10/30/2008
TTU AFS 4400/5500 1
Northern blot analysis
C c-9 t-10 C c-9 t-10 PRE PI PDMI PRE PI PDMI
An Wa
A di t
SCD
RNA
C c-9 t-10 C c-9 t-10 PRE PI PDMI PRE PI PDMI • Angus adipocytes expressed SCD higher than Wagyu adipocyte after short term (1 wk) of differentiation.
• Both c9 t11 and t10 c12RNA
PREPIM
PDMIC 5 20 40 100 uM C 5 20 40 100 uM
c-9, t-11 t-10, c-12
Both c9,t11 and t10,c12 CLA decreased SCD and FAS expression as the dose increased.
RNA
SCD
FAS
Northern blot analysis
• SCD PPARγ and LPL wereC Arg C Arg C Arg C Arg C Arg C ArgC Arg C Arg C Arg C Arg C Arg C Arg • SCD, PPARγ, and LPL were
greater in preadipocytes
incubated with arginine
than in control.
• t10,c12 CLA decreased
SCD i d
RNA
SCD PPAR γ C/EBP β LPL TNF α Pref ‐1
RNA
SCD PPAR γ C/EBP β LPL TNF α Pref ‐1
RNA
Control Arg CLA Arg + CLAControl Arg CLA Arg + CLA
SCD gene expression and
not overcome by co-
incubating with arginine.
SCD
RNA
PPAR γ
Control Arg CLA Arg + CLA
γ
Control Arg CLA Arg + CLA
10/30/2008
TTU AFS 4400/5500 2
Metabolic analysis
Fatty acid composition Lipogenesis
0 3
0.4
0.5
0.6
0.7
0.8
0.9SCD index
1
1.5
2
2.5 14C acetateincorpo rat ion
Fatty acid composition Lipogenesis
pmol
/cel
l/2h
f M
UFA
/SFA
(SC
D in
dex)
0
0.1
0.2
0.3
Control 5uM c9,t11
40uMc9,t11
5uMt10,c12
40uMt10,c12
0
0.5
Control 5uM c9,t11
40uM c9,t11
5uM t10,c12
40uM t10,c12
Ratio
o
• Models to study deposition
Postnatal Adipose Tissue
– Japanese Black vs. Angus• Beef marbling score: (Japanese) 0‐12 CAB carcasses approx. 4‐5 on Japanese scale
• 10‐12 equals greater than 20% extractable lipid in L.D. muscle vs. 12‐14 % EE for USDA Prime
10/30/2008
TTU AFS 4400/5500 3
Animal Growth Rate700
Corn-Angus
300
400
500
600Corn-Wagyu
Hay-Angus
Hay-Wagyu
Body W
eight, Kg
2nd group (hay)
3rd group (corn)
4th group (hay)
Target weightCorn: 1.36 kg/d to 325 kg/8mo
100
200
300
0 56 112 168 224 280 336 392 448 504 560 616 672Days on feed
U.S. Endpoint Jpn Endpoint
1st group (corn)
to 325 kg/8moHay: 0.9 kg/dto 325 kg/12mo
Carcass characteristics
• Angus fed corn had greater IML than hay
25
Corn Angus
Hay Angus) greater IML than hay and Wagyu fed corn.
• Hay-fed Wagyu (Jpnendpoint) had greater IML than other diet group.
10
15
20
Hay Angus
Corn Wagyu
Hay Wagyu
ntag
e of
fat c
onte
nt (I
ML
Choice +Prime -
Prime +
P-valueEndpoint : P<.01B*E : P<.05B*D : P<.05
• Wagyu had low final body weights.
0
5
6 8 10 12 14 16 18 20 22
Time on Feed (mo)
Perc
en
U.S. endpoint Jpn endpoint
Choice -
10/30/2008
TTU AFS 4400/5500 4
SCD enzyme activity vs. Time
• Increased between the 13
14
Corn Angus
H A Increased between the
U.S. and Japanese
endpoint, but not in the
hay-fed Angus steers.
• Increased most in hay-7
8
9
10
11
12
13 Hay Angus
Corn Wagyu
Hay Wagyu
aroy
l-CoA
desa
tura
seac
tivity
,m
olpe
r 7m
in p
er m
g pr
otei
n
P-valueEndpoint : P=.06D*E : P=.08B*D*E : P=.08
based Wagyu steers.
4
5
6
6 8 10 12 14 16 18 20 22
Time on Feed (mo)
Stea Nm
U.S. endpoint Jpn endpoint
SCD gene expression vs. Time
• Greater in corn-fed 0.7
Corn Angus
H A
steers
• Increased most in
Wagyu steers, but
decreased with time in 0.2
0.3
0.4
0.5
0.6Hay Angus
Corn Wagyu
Hay Wagyu
SCD
:28S
RN
A
P-valueDiet : P=.06Endpoint : P=.07D*E : P=.05B*E : P=.01
Angus steers.
0
0.1
6 8 10 12 14 16 18 20 22
Time on Feed (mo)
U.S. endpoint Jpn endpoint
10/30/2008
TTU AFS 4400/5500 5
– When looking at these animals to study intramuscular fat
Postnatal Adipose Tissue
intramuscular fat• larger adipocytes
• more adipocytes
• these were the predominant theories• they found that adipocyte size is actually less in Wagyu than Angusgy g
– Numbers of adipocytes are differentrate of preadipoc te proliferation as t ice as high in
Postnatal Adipose Tissue
• rate of preadipocyte proliferation was twice as high in both subcutaneous and intramuscular adipose tissue from Wagyu vs. Angus cattle
– Japanese Black have the ability to accumulate IM lipid seemingly indefinitely Angus have genetic limitations in preadipocytep p ydifferentiation/proliferation
10/30/2008
TTU AFS 4400/5500 6
– Numbers of adipocytes are differentrate of preadipoc te proliferation as t ice as high in
Postnatal Adipose Tissue
• rate of preadipocyte proliferation was twice as high in both subcutaneous and intramuscular adipose tissue from Wagyu vs. Angus cattle
– Japanese Black have the ability to accumulate IM lipid seemingly indefinitely Angus have genetic limitations in preadipocytep p ydifferentiation/proliferation
• Adipose tissue as an endocrine tissue‐now more hormones produced in adipose tissue than the
Hormone Production
hormones produced in adipose tissue than the anterior pituitary– Leptin
• 16 kDa protein secreted from white adipocytes
• belongs to a class of helical cytokines
• similar to IL‐2 and GH
• four alpha‐helical bundle structures
• 1995 – ob protein leptin administration could eliminate obesity in the ob‐/ob‐mouse (ob protein = leptin)
10/30/2008
TTU AFS 4400/5500 7
Hormone Production
Long-term signals regulating energy balance. Insulin and leptin are the two hormones that act as long-term regulators of food intake and energy balance. Both insulin and leptin act in the central nervous system to inhibit food intake and to increase energy expenditure Activation ofexpenditure. Activation ofthe sympathetic nervous system (SNS) is likely to contribute to the increase in energy expenditure.(Havel et al. 2000.)
Hormone Production
AgRP: Agouti-related proteinArc: arcuate nucleusCART: cocaine and amphetamine related transcriptMC4R: melanocortin 4 receptorNPY: neuropeptide YPOMC: proopiomelanocortinPVN: paraventricular nucleusPVN: paraventricular nucleusVMN: ventromedial nucleus.
10/30/2008
TTU AFS 4400/5500 8
“ob” mutation
– Receptorhi h ffi it
Leptin Receptor
• high affinity
• several splice variants of a single gene• “long form” contains 302 amino acids in intracellular domain
• expressed in various regions of the brain• categorized as a class I cytokine receptor (much like g y p (IL‐6, LIF)
10/30/2008
TTU AFS 4400/5500 9
– Signal transduction pathways of class I cytokine receptors
Leptin Signaling
cytokine receptors• typically lack intrinsic tyrosine kinase activity• activation usually occurs following formation of homodimers
• leptin actually forms homodimers aggregation activates JAK/STAT pathway
– JAK = Janus Kinase
– STAT = signal transducers and activators of transcription
Leptin
• Leptin treatment (exogenous addition)– dose‐dependent decrease in food intake
– loss of body weight
– loss of fat depots
– increased metabolic rate
10/30/2008
TTU AFS 4400/5500 10
Leptin
• Leptin is synthesized/secreted from white adipocytesadipocytes– example of adipose tissue acting as a an endocrine tissue acting on other tissues (brain e.g.)
– promoter of region of Leptin gene contains sites for C/EBP‐α and PPAR γ; therefore, adipose‐specific geneg
– increased leptin has a negative feedback loop on adipocytes to decrease the leptin production
Resistin
• discovered in 2001
• expression reduced by TZDs• expression reduced by TZDs
• C/EBP responsive gene
• Biological activities– impairment of glucose tolerance
antagonism of glucose uptake– antagonism of glucose uptake– inhibition of 3T3-L1 differentiation
10/30/2008
TTU AFS 4400/5500 11
Adiponectin
• discovered in 1995
• secreted by differentiated adipocytes• secreted by differentiated adipocytes
• models of insulin resistance have reduced adiponectin levels
• exogenous adiponectin can stimulate fatty acid oxidation by skeletal muscleoxidation by skeletal muscle
• secretion is stimulated by TZDs
TNFα• TNFα (pro‐inflammatory cytokine)
– synthesized in neutrophils, activated T and Bsynthesized in neutrophils, activated T and B lymphocytes AND adipocytes
• neutrophils, T and B lymphocytes endocrine fashion• adipocytes paracrine/autocrine effect
– polypeptide = 157 amino acids(human) = 17 kDa• species variation increased
– no signal peptide‐ but it is anchored to membrane and released in a soluble form
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TTU AFS 4400/5500 12
TNFα
• TNFα Receptors (two distinct)– TNFR‐I (55 kDa)
• single transmembrane glycoprotein
– TNFR‐II (75 kDa)• single transmembrane glycoprotein
– both have similar extracellular domain but highly distinct intracellular domains that lead to unrelated, different dignal transduction pathways
Satellite cell development and differentiation
FuseProliferation
Satellite cell
Transdifferentiation
10/30/2008
TTU AFS 4400/5500 13
In vitro marbling study
Satellite cell Proliferation
(Ciglitazone, Troglitazone, T-174) Thiazolidinedione(MUFA,PUFA) Long chain fatty acid Horse Serum
Long chain fatty acid (PUFA)
DifferentiationTransdifferentiation
Muscle‐derived cell
Transdifferentiation
Gene experession during transdifferentiation
Preadipocyte Adipocyte
Early Intermediate Late
Ligands
Transcriptional activation
PPAR RXRC/EBPβ
ADD1/SREBP1
C/EBPα
DifferentiationTransdifferentiation
Factors binding region
Adipose geneexpression
SCD
FASTNF‐α
Leptin
Functional Activation
10/30/2008
TTU AFS 4400/5500 14
BSC transdifferentiationA
rbit
. Rat
io
( P = .08)
C/EBP β
Arb
it. R
atio
( P < .01)
PPAR γ
Arb
it. R
atio
( P < .001)Myogenin
BSC transdifferentiation and growth promotants
Arbit. R
atio
* ( P < .03)
C/EBP β
E2 : Estradiol
MGA : Melanegesterol
Acetate
*
*
Treatment
M+E: MGA + E2
10/30/2008
TTU AFS 4400/5500 15
BSC transdifferentiation and growth promotants
Arbit. R
atio
* ( P < .05)
PPAR γ
E2 : Estradiol
MGA : Melanegesterol
Acetate
*
Treatment
M+E: MGA + E2
BSC transdifferentiation and growth promotants
Arbit. R
atio
* ( P = .05)
SCD
E2 : Estradiol
MGA : Melanegesterol
Acetate
Treatment
M+E: MGA + E2
10/30/2008
TTU AFS 4400/5500 16
BSC transdifferentiation and growth promotants
Arbit. R
atio
* ( P = .05)
Myogenin
E2 : Estradiol
MGA : Melanegesterol
Acetate
Treatment
M+E: MGA + E2
BSC transdifferentiation and growth promotants
Positive Control : Insulin, Oleic acid, and Ciglitizone
10nM estradiol
20nM 17β trenbolone 10nM Melengestrol acetate
10/30/2008
TTU AFS 4400/5500 17
Muscle Biopsy Study
Muscle Biopsy Study
10/30/2008
TTU AFS 4400/5500 18
BSC transdifferentiation in Biopsy study
40
50
60
300
400N
A,
danc
e
NA
, da
nce
bb
a a aa
a
0
10
20
30
40
Day 0 Day 7 Day 14 Day 280
100
200
Day 0 Day 7 Day 14 Day 28
500 400
C/E
BPβ
mR
rela
tive
abun
d
Muscle biopsy date
PP
ARγ
mR
Nre
lativ
e ab
und
Muscle biopsy date
e e
b b
a
0
100
200
300
400
Day 0 Day 7 Day 14 Day 280
100
200
300
Day 0 Day 7 Day 14 Day 28
SC
D m
RN
A,
rela
tive
abun
danc
e
Myo
geni
nm
RN
A,
rela
tive
abun
danc
e
Muscle biopsy date Muscle biopsy date
a
a
BSC transdifferentiation in Biopsy study
40
50
300
400
NA,
dance
A,
dance
bb
b
a a
a
a a
0
10
20
30
Cont E2 TBA T+E
0
100
200
Cont E2 TBA T+E
400 500
C/EB
PβmRN
relativ
e abun
d
Treatment
PPARγ
mRN
relativ
e abun
d
Treatment
e e
a
0
100
200
300
Cont E2 TBA T+E0
100
200
300
400
Cont E2 TBA T+E
SCD mRN
A,
relativ
e abun
dance
Treatment
MyogeninmRN
A,
relativ
e abun
dance
Treatment
b
aa
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