Download Objective Experimental Design

1/19/2016
Poultry Diet
Molecular mechanisms associated
with methionine deficiency in
meat-type chickens
Sammy Aggrey, Ph.D.
NutriGenomics Laboratory
Department of Poultry Science
University of Georgia, Athens GA
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Protein-The most expensive ingredient
Dietary CP requirement-misnomer
Requirement: Amino acid composition of protein
To meet protein requirement:
– limiting amino acids in the diet is supplied.
• USA: Diets are based on corn and soybeans
• First limiting amino acid in a corn soy diet:
– Methionine (MET)
Methionine
• Supplemented in diets by including either
DL-MET or MHA-FA (Analogue)
• Affects
– growth
– feathering
– Body composition
– feed efficiency,
– immunity
Objective
To study the molecular mechanisms in the
liver and Pectoralis major (P. major) muscle
in growing chickens when dietary MET
is restricted.
• Several studies with varying results
Strain: Ross708 males
Trt=2; Rep per Trt=4; N per rep=25
Experimental Design
TREATMENT
Basal Diet
CONTROL
CP (%)
ME (Kcal/g)
L-MET (%)
Starter (0-3 wk)
23
3.03
0.23
Grower (3-5 wk)
21
3.15
0.27
DEFICIENT
0-3 wk
Methionine (%)
0.63
0.63
3-5 wk
Methionine (%)
0.56
0.27
Starter diet: supplemented with 0.30% L-Met
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Growth and FCR during methionine
restriction phase
Transcriptome Analysis
• Tissues
CONTROL
TREATMENT
BWG3_4
601a
545b
BWG4_5
640a
612b
FCR3_4
1.67a
1.89b
FCR4_5
1.78
1.89
– Pectoralis major
– Liver
• Animals per tissue: 5 males
• Sex: Males
Sequence Mapping
Differential Expression
• Reads were inspected and trimmed for
adapter sequences
• Mapping>>Galgal4 using Tophat
• Genes and transcripts were detected
using Cufflinks v2.1.1
• Gene models: known and predicted genes
• Transcript abundance was measured in
fragment per kb of exon per million of
map reads (FPKM)
NGS vs RT-PCR
• 90-93% of reads were mapped
• About 12,596 (15,508) annotated mapped
R² = 0.9246
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0
-10
-8
-6
-4
-2
0
-5
2
Within tissue Analysis
Control vs Treatment (Restricted Met)
Gene-level differential analysis-Edge R
DE at FDR P<0.05
RT-PCR on 15 DE genes selected
randomly between liver and P. major
using StepOnePlus
Results
10
-12
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4
6
8
• Liver: 64 DE
• P. major: 919 DE
-10
-15
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Downregulated genes
Upregulated genes
P. Major
Liver
4
56
550
0
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Gene Ontology-P. major
Biological Process
P. Major
Liver
465
Gene Ontology-P. major
FDR
Molecular Functions
FDR
GO:0006955
Immune response
2.06E-11
GO:0016651
Oxidoreductase activity
0.015
GO:0006952
Defense response
4.64E-09
GO:0004908
Interleukin 1 receptor activity
0.015
GO:0045321
Leucocyte activation
7.17E-05
GO:0016491
Oxidoreductase activity
0.015
GO:0006954
Inflammation response
8.02E-05
GO:0003779
Actin binding
0.015
GO:0050853
B cell receptor signaling
2.40E-04
GO:0003954
NADH dehydrogenase activity
0.015
Gene Ontology-P. major
Cellular Components
Signaling Pathway Impact Analysis
FDR
GO:0071944
Cell periphery
2.80E-07
GO:0005886
Plasma membrane
8.10E-07
GO:0000323
Lytic vacuole
0.002
GO:0005764
Lysosome
0.002
GO:0002102
Podosome
0.002
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Classical probabilistic component
Magnitude of expression change
Position of DEG
Topography of pathway-gene interaction
Type of signaling between them
SPIA does not provide false positives
beyond the expected values
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KEGG Pathway
Size
NDE
pGFDR
Status
7
3.0E-4
Inhibited
What is going on during methionine deficiency?
Liver
Tight junction
134
Pectoralis major
Fc gamma R-mediated phagotocytosis
91
21
4.7E-5
Activated
Leukocyte transendothelial migration
118
23
4.7E-5
Activated
Inflammatory mediator regulation of TRP
channels
99
19
1.8E-4
ND
Osteoclast differentiation
131
22
4.6E-4
Activated
Natural killer cell mediation cytotoxicity
134
16
5.9E-4
Activated
B cell signaling pathway
72
17
9.2E-4
Inhibited
NF-kappa B signaling pathway
83
15
0.001
Activated
• Reduced skeletal and cardiac muscle contraction
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MYH13
MYH1
MYH3
MYH2
CKM2
-MYH6
-ACTN2
-MYL1
-MM-CK
• May affect walking ability
Major response to inflammation
NF-kB
TF that controls genes associated with inflammation response
• Fc gamma R-mediated phagocytosis pathway
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NCF1
SYK
PLCG2
VAV3
INPP5
CD32a
• B-cell signaling
• Inflammation mediators
– IL1R1, 5HTR and TRPA1
Osteoclast differentiation
• Bone homeostasis
– Bone forming osteoblast and bone resorbing
osteoclast
• Methionine deficiency>>osteoclast differentiation
– TF nuclear factor of activated T cells c1 is induced
• NF-kB
– Induction of osteoclast differentiation genes
– Prolong survival of osteoclast
– Increased resorption of bones
NFAM1
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Osteoclast differentiation
• Supportive mechanisms and/or genes
– Calcium signaling pathway (FDR=0.027)
– SYK, BTK, BLNK and PLC-gamma
• Osteoclastogenesis genes (DE)
– CTSK
– MMP9
– MMP7
Diet, D,L-Met
MHA-FA
Protein
What is going on during methionine deficiency?
Methylation Pathway
L-Methionine
THF
SAM
CH3
DMG
Re-methylation
Pathway
SAH
methylTHF
betaine
DNA, Proteins
Neurotransmitters
Phospholipids, etc
choline
Homocysteine
Transsulfuration Pathway
Cystathionine
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Changes cellular levels of SAM, SAH and Hcy?
Hcy>>>CYS (Transsulfuration)
CYS>>precursor of Glutathione (GSH)
Changes in cellular GSH levels?
Changes in ROS levels?
Cysteine
Taurine
Sulfate
Glutathione
Oxidant
Antioxidant
News Flash!!!
ROS
Oxidative
stress
Inactive
NFĸB
Active
NFĸB
INFLAMMATION
OSTEOCLAST DIFFERENTIATION
Dietary Methionine Deficiency
Excess ROS
or
GSH depletion ?
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Conclusion
Dietary methionine deficiency
• Negatively affect growth and feed efficiency
• Possibly reduce skeletal and cardiac muscle
contraction
• Potentially increase cellular stress
• Associated with muscular inflammation ????
• Compromised immunity?
• Reduced bone quality?
Acknowledgement
• Adisseo-France
• Dr. Fernando Gonzalez-Ceron
• Ms. Lindsey Rachett
Thank You
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