10/30/2008
Origin of Adipocyte
Mesenchymal precursor
Determination
Myoblast
Myoblastic cell
Chondrablast
Chondrablastic cell
Preadipocyte
Adipocyte
Differentiation
Preadipocyte Differentiation
Gerrard and Grant, 2003
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Cell line for Studying Mature Adipocyte
• Pluripotent
p
fibloblasts : 10T1/2,, OP9,,
Balb/c, 3T3, 1246, RCJ3.1, and CHEF/18.
- Convert to several cell types
• Unipotent fibroblasts : 3T3-L1, 3T3-F442A,
1429, Ob 1771, TA1, and 30A5.
- Undergone determination.
3T3‐L1 Cell
3T3‐L1, 0 day
3 day
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5 day
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Adipocyte
• 3T3‐L1 Preadipocytes differentiate into WAT‐like adipocytes
(postnatally)
– grown in culture; when confluency is reached, they withdraw from cell cycle (growth arrest)
– can be induced into differentiation with combination of FBS, insulin, dexamethasome, isobutylmethylxanthine(cAMP phosphodiesterase
inhibitor)
– this process is often called “adipose conversion”
– characteristics of the adipose conversion process
characteristics of the adipose conversion process
• cell rounding
• accumulation of lipid droplets
Adipocyte Conversion
• Changes in gene expression during adipose conversion (100‐150 genes)
– massive
massive accumulation of triglycerides
accumulation of triglycerides
• induction of many proteins (e.g. fatty acid binding protein, lipoprotein lipase, phosphofructokinase)
– Dramatic change in cell morphology
• altered expression of cytoskeletal and extracellular matrix proteins
• cytoskeletal proteins decrease
• extracellular matrix proteins
p
–
–
–
–
–
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type I and III collagen‐decrease
Fibronectin‐decrease
type IV and II collagen‐increase
Laminin‐increase
Entactin‐increase
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Transcriptional Regulation
• Transcriptional Control of Adipocyte Gene Expression
–
–
–
–
–
–
C/EBP family
CCAAT/enhancer binding protein (enhances transcripts of CCAAT genes)
absent in preadipocytes but induced during adipocyte conversion
necessary for adipocyte differentiation
ectopic expression of C/EBP‐α in fibroblasts causes conversion of adipose
C/EBP‐α gene knockout mouse (‐/‐) results in no TG synthesis
• involved in later part of adipose conversion cascade
– C/EBP
C/EBP‐β
β and δ
and δ are expressed immediately following the induction of are expressed immediately following the induction of
differentiation and up‐regulate PPAR‐γ
Transcriptional Regulation
• Transcriptional Control of Adipocyte Gene Expression
Evan D. Rosen et al. Genes Dev. 2000; 14: 1293-1307
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Transcriptional Regulation
• Transcriptional Control of Adipocyte Gene Expression
G3PDH, FAS, SCD
/
β
C/EBPβ
C/EBPδ
C/EBPα
PPARγ
0
Exponential ‐2
Growth Confluence Clonal expansion
2
4
Growth
arrest
6
8
10
Acquire adipocyte
Phenotype (Differentiation)
Transcriptional Regulation
– PPAR‐γ
p
peroxisome
proliferator activated receptors‐gamma
p
p
g
PPAR‐γ induces C/EBP‐α
members of steroid/thyroid hormone receptor super family
form heterodimers w/ RXR family of proteins to become active
• restricted to adipocyte cell types
• increase dramatically early in adipogenesis (also in BAT)
• 2 isoforms (different promoter, same gene)
•
•
•
•
– PPAR‐γ1 (31 more AA’s longer)
– PPAR‐γ2 (very adipose tissue specific)
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Transcriptional Regulation
• ligands for PPAR‐γ are lipogenic
• PPAR‐γ is
is activated by fatty acids (polyunsaturated FAs, activated by fatty acids (polyunsaturated FAs
linoleic acid) and by prostaglandins (15‐deoxy Δ12,14 PGJ2) • Thiazolidinedione (TZD) – has highest affinity to PPAR‐γ
(class of anti‐diabetic drug)
• PPAR‐γ is regulated by serine phosphorylation
(phosphorylation causes adipocyte to remain in proliferative state)
proliferative state)
• PPAR‐γ remains high in mature adipocyte
Hormone mediated differentiation
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Other factors for differentiation
• Up
Up-regulatory
regulatory factors : prostaglandin II,
II
thyroid hormone, sodium butyrate,
ascorbic acid, aldosterone, arachidonic
acid, AD4743, bezafibrate, pioglitazone, 3deazaadenosine.
• Down-regulated factors : TNFα, TGFβ,
retinoic acid.
Transcription Factors cont’d.
• Retinoic Acid
– Beta‐carotene Æ Vitamin A Æ Retinoic Acid)
– inhibits adipose conversion (differentiation process) of 3T3‐L1
– 3T3‐L1 and other preadipocytes express retinoic acid and mediate inhibitory response
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Functions of adipocytes
Bovine Adipose Tissue
• Bovine Adipose Tissue
– Prenatal (last 1/3 of gestation)
• cells
ll in
i state
t t off proliferation
lif
ti
• preadipocytes located in the stromal vascular
tissue go through many proliferative divisions
• preadipocytes later exit cell cycle and enter the
early phase of differentiation (one marker is SCD)
– during this early phase we observe initial
lipid filling
» In vivo, proliferation and differentiation
can occur concurrently even in more
mature cattle (14-18 mos.)
ÆÆreferred to as “bimodal distribution”
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Postnatal Adipose Tissue
• Subcutaneous Adipose Tissue
– Stearoyl Coenzyme A Desaturase (SCD) begins
increasing prior to weaning (5 mo)
– Subcutaneous adipose tissue in younger calffed have greater rates of preadipocyte
proliferation as compared to yearling-fed steers
– SCD activity was also greater in young calff d
feds
Stearoyl CoA desaturase
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Stearoyl CoA desaturase (SCD)
• May promote de novo fatty acid synthesis
and hypertropy of bovine adipocytes.
• Regulates TAG, CE, and VLDL synthesis.
• Controls membrane fluidity.
• Decreases lipid oxidation by activating
PPARα.
Stearoyl CoA desaturase
• SCD is sensitive to environmental factors:
- PUFA, cholesterol, Vit A, hormones
(insulin, glucagon), developmental
processes, temperature changes, and
thiazolidinedione.
• High SCD activity causes health disorders:
- diabetes, arteriosclerosis, cancer, and
obesity.
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SCD Protein Structure
Man, W. C. et al. J. Biol. Chem. 2006;281:1251‐1260
SCD activity
• Melting
16 : 0
point Increases.
• Hard fat.
CE
Stearoyl CoA
Desaturase
Microsome
18 : 0
• Palatability decreases.
(Camfield et al., 1997)
ACAT
DGAT
16 : 1
TAG
PL
• Melting
point decreases.
• Soft fat.
18 : 1
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• Palatability improves.
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Postnatal Adipose Tissue
• Intramuscular A.T.:similar but less
pronounced
• Intramuscular Fat (marbling, interfascicular)
– distinguishable from other fat deposits due to its location within perimysial connective tissue, along side myofibers
– rates of fatty acid biosynthesis are different (IM vs. SubQ)
– glucose contributes to a greater degree in I.M. vs. SubQ adipose tissue
– IM and SubQ are metabolically different
Postnatal Adipose Tissue
The contributions of
acetate, lactate,
and glucose to de
novo fatty acid
biosynthesis in
i.m. and s.c.
adipose tissues
(Smith and
Crouse, 1984)
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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
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)
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P-value
Endpoint : P<.01
B*E : P<.05
B*D : P<.05
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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
U.S. and Japanese
Hay Wagyu
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
Time on Feed (mo)
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P-value
Diet : P=.06
Endpoint : P=.07
D*E : P=.05
B*E : P=.01
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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
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