Rumen-protected methyl donors and the genome: beyond

nutrigenomics

Juan J. Loor, Zheng Zhou, and Mario Vailati-RiboniDepartment of Animal Sciences, Division of Nutritional Sciences,

and Illinois Informatics InstituteUniversity of Illinois, Urbana-Champaign, USA

XXI ASPA CongressJune 9‐12, 2015Milan, Italy

Outline

• Brief “history”

• Nutritional and physiological context

• Nutrigenomics

Met and choline

• Beyond nutrigenomics Can we “prime” cow and calf??

Nutritionally‐important methyl donors

Choline

Betaine(Tri-Methyl glycine)

Methionine

5-methyl-tetrahydrofolate(“folic acid”)

MAT1A

BHMTMTR GNMT

CBS

CTH

Antioxidants

Biochemistry of methyl donors is interrelated

[Modified from Mato et al. (2008)]

P-Ethanolamine

P-Choline

PEMT

SAH

SAHH

DNMT

Substrate =  e.g. CpG siteSAM = S‐adenosylmethionineDNMT = DNA methyl transferase

Methyl donor cycle regulation: non‐ruminants

MAT1A+

+

‐

ATP

MAT1A control:Genome levelPromoter methylation (Huh‐7 cells) (Tomasi et al., 2012)

Certain microRNA↓ mRNA (Yang et al., 2012)

Enzyme levelFeed‐forward activation by Met (sheep) (Xue and Snoswell, 1989)Feed‐back inhibition by SAM (sheep) (Xue and Snoswell, 1989)

(e.g. GNMT, DNMT)

ROM

‐

↑ Metabolism↑ Oxidative stress

BHMT+MTR +

Glutathione+

Oxidase

Methyl donor requirements differ during the life stages of ruminants

(Pinoti et al., 2002)

Adult ruminant  greater requirements due to ruminal degradation of dietary sources

Use of “rumen‐protection” technology important

(MTR)

(MTR)

History of rumen‐protection: amino acids and cholineJournal of Dairy Science papers:

1968 ‐ First paper in JDS: Griel, Patton, McCarthy and Chandler “Milk production response to feeding methionine‐hydroxy analog (MHA) to lactating cows”1970: Broderick, Kowalzyk and Satter “Milk production response to supplementation with encapsulated methionine per os or casein per abomasum” (Delmar Chemical, Ontario)2006: Rulquin et al. “Effect of different forms of methionine on lactation performance of dairy cows”2007: Cooke et al. “Supplemental choline for prevention and alleviation of fatty liver in dairy cattle”2011: Chen et al. “Effect of feeding different sources of rumen protected methionine on milk production and N utilization in lactating dairy cows”2011: Zom et al. “Effect of rumen‐protected choline on performance, blood metabolites, and hepatic triacylglycerols of periparturient dairy cattle”

Also,Noftsger and St‐Pierre (2003), Noftsger et al. (2005), Socha et al. (2005), 

St‐Pierre and Sylvester (2005), Ordway et al. (2009), Appuhamy et al. (2011), Lee et al. (2012), Osorio et al. (2013), Osorio et al. (2014ab)

Dietary methyl‐donors in dairy cows

SAM

SAHHomocysteine

Cysteine

Diet TissuesMilk

Liver health

DNA methylation Epigenetics

VLDLPC

Vit. B12 CH3

PE

rRNA complex Protein synthesis initiation

Choline

Betaine

Dimethylglycine

Diet

5-MTHF

Methionine

Diet

Folate+ + +

Cooke et al. 2007Zom et al. 2011

+

GlutathioneAntioxidants

Taurine+

EmbryoFetus

Pre‐calving Post‐calving

~3‐4 wk ~3‐4 wkCalving

(Block et al., 2001)

Energy balance

Body condition score

Energy intake

4% Fat corrected milk

(Ingvartsen, 2006)

Transition periodTissue mobilization

(Komaragiri and Erdman, 1997)

Both body fat and protein are mobilized

Increased milk by 3 kg/d with no change in intake

‐46

‐21

‐61

‐21

(Bertoni and Trevisi, 2013)

Inflammation and oxidative stress occur during transition

• Acidosis• Metritis• Retained placenta• High NEFA and

BHBA

(++)

Physiological context for rumen‐protected methyl donors

NEFA

Metabolism

OxidativeStress

Cell Damage

Inflammation

NutrigenomicsNutriepigenomics

Met, Choline

ProductionHealthFertility

Practical outcomes

Mechanistic outcomes

DMI (kg/d)

Day relative to calving Day relative to calving

Diet P = 0.67Time P <.001DxT P = 0.42

Diet P = 0.18Time P <.001DxT P = 0.78Met P = 0.06 

ControlMetaSmartSmartamine

Met = Control vs MetaSmart + Smartamine

~7 g Met/d supplemented ~10 g Met/d supplemented

Better performance with Methionine

Parameter

Diet

SE

P‐value

Control MetaSmart Smartamine Diet Met Par Time D×T

Milk yield (kg/d) 35.7 38.1 40.0 1.6 0.15 0.08 ‐‐ <0.01 0.86

Milk fat (%) 4.27 4.68 4.09 0.22 0.59 0.36 .05 <0.01 0.01

Milk protein (%) 3.04 3.26 3.19 0.08 0.13 0.05 ‐‐ <0.01 0.23

Milk fatyield (kg/d) 1.64 1.84 1.81 0.08 0.11 0.04 ‐‐ 0.04 0.01

Milk  proteinYield (kg/d) 1.11 1.23 1.24 0.05 0.08 0.03 ‐‐ 0.02 0.14

ECM (kg/d) 41.0 44.8 45.0 1.55 0.09 0.03 ‐‐ <0.01 0.07

Milk yield and components

Met = Control vs MetaSmart + Smartamine

(Osorio et al., 2013)

Day after parturition0 5 10 15 20 25 30 35

kg/d

10

12

14

16

18

20

22

24

26

Control MethionineCholine

Recent experiment confirms the benefit of rumen‐protected Methionine

• Dry matter intake during last 3 wk prepartumgreater (1‐2 kg/d) with Methionine 

• Milk protein % greater with Methionine

• Lower inflammatory and oxidative stress status

Day aftyer parturition0 5 10 15 20 25 30 35

kg/d

20

25

30

35

40

45

50

55

Control MethionineCholine

Dry matter intakeMilk productionMet P = 0.03Chol P = 0.41Day P < 0.01

Met P = 0.02Chol P = 0.90Day P < 0.01

(Zhou et al., 2015 Abs. 455, JAM Orlando, FL, USA)

Nutri....what??!!

“Nutrigenomics attempts to study thegenome‐wide influences of nutrition. From anutrigenomics perspective, nutrients are

dietary signals that are detected by the cellularsensor systems that influence gene andprotein expression and, subsequently,

metabolite production”Müller and Kersten, Nature Review, 2003

“Is a sub‐specialty of nutrition science which aims to understand how genome‐diet interactions influence individuals’ and populations’ responses to food, disease susceptibility, and population health” 

The Omics Ethics Research Group

genome‐widenutrients

dietary signalssensor systems

Definition of nutrigenomics

DNA

mRNA

Transcription

mRNA editing(splicing)

mRNA

Translation

Ribosomes(build proteins based on information in mRNA)

Proteinsynthesis

Newly‐formedprotein

Specific function inside (or outside) the cell

Enzymatic

Structural

Signaling

Transcription regulator

Transport

-10.0 7.0 21.0 -10.0 7.0 21.0 -10.0 7.0 21.0CO MS SM

1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

2

3

4

5

-10.0 7.0 21.0 -10.0 7.0 21.0 -10.0 7.0 21.0CO MS SM

1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

2

3

4

5

Smartamine +Control

MetaSmart +Control

Control(higher-energy,

“close-up” to calving)

Rumen-protected Methionine alters the liver transcriptome2,663 genes with diet  time effect

(Osorio et al., 2013)

The 12 most-impacted metabolic pathways with rumen-protected Methionine

Impact0 200 400 600 800 1000 1200 1400 1600

Cyanoamino acid metabolismTaurine and hypotaurine metabolism

Lipoic acid metabolismPropanoate metabolism

Pyruvate metabolismCysteine and methionine metabolism

Glyoxylate and dicarboxylate metabolismRiboflavin metabolism

Valine, leucine and isoleucine biosynthesisGlutathione metabolism

Glycolysis and gluconeogenesisCitrate cycle (TCA cycle)

Carbohydrate metabolism

Antioxidants

**These are key pathways responsible for the adaptations in liver to parturitionand rumen‐protected Methionine supplementation

1-Carbon metabolism pathway

Taurine

Transcription regulator Function FrequencyFBJ osteosarcoma oncogene (FOS) Regulates cell proliferation, 

differentiation, and transformation

Associated with apoptosis7 of 18

Ets homologous factor (EHF) Repressor of cellular differentiation 6 of 18Kruppel‐like factor 4 (KLF4) Repressor of cellular proliferation

Promotes cell survival6 of 18

Kruppel‐like factor 5 (KLF4) Transcriptional activator

Promotes and suppresses cell proliferation and cell growth

6 of 18

v‐myc avian myelocytomatosis viral oncogene homolog (MYC)

Regulates cell cycle progression, apoptosis, and cellular transformation

6 of 18

Top 5 most-frequently affected transcription regulators with rumen-protected Methionine

(Zhou et al., in review)

At day 7 postpartumEHF down-regulated 2.4-fold in cows fed Methionine vs. control

Biological meaning of transcription regulator networks?

At day 7 vs. -10 d postpartumEHF down-regulated 1.7-fold in cows fed Methionine

• Methionine could have direct anti-inflammatory role through EHF

Methionine, choline

Calf metabolism

Can methyl donors affect the developing calf in utero?

Or can they elicit an effect through colostrum?

Bioinformatics analysis revealed unique biological responses to rumen‐protected Met

Immune func on ↓ Nutrient metabolism ↑Endocrine signaling ↓

• Functional outcome still unknown

• Epigenomicalterations??

Methionine and mTOR signalling

Met?

Potential mechanisms:

• Binding directly to regulatory site on mTOR

• Binding to a co-regulator of mTOR(indirect)

• Stimulating a protein kinase

Not only protein‐coding genes...Epigenetic/epigenomic = “above” the genome

Epigenetic modifications are reversible modifications of DNA or histones that affect gene expression without altering the DNA sequence

Histone modifications

DNA methylation

microRNA target and repress mRNA

Targeted analysis of methylation status“Hypo‐methylation” or “Hyper‐methylation” status

Transcription regulators and target genes: PPARalpha (76 CpG sites)

Methylation status

mRNA expression

• These were newborn piglets

• Functional outcome on gluconeogenic flux was small

• Longer-term study needed e.g. through weaning transition

• Findings indicate a physiological benefit of supplementing rumen‐protected Met during the transition period

• Underscore the importance of maintaining an adequate Met:Lys balance

• Clear nutrigenomics effects • Likely epigenomic effects

Met : Lys

Conclusions

Perspectives• Interchangeable roles and interactions between methyl donors (e.g. choline, Met, betaine) should be further investigated at various life stages

• Molecular techniques will help  

Perspectives• Functional effects of increasing the methylation capacity through rumen‐protected Met or Choline supplementation need to be studied in cow and calf

Acknowledgements

Thank youjloor@illinois.edu