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Functional food genomics in Japan – State of the art

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Page 1: Functional food genomics in Japan – State of the art

Trends in Food Science & Technology 22 (2011) 641e645

* Corresponding author.

0924-2244/$ - see front matter � 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.tifs.2011.06.001

Functional food

genomics in

Japan e State

of the art

Yuji Nakaia,*, Akio Nakamurab

and Keiko Abea

aILSI Japan-Endowed Chair of Functional Food Science

and Nutrigenomics, Graduate School of Agricultural

and Life Sciences, The University of Tokyo, 1-1-1Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

(Tel.: D81 3 5841 1128; fax: D81 3 5841 1127;

e-mail: [email protected])bTechnical Research Institute R&D Center,

T. Hasegawa Co., Ltd., 29-7 Kariyado, Nakahara-ku,

Kawasaki-shi 211-0022, Japan

Nutrigenomics uses transcriptomics to comprehensively investi-

gate the effects of various nutrients on the body. In Japan, nutrige-

nomics has primarily been applied to functional food science.

However, human nutrigenomics data are quite complex, be-

cause they are inherently noisy, reflecting subtle changes in re-

sponse to nutrients. To detect minute changes in response to

such a complex input of genetic information, elaborate and effi-

cient experimental methods and statistics are necessary. This re-

view discusses the applicability of genomics for evaluating the

otherwise undeterminable effects of functional factors in the par-

ticular case of (R)-(-)-linalool as a useful stress suppressor.

IntroductionTwo decades of years ago, the science of functional foods

had humble beginnings in Japan as a national project entitled‘Systematic Analysis and Development of Food Functions’,sponsored by theMinistry of Education, Science and Culture(Arai et al., 2001). Research in functional foods traceda unique path of development, as Nature (Swinbanks &O’Brien, 1993) reported with the headline ‘Japan explores

the boundary between food and medicine’. In 2002, nutrige-nomics was launched as a comprehensive method to investi-gate the effect of nutrients on the body. In Japan, thismethodology has been applied mostly to functional food sci-ence. Meanwhile, the ILSI Japan-endowed chair ‘FunctionalFood Genomics’ was founded in the Graduate School of Ag-ricultural and Life Sciences at The University of Tokyo, in2003. The purpose of this endowed chair is to obtain scien-tific evidence on the effects of functional foods by analyzingthe gene expressions evoked in the body, and 32 representa-tive food companies participated in the first stage(2003e2008). In collaboration with university staff, thesecompanies have been employing transcriptomics to evaluatethe physiological functionalities of food components, in-cluding polyphenols, carotenoids, tocopherol, lignan, aminoacids, dietary fibers, some fermentation products, and essen-tial minerals. The target tissues include the liver, intestine,adipose tissues, muscle, animal immune system, and humancell cultures.

The second stage (2008e2013) is in progress, and its ac-tivities are aimed at scientific, social, and administrative tar-gets. Scientifically, the chair seeks to (1) find key moleculesthat may act as triggers for physiological functionalities; (2)elucidate their modes of interaction with the food compo-nents of interest; (3) define the strength and continuity offunctional food components in the body, as well as the mech-anisms evoked in target tissues of normal, impaired, and KOmice; (4) construct useful assay systems for evaluating thetotal effect of each multifunctional food component; and(5) explore the area of genomics-based food safety assess-ment, with special emphasis on identifying risk factors fordeficient or excessive food intakes.

Characteristics of nutrigenomics studiesNutrigenomics aims to detect minute changes in the hu-

man body in response to daily-ingested food components.These changes are assessed by closelymonitoring alterationsin the gene expression profiles. In a nutrigenomics study, theexperimental system must be optimized to detect smallchanges. Using the restricted-feeding method for a fastingexperiment, we found that genes related to the ubiquitin-proteasome system were upregulated in the brown adiposetissue of rats fasted for 24 h (Nakai et al., 2008). When therat was allowed food for 6 h after the 18-h fast, genes for im-munoproteasome in the rat liver were upregulated (Ushiamaet al., 2010).

Page 2: Functional food genomics in Japan – State of the art

642 Y. Nakai et al. / Trends in Food Science & Technology 22 (2011) 641e645

The above findings required both optimized experimen-tal design and optimized data analysis methods. For exam-ple, there are many methods for Affymetrix-type DNAmicroarray data quantification and these can lead to differ-ent results (Garosi et al., 2005; Kadota, Ye, Nakai, Terada,& Shimizu, 2006; Kadota, Nakai, & Shimizu, 2008). There-fore, it is important to carefully select the most appropriateDNA microarray analysis method for a given study. Tomaximize the advantages of our comprehensive analysis,we applied recent guidelines for combined data quantifica-tion and two-group comparison methods, which were de-veloped to improve the detection of differentiallyexpressed genes in an Affymetrix-type DNA microarray(Kadota, Nakai, & Shimizu, 2009).

Nutrigenomics and stress study‘Stress’ can be defined as a state in which the body re-

ceives some mental or physical load. In general, our bodycan keep homeostasis against external stress. However, ifthe stress continues for a long time and/or is too strong,

neutrophils lymphocytes

Control

Restraint

(R

)-(-)-Linalool+R

estraint

0

20

40

60

80

Rel

ativ

e po

pula

tion

(%)

Control

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)-(-)-Linalool+R

estraintA

3

2

10.8

0.6C

Nor

mal

ized

inte

nsity

(log

scal

e)

Nor

mal

ized

inte

nsity

(log

scal

e)

1.5

10.8

0.6

RestraControl

0.4

0.3

B

C

Fig. 1. Effect of (R)-(-)-linalool inhalation on (A) leukocyte populations, (B) rewhole blood. (A) Data are expressed by means � SEM (n ¼ 4). *p < 0.05 cprofiles of 73 stress-upregulated and 42 stress-downregulated genes in wholintensity against the control group for (R)-(-)-linalool-up (red) or -down (bluegroup (n ¼ 4), the stressed group in which rats were exposed to restraint streduring the stress ((R)-(-)-linalool þ restraint). (For interpretation of the refere

version of this

organisms are unable tomaintain homeostasis. This responseis known as stress disease, and the etiology of stress diseaseis called a stressor. Various mental and/or physical stressorsexist, and major stress diseases include psychosomatic disor-der, depression, and neurosis. Metabolic syndrome is a stressdisease in a broad sense, particularly when regarding nutri-tional deflection as a stressor. However, because the develop-mental process of stress disease involves the accumulation ofminute changes and because the causes are very compli-cated, the stress research field remains enigmatic.

Nutrigenomic and stress studies are expected to detectminute outputs from a complicated input. We successfullydetected the effect of various stresses on gene expressionprofiles by applying a nutrigenomics method to stress studies(Motoyama et al., 2009; Nakamura et al., 2010; Nakamura,Fujiwara, Matsumoto, & Abe, 2009). Motoyama et al.(2009) showed that the gene expression profile in the liverof mice stressed by a 30-day isolation changed to allow fataccumulation. Male BALB/c mice were housed at fivemice per cage for 10 days and then were exposed to isolation

(R)-(-)-Linalool+RestraintRestraintontrol

(R)-(-)-Linalool+Restraintint

straint-upregulated and (C) restraint-downregulated gene expression inompared to the control group (Student’s t test). (B) and (C) Expressione blood, respectively. Each line plot represents changes in normalized)-regulated gene. The intensities represent mean values in the controlss (n ¼ 4), and the group (n ¼ 4) in which (R)-(-)-linalool was inhalednces to colour in this figure legend, the reader is referred to the webarticle).

Page 3: Functional food genomics in Japan – State of the art

643Y. Nakai et al. / Trends in Food Science & Technology 22 (2011) 641e645

stress (one mouse per cage) for 30 days. The control micewere kept five per cage for 30 days. The isolation stressgroup did not show a significant change in the white adiposetissue weight, while the gene expression profiles were quitedifferent between the control and the stress groups. These re-sults indicate that gene expression changes can be detectedbefore phenotypic changes are observed. It was not untilDNA microarray analyses were performed that the changesin the gene expression profile were found. This is the firstcase in which the nutrigenomics methodology has been ap-plied to a stress study successfully.

Effect of an odorant on the gene expression profiles inrat blood and hypothalamus under restraint stress

A large number of studies have been directed at certainkinds of psycho-physiological effects that are elicited byodorants (Buchbauer, Jirovetz, Jager, Dietrich, & Plank,1991; Buchbauer, Jirovetz, Jager, Plank, & Dietrich,1993; Heuberger, Hongratanaworakit, Bohm, Weber, &Buchbauer, 2001; Nagai, Wada, Usui, Tanaka, & Hasebe,2000; Tsuchiya, Tanida, Uenoyama, & Nakayama, 1992).However, research assessing these effects in vivo is stillquite limited. Applying the nutrigenomics experimental de-sign and data analysis method, Nakamura et al. (2009;2010) elucidated a new functional aspect of odorant.

These authors first examined the effect of the odorant(R)-(-)-linalool on the gene expression profile in rat blood un-der the condition of restraint stress (Nakamura et al., 2009).

Ntrk2

Grin3A

Pgrmc1

Map1B

Ank3

Rnf6

Dpysl3

Tgfb2

Prg1

Mtap2

Nrxn3

Sox11

Slitrk1

Tiam1

Bbs4

B3Gnt1

Foxg1

neurite

development

Nlgn1 Dlgh4

neuron

development

neuron differentiation

Btg2 Nr4A2

Cebpb

Ntrk2

Grin

Pgrm

Map

Ank3

Dpys

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BA

Fig. 2. Effect of (R)-(-)-linalool inhalation on neuron-differentiation-related gassociation of neuron-differentiation-related genes with multiple Gene Ontotistically identified by rank products between stressed group (restraint) and (Rmap of expression levels of the 22 genes. The colors indicate higher (red) andin which rats were exposed to neither stress nor odor, while the (R)-(-)-linaloo

(R)-(-)-linalool inhalation under 2 h restraint, but not regula

Linalool, 3,7-dimethyl-1,6-octadien-3-ol, is found in numer-ous foods and flowers (Bazemore, Rouseff, & Naim, 2003;Hunter & Moshonas, 1966; Ito, Sugimoto, Kakuda, &Kubota, 2002; Kim & Lee, 2002), and its characteristic floralodor is important for the formulation of a variety of fruit-likeflavors and fragrances. The psycho-physiological effects eli-cited by this odorant have also been investigated(Elisabetsky, Marschner, & Souza, 1995; H€oferl, Krist, &Buchbauer, 2006; Kuroda et al., 2005; Re et al., 2000; Shenet al., 2005; Tanida, Niijima, Shen, Nakamura, & Nagai,2006).

In recent years, particular interest has been paid to theeffects of inhaled odorants on health maintenance and pro-motion. However, the molecular mechanisms that underliethe effects elicited by odorants such as linalool remain un-clear. To investigate the effects of odorants quantitatively,the gene expression profiles of rat blood cells were deter-mined under a restraint-stressed condition, with or withoutinhaled (R)-(-)-linalool. Male Wistar rats were divided intofour groups (control, stressed, stressed þ odorant-inhaled,and odorant-inhaled groups). Rats in the stressed groupwere placed in a restraining plastic tube for 2 h. Twenty mi-croliters of (R)-(-)-linalool (92% ee) were evaporated andallowed to spread throughout a 40-L box containing re-strained or non-restrained rats.

Two major findings were obtained by profiling the wholeblood cells of the rats. First, significant changes in neutrophilsand lymphocytes caused by the restraint were repressed by

3A

c1

1B

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2

2

3

1

1

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1

4

2

b

1

1

(R

)-(-)-Linalool

Restraint

Control

(R

)-(-)-Linalool+R

estraint

ene expressions in hypothalamus. (A) Euler diagram representing thelogy (GO) terms. The 22 genes represented as gene symbols were sta-)-(-)-linalool þ restraint group exposed to both stress and odor. (B) Heatlower (green) levels of gene expression. The control represents a groupl group has rats exposed to odor only. These genes are upregulated byted by (R)-(-)-linalool under the non-stress condition.

Page 4: Functional food genomics in Japan – State of the art

644 Y. Nakai et al. / Trends in Food Science & Technology 22 (2011) 641e645

exposure to the odorant (Fig. 1A). Second, inhalation causedsignificant changes in the restraint stress-induced expressionlevel variations of 115 genes (Fig. 1B and C; 70 out of 72stress-upregulated genes were downreglated and 39 out of43 stress-downregulated genes were upregulated, respec-tively). These findings show that the (R)-(-)-linalool inhalationrepressed stress-induced effects on the blood cells and geneexpression profiles. The results also suggest the possibilitythat odorant-induced effects can be quantitatively evaluatedby analyzing the blood cell and gene expression profiles.

Next, to explain one of the molecular mechanisms of stressrelaxation by (R)-(-)-linalool inhalation, gene expression pro-filing was performed with a hypothalamus sample as a stressresponse center (Nakamura et al., 2010). Inhalation of thisaroma under a restraint stress-added condition upregulatedseveral neuron differentiation-related genes towards activat-ing neuronal maturation processes (Fig. 2A and B). Inhalationalso upregulated restraint stress-inducible heat shock protein-related genes associated with the suppression of stress-causedapoptosis. Finally, the (R)-(-)-linalool inhalation returned thestress-elevated levels of neutrophils and lymphocytes backto near-normal levels, and reduced the activity of more than100 stress-responsive genes. These findings elucidate thephysiological effects of the aroma through an in-depth analy-sis of gene expression levels. The results may also aid in thedevelopment of a newmethod for evaluating the in vivo effectscaused by odorants to cope with stresses.

ConclusionA method developed to detect minute gene expression

profile changes in nutrigenomic research can be appliedto other fields, such as stress study. To maximize the advan-tages of this comprehensive analysis, it is important to usethis toolbox in conjunction with sophisticated experimentaldesigns and appropriately selected data analysis methods.

AcknowledgementsWe thank Drs. K. Shimoi and K. Motoyama for collabora-

tion in the mouse isolation stress study.

References

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