EPIGENÉTICA DE LA NUTRICIÓN Y LA OBESIDAD” · “ epigenÉtica de la nutriciÓn y la...

“ 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?

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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

http://www.youtube.com/watch?v=eYrQ0EhVCYA&feature=related

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


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)


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)


2

4

6**

HOM

A


100

200

300

400

500**

Fina

l Bo

dy W

eigh

t (g

)


50

100

150

200

250

300

350 **

Food

Int

ake (

Kca

l/day

)


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


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.



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



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.



b



b b


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:




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


Retroperitoneal adipose tissue

gDNA Extraction

Sodium Bisulfite treatment(converts cytosine to uracyl)

SequencingMethylation-amplification PCR

U

Adipocyte Isolation(colagenase digestion)

Caf C

EXPERIMENTAL DESIGN


0.5

1.0

1.5

2.0

2.5 *

Lept

in m

RNA

expr

essi

on(R

etro

perit

onea

l W

AT)


2.5

5.0

7.5

10.0 **Le

ptin

(ng

/ml)


5

10

15

20

25 **

Retro

perit

onea

lW

hite

adip

ose t

issu

e (g)


2

4

6**

HOM

A


100

200

300

400

500**

Fina

l Bo

dy W

eigh

t (g

)


50

100

150

200

250

300

350 **

Food

Int

ake (

Kca

l/day

)


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


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.



Wonders:




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


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.


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)


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.



602 CpG hypermethylation

432 CpG hypomethylation


(>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)



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


* *

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




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




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


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


DNA METHYLATION

I. INTRODUCCIÓN

↑ ↓

DNA METHYLATION INHIBITS GENE EXPRESSION

↑ ↓

↓ ↑


I. INTRODUCCIÓN

↑ ↓



Ng S-F et al. Nature 2010; 467: 963-6.



b


a, Blood glucose during glucose tolerance test b, Plasma insulin during glucose tolerance test


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)

http://www.ncbi.nlm.nih.gov/pubmed/18667010?ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum




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


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



Sequencing by synthesis +DNA polymerase incorporates dNTPs +releases pyrophosphate (PPi) ATP



Chain-termination methods +thin-layer electrophoresis +fluorescent detection of dNTPs


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


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.


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?

http://www.ncbi.nlm.nih.gov/entrez/utils/fref.fcgi?PrId=3094&itool=AbstractPlus-def&uid=11875497&db=pubmed&url=http://dx.doi.org/10.1038/nm0302-262


Many genes related to obesity are regulated by DNA methylation


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


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.)


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


Taurine, glycine Micronutrient restriction

Iron, CalciumEnvironment Ambient temperature

HyperthermiaCold


SmokingExperimental/therapeutic drugs

GlucocorticoidsEstrogensStreptozotocin



Organ system Organ size/cellular phenotye Heart



Brain Liver


Isletsbcells


Lung Fat



Bone


PathologyCardiovascular disease Coronary artery disease Stroke


Maternal

Placental

Fetal

Neonatal

Postnatal



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

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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 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

Hales & Barker. 2001

Type 2 Diabetes Metabolic Syndrome



•BMI

•BMI

•WEIGHT

• WEIGHT• HEIGHT

• HEIGHT

THE THRIFTY PHENOTYPE



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


Taurine, glycine Micronutrient restriction

Iron, CalciumEnvironment Ambient temperature

HyperthermiaCold


SmokingExperimental/therapeutic drugs

GlucocorticoidsEstrogensStreptozotocin



Organ system Organ size/cellular phenotye Heart



Brain Liver


Isletsbcells


Lung Fat



Bone


PathologyCardiovascular disease Coronary artery disease Stroke


Maternal

Placental

Fetal

Neonatal

Postnatal





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?

http://www.ncbi.nlm.nih.gov/entrez/utils/fref.fcgi?PrId=3094&itool=AbstractPlus-def&uid=11875497&db=pubmed&url=http://dx.doi.org/10.1038/nm0302-262

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

EPIGENÉTICA DE LA NUTRICIÓN Y LA OBESIDAD” · “ epigenÉtica de la nutriciÓn y la...

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