Rumen protected methyl donors and the genome beyond nutrigenomics
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Transcript of Rumen protected methyl donors and the genome beyond nutrigenomics
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)
“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
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
Thank [email protected]