Endowed Chair of “Food for Life”Organization for Interdisciplinary Research ProjectsThe University of Tokyo

1

Workshop Argentina-Japan“Bioscience and Biotechnology for the Promotion of

Agriculture and Food Production”-August 3rd to 7th 2009-

Hisanori Kato

2

Life Expectancy at Birth

(2008, by the Japanese Ministry of Health, Labor, and Welfare)

Female: 86.05 years (the longest in the world)

Male: 79.29 years (the 4th in the world)

Japan 日本

3

JapaneseBlessed with a Variety of Healthy Traditional Foods

Abundant Experience on Functional Food Factors

Accumulated research data and experience will help health promotion of other countries!!

Nature, 364, 180 (1993)

Functional Food in Japan – Food for Specified Health Uses (FOSHU)

Omics Analyses in Food and Nutrition Science

700 1260 1820 2380 2940 3500Mass (m/z)

0

2.4E

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

Spec #1[BP = 829.7, 24053]829.7285

881.7770

1378.3245 2191.8588

2222.00811799.7345

785.68651014.01261060.6566

833.5817 2279.1555

1927.83662145.81691088.0897 1696.5836871.5511 2198.89811051.0375 2483.3095 2869.50201679.55641881.8104709.6359 1412.3932 2175.0235

Transcriptomics

Proteomics

Metabolomics

12C PF0

1

2

3

4

5

6

Effects of Dietary Protein Deprivation and Amino Acid Deprivation on mRNA Levels of IGFBP-1 Gene

（12 % Casein Diet）（Protein-free Diet）

0

1

2

3

4

5

Rel

ativ

e m

RN

A L

evel

s

AA+ AA-CompleteMedium

Amino Acid-Deprived (12 h)

Rat Liver Hepatoma Cells

Rel

ativ

e m

RN

A L

evel

s

GROWTH RETARDATION

6

x1/2x2

x5x10

x1/5x1/10

Transcriptome Analysis of the Effect of Food

DNA Microarray Analysis

RNA Extraction Exhaustive Analysis of Gene Expression

Treated

Control

Control

Animals

Cells

・Intake of Nutrients

・Excessive Intake・Functional Food Factors

Food

Comprehensive Evaluation of the Function and Safety

The World of OMICS

8

Omics Discipline

Genomics

Transcriptomics

Proteomics

Metabolomics

·····

Nutritional OmicsNutrigenomics(narrow sense)Nutritranscriptomics

Nutriproteomics

Nutrimetabolomics

·····

Target

Genes

Transcripts

Proteins

Metabolites

····· Nutrigenomics

Nutriomics

Other OMICSEpigenomics (Chromatin Modification)

Interactomics (Protein-Protein Interaction)

Toponomics (Cellular Localization)(Localizomics)

Fluxomics (Cellular Flux of Metabolites)

Lipidomics (Lipids)

Glycomics (Sugars)

··· CAUTION: Economics and Comics Do NOT

Belong to the Omics Family

10

Use of DNA Microarray 1.Nutritional Intervention

Understanding Whole Response

2.Known Function of a Food Factor

UnderstandingMechanisms

3. A Food Material or Component of Interest

Unknown Functionality

DNA Microarray and Other Omics Analyses

IleLeuLys

Met+Cys

Phe+Tyr

ThrTrpVal

100 150%0 50

Essential Amino Acid Pattern of Gluten

86420-2-4120

140

160

180

200

220

240

PF

12C12G

c

Rat Body Weight

12% Casein（12C)

12% Gluten（12G)

Protein Free（PF)

CaseinGlutenCornstarchMineral MixVitamin MixCelluroseCholine chloride

140 (+Met 2)0

6565040101002

01506485040101002

00

7985040101002

11

12% Casein

Protein Free

12% Gluten

Object

MethodMale Wistar rats ( 5 wk old)

RNA extraction(Liver, Muscle,,,)

- 6 0 7 (Day)Feeding (10:00~18:00)

To obtain a comprehensive view of the changes in gene expression in response to the alteration of protein nutrition

12% Casein

Rat U34A GenomeArray (Affymetrix)

12

Ingestion of Gluten UpIngestion of Gluten Up--regulates Genes for regulates Genes for Both Synthesis and Catabolism of CholesterolBoth Synthesis and Catabolism of Cholesterol

2 Acetyl-CoA

Acetoacetyl-CoA

HMG-CoA

Mevalonate

Squalene

Squalene epoxide

HMG-CoA synthase(5.4)

HMG-CoA reductase(4.4)

Squalene synthetase(4.4)

Squalene eposidase(5.8)

Lanosterol 14-demethylase

(3.2)

Dehydrocholesterolreductase

(3.2)

Cholesterol7-α hydroxylase

(2.5)

Dehydrocholesterol

Lanosterol

14-demethyl lanosterol

Cholesterol

7α-Hydroxycholesterol

-4 -3 -2 0 1 7 812% Casein Diet 12C

12% Gluten + Lys +Thr (12GLT) 12GLT

12% Gluten (12G) 12GPellet Diet

12% Casein

Diet

12% Casein

Diet

Protein- Free Diet (PF) PF

<day>

Day

10:00～18:00

Excision of Skin

DNA Microarray(n=4)

1.5 h24 h

Protein Nutrition and Skin Gene ExpressionProtein Nutrition and Skin Gene Expression

Body Weight

15

Professional Program for Agricultural Bioinformatics (Tokyo Univ.)

DFW

RMA qFARMS

MASBody Weight

Choice of Normalization MethodChoice of Normalization Method

Other Ongoing Projects (transcriptomics)

Anti-obesity Effect of Dietary Amino Acid Supplementation

Deficiency and Excess of Amino AcidsSpecific Proteins and Pepetides

Protein Intake Levels at Various Life Stage

Effect of Dietary Factor on Longevity, Diabetes, Hypertension

Effect of Biolgical Rhythm and Exercise on Gene Expression

Insulin-mimetic Activity of Soy IsoflavoneHypocholesteroleamic Effect of Sulfur-Containing Amino Acid

【Cells】 L6 myotubes (from rattus)

【Methods】Differentiation for nine to eleven days

Precultivation for 2 hours

Cultivation for 4 hours

Stimulation by 50 μM genisteinCultivation in DME medium

Stimulation of Glucose Uptake by a Soy Isoflavone, Genistein, in L6 Myotubes

DNA Microarray and Pathyway Analysis

Insulin

Activation of some protein kinases including IRS-1

GLUT4

VAMP2Translocation

Glucose

The activation of AMPK

GLUT4

VAMP7 Translocation

Glucose

Insulin receptor

Exercise

The rise of the ratio of AMP to ATP

Genistein Seems to Activate Two Pathways Leading to GLUT4 Translocation

20

Functional CategoryPathway Analysis

Comparison with Other Data, Database

Normalization

A Typical Flow of DNA Microarray Data Analysis for Food and Nutrition Science

Cluster Analysis, Principal Component Analysis, etc.（Tendency, Extraction of Marker Genes）

Raw Data

Each Responder Genes（Rank Analysis, Statistical, Fold change…)

Filtering

AnnotationInterpretationDiscussion

21