiological Function
NUTRIOMICS
Nutriomics