10/30/2008
Northern blot analysis
An
C
c-9
t-10
C
c-9
t-10
PRE
Wa
PI
PDMI
PI
PRE
PDMI
RNA
• Angus
A
adipocytes
di
t
expressed SCD higher than
Wagyu adipocyte after
SCD
short term (1 wk) of
PRE
c-9, t-11
PDMI
PIM
C
5
20
t-10, c-12
40
100 uM
C
5
20
40
differentiation.
100 uM
• Both c9
c9,t11
t11 and t10
t10,c12
c12
RNA
CLA decreased SCD and
SCD
FAS expression as the
dose increased.
FAS
Northern blot analysis
C
Arg
C
Arg
C
Arg
C
Arg
C
Arg
C
Arg
• SCD,
SCD PPARγ
PPARγ, and
and LPL were
RNA
greater in preadipocytes
SCD
Control
PPAR γ
C/EBP β
Arg
LPL
TNF α
CLA
Arg
Pref ‐1
incubated with arginine
+ CLA
than in control.
• t10,c12 CLA decreased
RNA
SCD gene expression
i and
d
SCD
Control
RNA
Arg
CLA
Arg
+ CLA
not overcome by coincubating with arginine.
PPAR γ
TTU AFS 4400/5500
1
10/30/2008
Metabolic analysis
Fatty acid composition
Fatty acid composition
2.5
SCD index
14C acetate
inc orpo ration
0.8
2
0.7
pmol/cell/2h
Ratio of MUFA/SFA (SCD index)
0.9
Lipogenesis
0.6
0.5
0.4
1.5
1
03
0.3
0.2
0.5
0.1
0
Control
5uM
c9,t11
40uM
c9,t11
5uM
t10,c12
40uM
t10,c12
0 Control
5uM
c9,t11
40uM
c9,t11
5uM
t10,c12
40uM
t10,c12
Postnatal Adipose Tissue
• Models to study deposition
– 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
TTU AFS 4400/5500
2
10/30/2008
Animal Growth Rate
700
Corn-Angus
Corn-Wagyu
600
Hay-Angus
Body Weight, Kg
Hay-Wagyu
500
4th group (hay)
400
3rd group (corn)
Target weight
Corn: 1.36 kg/d to 325 kg/8mo
to 325 kg/8mo
Hay: 0.9 kg/d
to 325 kg/12mo
2nd group (hay)
300
1st group (corn)
200
U.S. Endpoint
Jpn Endpoint
100
0
56
112
168
224
280 336
Days on feed
392
448
504
560
616
672
Carcass characteristics
• Angus fed corn had
25
Percen
ntage of fat content (IML)
Corn Angus
greater IML than hay
Hay Angus
Corn Wagyu
20
and Wagyu fed corn.
Hay Wagyu
• Hay-fed Wagyu (Jpn
15
endpoint) had greater
Prime +
IML than other diet
10
Prime -
group.
Choice +
5
• Wagyu had low final
Choice -
U.S. endpoint
body weights.
Jpn endpoint
0
6
8
10
12
14
16
18
Time on Feed (mo)
TTU AFS 4400/5500
20
22
P-value
Endpoint : P<.01
B*E : P<.05
B*D : P<.05
3
10/30/2008
SCD enzyme activity vs. Time
14
• Increased between the
Corn Angus
Stea
aroyl-CoA desaturase activity,
Nm
mol per 7min per mg protein
13
H A
Hay
Angus
Corn Wagyu
12
Hay Wagyu
U.S. and Japanese
11
endpoint, but not in the
10
9
hay-fed Angus steers.
8
• Increased most in hay-
7
6
based Wagyu steers.
5
U.S. endpoint
Jpn endpoint
4
6
8
10
12
14
16
18
20
22
P-value
Endpoint : P=.06
D*E : P=.08
B*D*E : P=.08
Time on Feed (mo)
SCD gene expression vs. Time
0.7
• Greater in corn-fed
Corn Angus
H A
Hay
Angus
0.6
Corn Wagyu
steers
Hay Wagyu
SCD:28S RNA
0.5
• Increased most in
0.4
Wagyu steers, but
0.3
decreased with time in
0.2
Angus steers.
0.1
U.S. endpoint
Jpn endpoint
0
6
8
10
12
14
16
18
20
22
P-value
Diet : P=.06
Endpoint : P=.07
D*E : P=.05
B*E : P=.01
Time on Feed (mo)
TTU AFS 4400/5500
4
10/30/2008
Postnatal Adipose Tissue
– When looking at these animals to study intramuscular fat
intramuscular fat
•
•
•
•
larger adipocytes
more adipocytes
these were the predominant theories
they found that adipocyte size is actually less in Wagyu
gy than Angus
g
Postnatal Adipose Tissue
– Numbers of adipocytes are different
• rate
rate of preadipocyte
of preadipoc te proliferation was twice as high in proliferation as t ice 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 preadipocyte
p
p y
differentiation/proliferation
TTU AFS 4400/5500
5
10/30/2008
Postnatal Adipose Tissue
– Numbers of adipocytes are different
• rate
rate of preadipocyte
of preadipoc te proliferation was twice as high in proliferation as t ice 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 preadipocyte
p
p y
differentiation/proliferation
Hormone Production
• Adipose tissue as an endocrine tissue‐now more hormones produced in adipose tissue than the
hormones produced in adipose tissue than the anterior pituitary
– Leptin
•
•
•
•
•
TTU AFS 4400/5500
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 Æ leptin administration could eliminate obesity in the ob‐/ob‐ mouse (ob protein = leptin)
6
10/30/2008
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 of
expenditure.
the sympathetic nervous
system (SNS) is likely to
contribute to the increase in
energy expenditure.
(Havel et al. 2000.)
Hormone Production
AgRP: Agouti-related protein
Arc: arcuate nucleus
CART: cocaine and
amphetamine related
transcript
MC4R: melanocortin 4
receptor
NPY: neuropeptide Y
POMC: proopiomelanocortin
PVN: paraventricular nucleus
VMN: ventromedial nucleus.
TTU AFS 4400/5500
7
10/30/2008
“ob” mutation
Leptin Receptor
– Receptor
• hi
high affinity
h ffi it
• several splice variants of a single gene
• “long form” contains 302 amino acids in intracellular domain
• expressed in various regions of the brain
g
y
p (
• categorized as a class I cytokine receptor (much like IL‐6, LIF)
TTU AFS 4400/5500
8
10/30/2008
Leptin Signaling
– Signal transduction pathways of class I cytokine receptors
cytokine receptors
• typically lack intrinsic tyrosine kinase activity
• activation usually occurs following formation of homodimers
• leptin actually forms homodimers Æ 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
TTU AFS 4400/5500
9
10/30/2008
Leptin
• Leptin is synthesized/secreted from white adipocytes
– 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 g
gene
– 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
– inhibition of 3T3-L1 differentiation
TTU AFS 4400/5500
10
10/30/2008
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 muscle
oxidation by skeletal muscle
• secretion is stimulated by TZDs
TNFα
• TNFα (pro‐inflammatory cytokine)
– synthesized in neutrophils, activated T and B synthesized in neutrophils, activated T and B
lymphocytes AND adipocytes
• neutrophils, T and B lymphocytes Æ endocrine fashion
• adipocytes Æ 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
TTU AFS 4400/5500
11
10/30/2008
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
Satellite cell
Transdifferentiation
Proliferation
TTU AFS 4400/5500
Fuse
12
10/30/2008
In vitro marbling study
Proliferation
Satellite cell
(MUFA,PUFA) Long chain fatty acid
(Ciglitazone, Troglitazone, T-174) Thiazolidinedione
Horse Serum
Long chain fatty acid (PUFA)
Transdifferentiation
Differentiation
Gene experession during transdifferentiation
Muscle‐derived cell
Transdifferentiation
Differentiation
Preadipocyte
Early
Adipocyte
Intermediate
Late
Ligands
ADD1/
SREBP1
C/EBPα
Transcriptional activation
C/EBPβ
PPARÉ
RXR
Factors binding region
Leptin
SCD
Adipose gene
expression
FAS
TNF‐α
Functional Activation
TTU AFS 4400/5500
13
10/30/2008
Myogenin
( P < .001)
Arbit. Ratio
Arbit. Ratio
C/EBP β
( P = .08)
Arbit. Ratio
BSC transdifferentiation
PPAR γ
( P < .01)
BSC transdifferentiation and growth promotants
*
Arbit. Ratio
*
C/EBP β
* ( P < .03)
E2 : Estradiol
MGA : Melanegesterol Acetate
M+E: MGA + E2
Treatment
TTU AFS 4400/5500
14
10/30/2008
BSC transdifferentiation and growth promotants
Arbit. Ratio
*
PPAR γ
* ( P < .05)
E2 : Estradiol
MGA : Melanegesterol Acetate
M+E: MGA + E2
Treatment
Arbit. Ratio
BSC transdifferentiation and growth promotants
SCD
* ( P = .05)
E2 : Estradiol
MGA : Melanegesterol Acetate
M+E: MGA + E2
Treatment
TTU AFS 4400/5500
15
10/30/2008
Arbit. Ratio
BSC transdifferentiation and growth promotants
Myogenin
* ( P = .05)
E2 : Estradiol
MGA : Melanegesterol Acetate
M+E: MGA + E2
Treatment
BSC transdifferentiation and growth promotants
Positive Control : Insulin, Oleic acid, and Ciglitizone
20nM 17β trenbolone
TTU AFS 4400/5500
10nM estradiol
10nM Melengestrol acetate
16
10/30/2008
Muscle Biopsy Study
Muscle Biopsy Study
TTU AFS 4400/5500
17
10/30/2008
BSC transdifferentiation in Biopsy study
60
a
a
a
a
PPARγ mRN
NA,
relative abund
dance
C/EBPβ mRNA,
dance
relative abund
50
40
b
400
b
30
20
10
0
300
a
a
200
100
0
Day 0
Day 7
Day 14
Day 28
Day 0
500
b
a
a
300
Day 14
Day 28
400
Myogenin mRNA,
e
relative abundance
SCD mRNA,
e
relative abundance
b
400
Day 7
Muscle biopsy date
Muscle biopsy date
200
100
0
300
200
100
0
Day 0
Day 7
Day 14
Day 0
Day 28
Muscle biopsy date
Day 7
Day 14
Day 28
Muscle biopsy date
BSC transdifferentiation in Biopsy study
a
a
400
a
40
b
30
20
10
PPARγ mRNA, dance
relative abund
C/EBPβ mRN
NA, relative abund
dance
50
a
a
b
300
b
200
100
0
0
Cont
E2
TBA
Cont
T+E
E2
T+E
500
a
a
300
a
b
200
100
0
Myogenin mRNA, e
relative abundance
SCD mRNA, e
relative abundance
400
400
300
200
100
0
Cont
E2
TBA
Treatment
TTU AFS 4400/5500
TBA
Treatment
Treatment
T+E
Cont
E2
TBA
T+E
Treatment
18