NUTRIGENOMICS; AN EMERGING TOOL IN NUTRITION

SPEAKER: Siddhartha S. Pathak

Introduction

• Nutrition ; induce metabolic changes in muscle,being reflected in tissue remodeling, increasedprotein turnover, and muscle atrophy.

• From a production point of view, these changesinfluence efficiency of production and meat quality.

• Tremendous economic importance.

Contd…

• Relationship of nutrition with health and productionis long known.

• But how the components exert their effect-graduallyknown.

• Expression of genes; Coding of certain protein in thegenome.

Contd…

• Constant switching on or off of gene; Physiologicalprocesses and pathological events.

• Bioactive feed components; key environmentalfactors regulating gene expression

(Mead, 2007)

• Nutrient- gene interaction wide spread and wellknown.

Contd…

• Advent of molecular tools and analysis systemsrevealed, mechanism by which individual sensesnutrient status and their metabolism.

• With these knowledge a new area of study called

“NUTRIGENOMICS” has emerged.

(Roche et al 2006)

We need to change the way we evaluate

We need to make a Change what we think of feed

A time for New Nutritional Strategies

HISTORY

• 400BC : Hippocrates speculated the hypothesis thatthe warm body temperature was innate

• 19th century : Liebig identified carbohydrates,proteins, lipids, and other macronutrients thatreleased heat.

• 18th and 19th centuries : “Chemical and AnalyticalEra of Nutrition”

Contd…• 20th century :“Biological Era” studies on metabolism

and chemistry of the food.

• 21st century : “Post- Genomic Era” characterized bythe integration of three fields: biological, social, andenvironmental, scientific discoveries on nutritionalmetabolism are included.

• After the Human Genome Project: evolution ofbioinformatics provided advances in “omics” scienceresearch.

• Leading to the development of “NUTRIGENOMICS”.

What is a gene??...

• Variations in the DNA; results inthe changes to the structure andfunction of the protein.

• Functional and physical unit ofheredity passed from parent tooffspring.

• Genes are segments of DNA thatcontain the information for makinga specific protein.

What is Nutrigenomics??

• “Nutrigenomics” refers to study of the effects ofnutrients/bioactive food on gene expression of anindividual.

• The study of how genes and gene products interactwith dietary chemicals to alter phenotype and,conversely, how genes and their products metabolizenutrients is called nutritional genomics or“nutrigenomics” (Kaput et al., 2005).

Contd…

• A new science seeking to understand the influence ofdietary components on the Genome, Transcriptome,Proteome, and Metabolome (Ronteltap et al., 2009).

• Simply, “It is a science which study the effect ofnutrients on the gene expression.

OMICS SCIENCES

Why NUTRIGENOMICS ????

• Focus of most nutritional research has been onparameters related to

bird growth performance

reproduction

intestinal health or

the utilization of specific nutrients as a mean toreduce excess waste.

Contd…

“N”; individual animals/birds statistical differences

Closely interrelated measured parameters

Considerable time

DIFFICULTIES

Replication of treatments ; statistical differences between

individual treatments

Contd…

For example, damage in the intestine as a result ofbacterial overgrowth will result in reduced nutrientabsorption and, subsequently, reduced animalperformance.

The question remains if the excess availability ofnutrients leads to more bacterial growth or if bacterialgrowth leads to a lower availability of nutrients

Contd…

Relationship “specific nutritional interventionand the impact on health and performance” lies in adeeper understanding of the impact of thesenutrients on the expression of specific genes orspecific metabolic pathways.

Development of molecular tools; enableresearchers to study the effects of specific nutrientson the whole genome

(Muller and Kersten, 2003).

• Selenium deficiency shown to alter protein synthesisat transcriptional level (Rao et.al., 2001).

• Results; adverse effect like enhancement of stressthrough up-regulation of specific gene expressionand signaling pathway.

• Genes responsible for detoxification mechanism andprotection from oxidative damage were hampered,

• Alteration of phenotypic expression

Contd…

• Hence, it is apparent that possibly “nutrigenomics”identify the specific markers to manipulate geneexpression through use of nutrients.

• Path breaking tool through identification of pathwaysresponsible for dietary induced diseases.

NUTRIGENOMICS: HOW DOES IT WORK???

Fig. Central Dogma (Francis Crick, 1956)

Contd…• These processes are controlled by the basic genetic

make-up, external factors such as

Disease

Environmental toxins or specific nutrients

• Rather than measuring the effect of a specificnutrient on animal performance or the physiologicalresponse at the end of the experiment, researcherscan extract “specific messenger RNA (mRNA)” fromtissue at any stage.

Contd…

• Amount of mRNA present relates to the relativeamount of copies of known genes, measured byusing gene chips.

• Using contrasting colour labels, it can be determinedif a gene is “up-regulated”, “down-regulated” or“unaffected” as a result of a specific dietarymanipulation.

NUTRIENT REGULATION OF GENE EXPRESSION

• Feed had basically one job: get metabolized toprovide energy for the cell.

• Indeed this happens to most dietary chemicals- butnot all.

• Some don’t get metabolized at all.

Contd…moment they are ingested

they are peel off

become ligands-molecules

binds to protein

Involved in turning on certain genes to one degree or another.

Contd…

• Nutritional regulation of gene expression in eukaryoticcells; considerably slower due to the

(Sanderson, 1998).

Complexity of mechanisms controlling geneexpression epithelium (IE) cells respond

Difficulty in identifying specific metabolitesto which the intestinal epithelium (IE) cellsrespond

• Exogenous nutrients can affect gene expression directly(A) or indirectly (B and C in Figure).

EFFECT OF CARBOHYDRATE ON GENE EXPRESSION

• In the liver, glucose, in the presence of insulin, induces expression of genes encoding glucose transporters and glycolytic and lipogenic enzymes, eg. acetyl-CoA carboxylase (ACC)

• Although insulin and glucagon were long known as critical in regulating gene expression, it is only recently that Glucose also have been shown to play a key role in transcriptional regulation.

REGULATION OF GENE EXPRESSION BY DIETARY FAT• Plays role as an energy source and its effects on

membrane lipid composition, dietary fat hasprofound effects on gene expression.

• The effects of dietary fat on gene expression reflectan adaptive response to changes in the quantity andtype of fat ingested.

• Specific fatty acid–regulated transcription factorshave been identified in bacteria, amphibians andmammals

Contd…• PPARs (Peroxisome proliferator-activated receptors )

• Regulated either by direct binding of (oxidised) fattyacids, fatty acyl–coenzyme A or oxidised fatty acid(eicosanoid) regulation of cell surface receptors andthat of intracellular calcium levels as well asactivation of signaling cascades (Jump, 1999).

• At the cellular level, the physiological response tofatty acids will depend on the quantity, chemistryand duration of the fat ingested

Contd..“Short-term (2-day) feed restriction” no change either in the number or the size of fat cells in

white adipose tissues

Declined mRNA expression of PPARγ2, glucocorticoidreceptors (GR)

“Long-term (14-day) feed restriction”

an increased number of smaller adipocytes

Recovered expression of PPARγ2 and glucocorticoid.

EFFECT OF PROTEIN ON GENE EXPRESSION

• Normal insulin secretion is influenced by level ofProtein Kinase C (PKC), K+ channel protein, calcium ion(Ca 2+).

• Increased ATP to ADP ratio achieved through glucosemetabolism, close the K+ ATP channel, which leads todepolarization of -cells.

• Depolarized -cells opens the voltage dependent Ca 2+channels which results in influx of calcium leads toexocytosis of insulin granules.

Contd…

• Protein-rich diets cause a shortage of mRNAnecessary for expression of the fatty acid synthasegene in the adipocytes, resulting in the moderationof total body fat.

EFFECT OF MINERALS ON GENE EXPRESSION

• As similar to other nutrients, mostly minerals areinvolved in several gene expressions

• “Bivalent metals”; strong influence gene expression.

• Both parenteral and oral zinc or cadmium applicationenhance the transcription rate of the metallothionein(MT) gene in intestinal tissue.

(Ouellette et al., 1982)

Cadmium

• Prolongsthe half-life of MT mRNA in hepatocytes.

• This effect on the half-life prolongation of MT mRNAtissue specific.

• Influence of cadmium is stronger than that of zinc,and the intensity of effect in spermatocytes andspermatids is higher than in hepatocytes andfibroblasts (De et al., 1991)

Zinc

• Improvement of zinc status results in a significant reduction of COX mRNA abundance.

(Fong et al. (2005)

• Zinc oxide supplementation: increased insulin-like growth hormone I (IGF-I) and IGF-I receptor gene expression in the small intestine of weanling piglets

(Li et al., 2006)

EFFECT OF VITAMINS ON GENE EXPRESSION

• Vitamins are micronutrients needed in very smallquantity and are involved in gene expression.

• Biotin is involved in various essential proteins(enzymes) synthesis at gene level.

• Vitamin C is involved in hepatic gene expression

Vitamin A

• Vitamin A exerts its regulatory function in the form ofretinol and retinoic acid.

• The most important target tissues are in the adrenalglands, testes, cerebellum, kidneys, prostate, cerebralcortex, skin and the viscera.

• After retinoic acid binds to its receptor, it will stimulatethe transcription and translation of vitamin A-responsive genes, including some involved in celldifferentiation (growth hormone, glycerolphosphatedehydrogenase and leptin production among others.

Contd…

• Deficient vitamin A status was found to negativelyinfluence hepatic PEPCK gene expression in mice. Bythe oral application of retinoic acid that expressioncould be restored

(Scribner et al., 2005)

BIOACTIVE FEED COMPONENTS

RESEARCHERS EXPERIMENT

Averous et al.

2003

Amino acids by themselves or along with hormones can

play an important role in the control of gene expression

Oullette et al.

1982

Bivalent metal ions like zinc or cadmium enhances the

transcription rate of the metallothionein gene in intestinal

tissue

Bremner and

Beattie 1990

Iron influences transferrin and ferritin concentrations by

exerting an effect on mRNA stability and the translation

rate.

Yuchi et al 1996

Hyperammonaemia due to significant depression of

ornithine transcarboxylase gene expression is seen in biotin

deficient individuals

FUNCTIONAL GENOMICS

• Measures the level of expression of all or a selectedsubset of genes.

• The most powerful tool available is DNA arraytechnology.

• Using one array the expression level of more than80,000 genes can be measured in parallel, and tensof samples can be screened per day.

Contd…

• In addition, high per-analysis cost seriously reducesthe number of measurements performed per study.

• Furthermore, different platforms are used, e.g.cDNA, oligonucleotide array and printed microarrays,on chip synthesis.

PROTOCOLS INVOLVED IN NUTRIGENOMICS

Transcriptomics

Proteomics

Metabolomics

TRANSCRIPTOMICS

• “Transcriptome” Complete set of RNA that can beproduced from the genome.

• Study of the transcriptome, i.e. gene expression atthe level of the mRNA

• Several methods to contour gene expression

RT-PCR

cDNA

Microarray

• Technology (also called DNA chip technology) iscurrently the most powerful.

• Tool in transcriptomics, which enables themeasurement in parallel and in tens of samples, ofthe expression of up to 50,000 transcripts.

• Problems such

Standardization of the data

Sharing of results

DNA ARRAY

DNA Microarray technology

GENE EXPRESSION PROFILING

PROTEOMICS

• Proteins are the ‘‘molecular robots’’ that do all thework. (Kussmann et al 2009)

• Proteomics ; studies related to

Structure of proteins,

Expression levels,

Biochemical activity

• Identify and quantifies bioactive proteins andpeptides and addresses questions of nutritionalbioefficacy (Kussmann et al 2006)

TECHNIQUES

• Western blot

• Immune histochemical staining

• Enzyme linked immunosorbent assay (ELISA)

• Mass spectrometry.

APPLICATION OF PROTEOMICS IN ANIMAL AND FEED SCIENCES

• Assessment of the influence of dietary componentson the proteomes of selected organs, for example,the liver.

• “Proteome” represents the protein equivalent of thegenome, which is determined by the sequence, thetype and number of its nucleotides.

• Evaluate the effect of dietary methionine on breast-meat accretion and protein expression in skeletalmuscle of broiler chickens

(Corzo et al., 2006)

• “Tandem mass spectrometer” a total of 190individual proteins were identified from Pectoralimajor muscle tissue;

• Three of them were recognized which differeddistinctly between the treatment proteome;Potential biomarkers regulated by a methioninedeficiency in broiler chickens.

METABOLOMICS

• Study of chemical processes involving in the metabolites

• Examine the whole metabolism

• Reflects the behavior of different patterns of genes ,

• Investigates the metabolic regulation, in response to specific environmental changes.

TECHNIQUES

• Nuclear Magnetic Resonance (NMR) spectroscopy,

• High performance liquid chromatography (HPLC)

• Gas chromatography-mass spectrometry (GC-MS).

• Main characteristics of these new technologies wereminiaturization, automation, high throughput andcomputerization

APPLICATIONS

1. To develop feed matching to its genotype

2. To select nutrients fine-tuned with genes of bird

3. To understand role of nutritional management inperformance (production/disease) of bird

4. Immune system

5. Diseases

TO DEVELOP FEED MATCHING TO ITS GENOTYPE

• “Goal” develop feeds, matched to genotypes of birdsto benefit health and enhance normal physiologicalprocesses.

• “gene chips” that contain the genetic code of bird;measure the effects of certain nutritionalsupplements, and how they alter the geneinteractions of the body.

TO SELECT NUTRIENTS FINE-TUNED WITH GENES OF BIRDS

• Nutrigenomics, not alter the genetics of an animal norto genetically modify the animal rather altering theactivity of genes, “switching on good genes andkeeping bad ones switched down” .

• Selection of nutrients for fine-tuning genes and DNApresent in every cell and every tissue of an animal.

• Example: stress response genes

ROLE OF NUTRITIONAL MANAGEMENT IN PERFORMANCE (PRODUCTION/DISEASE) OF BIRDS

• Gene expression studies will allow for theidentification of pathways responsible foreconomically important traits.

• Dietary manipulations and nutritional strategies arekey tools for influencing production.

• Provides new tools; clearly understand hownutritional management can be applied to addressdisease, performance and productivity in birds.

NUTRIGENOMICS AND IMMUNE SYSTEM

• Nutrition is the “key element” of health maintenance,particularly for the immune system.

• A deficiency of an essential nutrient will eventuallyaffect the body’s performance.

• Immune system is particularly sensitive to deficiencies,and once the immune system is compromised,negative consequences follows.

Contd…

• Deficiency is now rare in modern livestockproduction systems.

• Move to the next stage rather than merelypreventing deficiency, we can strive to actually meetthe bird’s exact requirements from its diet, in orderthat it can meet its genetic potential.

NUTRIGENOMICS AND DISEASES

• DNA microarrays; simultaneous assessment, of thetranscription of thousands of genes and of theirrelative expression between normal cells anddiseased cells or before and after exposure todifferent dietary components.

• Assist in the discovery of new biomarkers for diseasediagnosis and prognosis prediction and of newtherapeutic tools.

Contd…• Functional integrity of gene; depends on metabolic

signals that the nucleus receives from internalfactors, e.g.

hormones, and

external factors, e.g. nutrients

• Genomes evolve in response to many types ofenvironmental stimuli, including nutrition.

• Therefore, the expression of genetic information canbe highly regulated by nutrients found in feed.

FUTURE OF NUTRIGENOMICS

• Nutrigenomics; new tool for nutritional research inmitigating the problems related to bird’s health andproduction.

• In coming year’s innovations in nutrition research with useof various molecular technologies will indubitably updateour basic understanding of nutrient gene interrelationshipand help to define new methods for managing production.

• Finally by targeting the specific gene through nutritionalmanipulation, it may be possible to get the desiredperformance in terms of health as well as production.

• Discovery of “gene markers” related to economicallyimportant traits; improved by dietary regimens

• Information from nutrigenomics; lot of knowledge,understand the role of proper nutrition onperformance, health and disease in birds.

• Molecular findings with information from whole-animal studies, such as weight gain or eggproduction, we can better understand how nutrientsspecifically elicit their effects on animal health andproduction.

CONCLUSION

Contd…

• Widely utilized microarray or DNA chip technology in nutrigenomics research enables

Not only the screening of large numbers of genes

Simultaneously, giving a comprehensive picture of the variation of gene expression

Patterns, but will also provide explanations for complex regulatory interactions

Contd…

• “Nutrigenomics” is still young and emerging tool forrapid and effective evaluation of nutritionalstrategies.

• Provide a valuable tool for producers to differentiateand identify nutritional strategies to maximize notonly animal performance but ultimately profitability.

THANK YOU