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

10/30/2008

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