NUTRIGENOMIC EFFECTS OF ZINC AND SELENIUM

Aadil Majeed KhanM.V.Sc Scholar (M-5628)ANIMAL NUTRITION DIVISION

2

OUTLINE

Introduction

Nutrigenomics and Nutrigenetics

Zinc : Nutrigenomic perspective

Selenium : Nutrigenomic perspective

Conclusion

3 Nutrigenomics

Nutrigenomics is the study of molecular relationships between nutritional stimuli and the response of the genes (Chavez et al., 2003).

Term described by Nancy Fogg Johnson and Alex Meroli (1999).

Nutritional genomics is a science which deals with role of nutrient in gene expression or their inter-relationship (Della Penna, 1999).

4 Cont...

Advancement in nutritional science discovered that nutrients or their metabolites can regulate various body functions directly or by stimulating or inactivating specific regulators (Mariman, 2006).

New areas of research like genomics, proteomics, metabolomics & bio-informatics (Daniel, 2002).

Emerging face of molecular nutrition to improve animal health and production.

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Nutrigenomics & Nutrigenetics: Two Sides of a Coin

Nutrigenomics: the application of genomics in nutritional research, eg. The way in which food ingredients influence the gene

Nutrigenetics: study of individual differences at the genetic level influencing genetic response.

Differences at SNP level

Mutch , et al. (2005)

6NUTRIGENOMICS: HOW DOES IT

WORK???

(Francis Crick, 1956)

7

Contd…

These processes are controlled by the basic genetic make-up, external factors such as

Disease Environmental toxins or specific nutrients

Effect of a specific nutrient on animal performance/physiology Vs. specific messenger RNA (mRNA) from tissue at any stage.

Amount of mRNA present relates to the relative amount of copies of known genes, measured by using gene chips.

Using contrasting colour labels, it can be determined if a gene is “up-regulated”, “down-regulated” or “unaffected” as a result of a specific dietary manipulation.

8BASIC TOOLS

TRANSCRIPTOMICS Approach in which mRNA, and consequently gene expression is

analyzed in a biological sample under certain conditions at given point of time

METABOLOMICSQuantitative analysis of all metabolites in a biological

system such as cell, tissue or biological fluid (blood, plasma

and saliva)

PROTEOMICS Take this analysis further and

aims to characterize all proteins in a biological sample at the

functional level

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Transcription-factor Pathways Mediating Nutrient-gene Interaction

(Paiwaiya et al., 2011)

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Zinc is required for the structural and functional integrity of over 2000 transcription factors (Kwun,2004)

The most important functions in livestock are those that become limiting to health and production when they are deprived of zinc. There are four outstanding, interconnected candidates.

1) Gene expression

2) Appetite control

3) Fat absorption

4) Antioxidant defence (Suttle,2005)

Nutrigenomic perspective

11Metallothionein 

Cysteine-rich cytosolic proteins Low molecular weight (MW ranging from 500 to14000 Da) proteins. Localized to the membrane of the Golgi apparatus Bind both physiological (such as zinc, copper, selenium)

and xenobiotic (such as cadmium, mercury, silver, arsenic) heavy metals There are four main isoforms expressed; MT1 (subtypes A, B, E, F, G, H,

L, M, X), MT2, MT3, MT4 Mammalian MT binds three Zn(II) ions in its beta domain and four in the

alpha domain.

12 Zinc: Role in genome stability

Zinc plays pivotal role in maintenance of genomic stability, genetic expression and apoptosis modulation (Almendro et al., 2011).

Deficiency of Zn can cause changes into the DNA similar to what is seen after radiation exposure (Rosa et al., 2011).

Zn is integral part of DNA repair protein OGG1 which repairs oxidized guanine in DNA. Its dysregulation leads to point mutations and down regulation of gene expression (Thomas et al.,2015).

13Zn is involved in regulation of small intestinal, thymus

and hepatocytes gene expression

(Blanchard and Cousins, 2000)

Regulation of Intestinal Gene Expression by Dietary Zinc: Induction of Uroguanylin mRNA by Zinc Deficiency

Rats, 15 d duration

14Effect of zinc deficiency on the mRNA expression

pattern in liver and jejunum of adult rats

(Pfaffl et al., 2003)

Zn deficiency: Semi-synthetic diet; 2 µg Zn/g; n=6Control: 58 µg Zn/g; n=7 After 29 days entire jejunum and liver total

RNA was extracted.

15

Cont…

Gene expression pattern captured as an image of a cDNA-array hybridized with Cy3-labeled control sample (green) and Cy5-labeled sample (Zn deficiency in red).

16Effect of Zinc Deficiency and Supplementation on

Insulin Signalling in Chickens

Gr. IR(ng/ml)

IRP(ng/ml)

IRS(ng/ml)

AKT(ng/ml)

PI3K(ng/ml)

GLUT4(ng/ml)

IR gene expression(arbitrary unit)

IRS1gene expression(arbitrary unit)

1 23 ± 2.6

4.3 ± 1.2 33.3±1.5

2.2 ± 0.3 16.3 ± 1.5 2.5 ± 0.2

3.1 ± 0.62 11.3 ± 1.32

2 25 ± 6.1

2.5 ± 0.5a

32±2 1.5 ± .2a 6.3 ± .5a 1.3 ± 0.3a

2.9 ± 0.71 10.6 ± 1.22

3 21 ± 2.1

5.3 ±0.8b

34.7±1.5

3.2 ± 0.3cd

25.3 ±2.5cd

4 ± 1.0b 3.2 ± 0.42 12.3 ± 2.45a: Gr. 2 Vs. Gr. 1 (P<0.05 ); b: Gr. 3 Vs. Gr. 2 (P<0.05) c: Gr. 3 Vs. Gr. 1 (P<0.05); d: Gr. 3 Vs. Gr. 2 (P<0.01 )

(Alkaladi, 2013)

21-d experiment(1)Control group,(48.37 ppm) Zn .(2) Zn deficient group(28ppm) Zn.(3) Zn supplemented group (60ppm) Zn

one-day-old Hubbard male broiler

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Effect of dietary Zinc oxide on expression of genes associated with inflammation

(Sargeant, 2010)

a,b,c differ (P<0.01)

Three days post-weaning orally challenged with 109 CFU ETEC (K88), n=12 each group

ZnO-supplemented (ZnO at 3.1 g/kg) NS non-supplemented

32 day experiment in piglets

Relative gene expression of MT1

18Zinc increases the expression of Th1 cytokines mRNA in mice infected with Leishmania major

(Afshari et al.,2016)

Untreated group (a)Zn group=(a)+oral 50 µl Zn sulphate (0.22 mg Zn)Glucantime group=(a)+50 µl (i/p) (1.5 g/5 ml)n=6

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(Afshari et al.,2016)

Cont…

***p≤0.001 **p≤0.01

20Epigenetic effects of dietary zinc on the

porcine ZIP4 gene expression

(Karweina et al., 2014)

n=10 per groupPeriod of study: 04 weeks

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Effect of zinc source on the expression of ZIPII transporter genes in goats

Tissue Control ZnSO4 Zn-AAAbomassum 1.02±0.32* 1.22±0.28 0.47±0.o8*Proximal duodenum

1.01±0.22 0.49±0.25 0.66±0.35

Middle duodenum

1.07±0.53* 5.74±2.03* 1.45±0.45*

Distal duodenum

1.07±0.23 1.69±0.78 1.03±0.49

Jejunum 1.00±0.18 1.12±0.40 1.13±0.13Ileum 1.00±0.03 1.23±0.21 0.75±0.09Liver 1.00±0.02* 0.44±0.16* 1.60±0.23*

mRNA(ZIPII)

*(p<0.05)

Relative abundance of ZIPII mRNA in the main digestive tissues and liver of goats fed different sources of zinc.

(Wang, et al.,2014)

Control group =basic diet with no additional zinc.Second group= basic diet with 60 mg/kg of zinc sulphateThird group=basic diet with zinc amino acid chelate (Zn-AA)

7 day experimentn=6 in each group

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Effect of in vitro zinc supplementation on HSPs expression and IL10 production in heat treated MNC of Sahiwal and Karan Fries cows

(Sheikh et al.,2016)n=12 each(P<0.05)

23Relative expression of HSP90α mRNA at different levels of

exposure in transition Sahiwal and KF cows

Zinc (0.01 mM), used in its salt form, Zinc sulfate heptahydrate

(Sheikh et al.,2016)

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Nutrigenomic perspective

Beneficial in preventing liver necrosis in rats (Schwarz and Foltz, 1957).

Prevent exudative diathesis in chicks (Patterson et al., 1957).

Prevent liver necrosis in swine (Eggert et al., 1957).

Prevent NMD in calves (Muth et al., 1958).

Essential component of glutathione peroxidase (Rotruck et al., 1973).

Selenium has an important role in the expression of the APC gene, which is related to cell cycle regulation (Mauro et al.,2011).

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Transcriptomics, proteomics, Metabolomics, Gene knock-

down/outIdentification of SNPs in Se metabolic pathways

New functional biomarkers and understanding

mechanism of Se action

Functional SNPs: genetic factor that

influence Se metabolic pathways

Selenoproteins

Downstream targets

Se requirements: Se intake and susceptibility to disease

Selenium Gene Interaction

How selenium intake affects gene expression How genetic factors influence Se metabolism

26Selenome

Biochemical unit that determines much of the functional activity of Se

It includes proteins involved

a. In synthesis of selenocysteine tRNA

b. In incorporation of selenocysteine during translation

c. In transport of selenium in the blood

Se is incorporated into 25 selenoproteins

27Selenoproteins & their synthesis

Major function: Protection from oxidative stress, roles in redox state, thyroid metabolism, stress response, inflammation

Recoding of the UGA codon to incorporate Sec requires a specific stem-loop structure ‘SECIS’

Se incorporation requires specific selenocysteyl-tRNA (tRNA-Sec) Sec is synthesized directly on tRNA from selenide, ATP & seryl-

tRNA

28Function & catalytic role of Selenoproteins

Selenoproteins Catalytic reaction Role of Sec

Glutathione peroxidases

Reduction of hydroperoxides

An ionized selenolate reacts with H & O

Iodothyroninedeiodinases (D1– 3)

Conversion of T4 to T3 (D1, D2),degradation of T3 and T4 (D1, D3)

D1-iodine acceptor forming a selenenyliodide intermediate D2, D3 no selenenyliodide intermediate

Thioredoxin reductases(TrxR1, TrxR2, TGR)

NADPH-dependent reduction of Trx and other substrates

Part of a redox active selenylsulfide-selenolthiol motif

Glycine reductase

Conversion of glycine to acetyl phosphate

Se-carboxymethyl-selenoether intermediate

29Se modulates transcription factors

Se modulates cellular activities by acting on proteins important for signal transduction

Reproductive organ appears to be a priority tissue for Se accumulation

Groups Se Status

Diet

Gr.I Deficient Yeast based diet (0.02 ppm Se)Gr.II Adequate Yeast based diet + 0.2 ppm (Se as Sod.

Selenite)Gr.III Excess Yeast based diet + 1 ppm (Se as Sod.

Selenite)

(Ranawat and Bansal, 2010)

Study was conducted for eight weeks in mice

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Selenium levels and glutathione peroxidase activity in mice testis

Parameters Se deficient

Se adequate Se excess

Testes Se (µg/g tissue)

0.47±0.02a

0.75±0.02b 0.96±0.03c

Testis GPx (µmoles NADPH oxidized/min/mg protein)

49.8±1.10a

116±3.52b 143±1.43c

abc(P<0.001)

Serum Testosterone, LH and FSHSe deficient Se adequate Se excess

T (ng/ml) 0.44±0.01a 0.68±0.01b

0.46±0.02a

LH (ng/ml)

1.2±0.01a 2.4±0.03b 1.5±0.01a

FSH (ng/ml)

0.4±0.02a 0.58±0.02b

0.46±0.001a

abc(P<0.001)

31Effect of Se on protein expression of CREB & CREM

Parameters Se deficient

Se adequat

e

Se excess

CREB A(405nm)

0.281±0.01

0.642±0.01

0.314±0.01

CREM A(405nm)

0.235±0.01

0.762±0.01

0.278±0.01

mRNA Expression

ProteinExpressio

n

(Ranawat and Bansal, 2010)

32Selenium affects Selenoprotein gene expression &

antioxidative capacity in mice

Groups

Diet

I Basal diet (0.045 mg/kg Se)II Basal diet (0.1mg/kg Se)III Basal diet (0.4 mg/kg Se)IV Basal diet (0.8 mg/kg Se)

7 week old male mice (n=80) Experimental period 56 days

(Zhang et al., 2013)

Nutrigenomics has been implicated to study the effect of Se deficiency or excess on gene expression of Se dependent GPx and Txn-rd enzymes

33Effects of dietary selenium on liver

antioxidant capacityParameter Gr.I

(0.045 ppm)

Gr. II (0.1ppm)

Gr. III (0.4 ppm)

Gr. IV (0.8 ppm)

GSH-Px (U/mg protein)

333 686 751 525

GR (U/mg protein) 34 46 41 24SOD (U/mg protein)

781 802 804 757

CAT (U/mg protein)

33 45 41 26

Antioxidant enzyme activities were increased in Se adequate & super-nutrition status

(Zhang, et al., 2013)

34 Glutathione peroxidase gene expression in tissues

35Effects of gastrointestinal microbiota on

selenoprotein expression

(Juliane et al., 2009)

5 week experiment in miceSelenium deficient=0.086ppmSelenium-adequate=0.15ppm

36Effects of organic and inorganic dietary selenium

supplementation on gene expression in oviducts of hens

(Brennan et al.,2011)

43 weeks experimental periodControl=basal semi-purified diet SS=basal diet + 0.3 ppm Se as Sodium seleniteSP=basal diet + 0.3 ppm Se as Sel-Plex,SP

37Nutrigenomics study on SepS

expression in acute phase response

• Ample Se is needed as a substrate in the form of selenocysteine-loaded tRNA in order to support the biosynthesis of selenoproteins

• SEPS is connected to physiological pathways that modulate inflammation e.g. NF-kB, JNK activation & induction of acute phase proteins

• Se deficiency impair SEPS expression

Dietary Selenium controls selenoprotein expression in acute phase response

(Stoedter et al., 2010)

38 Selenium control selenoprotein expression in acute phase response

(Stoedter et al., 2010)

Experiment 1. To confirm the effect of LPS on SepS expression

Experiment 1 o

o

Selenium control selenoprotein expression in acute phase response

(Stoedter et al., 2010)

Experiment 2. Effect of Se supplementation on LPS induced acute phase response

40Regulation of hepatic SepS in response to Selenium and LPS

Se supplementation mitigate sex specific LPS dependent rise in cytokines with parallel specific changes in hepatic selenoprotein expression

(Stoedter et al., 2010)

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Time (Day)

Gr. 1 (Se NP)

Gr.2 (Sodium selenite)

0 100.0±3.5 100±3.510 115.1±9.0a 129.8±6.7a

20 123.7±8.1ab 89.3±5.7ac

30 64±4.8ac 69.1±6.4ac

(a)compared to day 0, p<0.05(b) compared to group 2, , p<0.05(c) compared to day 10 and day 20

p<0.05 Groups 1 Se NPs=1ppmGroup 2 Sodium Selenite=1ppm30 day experiment

Effect of sodium selenite and selenium NP on transferrin gene expression in sheep

Relative expression of transferrin(% control)

(Kojouri et al.,2011)

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Enhanced water-holding capacity of meat was associated with increased Sepw1 gene expression in pigs fed selenium-enriched yeast (Gang et al.,2010).

Selenium NP increases the expression of HSP90 gene in myocytes after an intense exercise (Ali et al.,2013).

Selenium deficiency induced damages and altered expressions of metalloproteinases and their inhibitors (MMP1/3, TIMP1/3) in the kidneys of growing rats (Jing et al.,2015).

43Conclusion

Nutrigenomics is an emerging face of molecular nutrition to improve animal health and production

Nutrigenomic application unravelled the mechanism involved in Zn-gene and Se-gene interaction that can potentially influence the pattern of mettalothonien and selenoprotein synthesis respectively.

Transcriptomic studies shows that Zinc alters expression of genes as well as plays a pivotal role in genome stability.

Transcriptomic studies shows that Selenium alters expression of genes involved in apoptosis and cell cycle control.

Nutrigenomics studies related to Zinc and Selenium are required in different class of animals to unravel various mechanisms in order to improve health and production of animals.

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Alteration of gene expression by Zinc oxide nanoparticles or Zinc Sulfate

(Zhang et al., 2016)28 d experiment in adult hens

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Arrows indicate some key features1)Apoptosis 4)Antioxidant activity 2)ECM part 5)Channel regulator activity3)Synapse 6)Receptor regulator activity

(Zhang et al., 2016)