Adiponectin

Adiponectin
‣ ADIPOQ gene product
‣ 244 amino acid
‣ Specifically and highly expressed
in adipose tissue
‣ The most abundant secretory
protein of adipose tissue in human
plasma
2

Plasma adiponectin levels
Negatively correlated
‣ fasting plasma insulin
‣ triglyceride
‣ glucose
‣ postprandial glucose
levels
Positively correlated
‣ insulin-stimulated
glucose utilization
‣ lipoprotein metabolism
‣ insulin-mediated suppression
of postprandial FFA release
“insulin sensitivity”
adiponectin - obese humans
- type 2 DM patients
3

Adiponectin actively
influences metabolic
processes
‣Increases
fatty acid oxidation (Fruebis et al.)
glucose utilization (Yamauchi et al.)
‣Decreases
insulin resistance
hepatic glucose production
4

Kissebah et al.
Chromosome 3q27
(adiponectin gene located)
Vionnet et al.
Insulin resistance syndrome
(Europeans)
Diabetes susceptibility locus in 3q27
(native French)
5

Endothelin-1
6

Signal transduction pathways
of Endothelin-1
short-term:
DAG
long-term: ET-1 may act through MAPK cell proliferation
& differentition
7

Endothelin-1
‣ Regulation of metabolic events
‣ inhibits adipocyte differentiation
‣elevated ET-1 plasma levels
• diabetes
• obesity
• insulin resistance
Hypothesis: downregulation of
adiponectin expression
8

Objective
To explore the regulatory
effects of ET-1 on
adiponectin expression
and secretion and the
underlying mechanisms in
3T3-L1 adipocytes
9

Materials
and Methods
10

Cell culture
3T3-L1 fibroblasts
(American Type Culture Collection, Rockville, MD)
adiponectin
gene expression
assay
(10 cm Petri dishes)
adiponectin
secretion
assay
(12-well plates)
grown and maintain in
Dulbecco’s modified Eagle (DME)
High glucose, 100 units/ml of penicillin,
100 µg/ml of streptomycin, 10% fetal bovine serum
and 10% CO 2
11

Differentiation procedure
Cell were grown to 2 days
IBMX
Dexamethasone
Insulin
Incubation (3 days)
Incubation (3 days)
Insulin
Changed medium every 3 days
determined by staining Oil Red O
12

Effect of ET-1 on adiponectin secretion
3T3-L1
incubated 6 h in DMEM-BSA
(absence of serum)
washed
10-7 M ET-1
10-7 M ET-1
1-24 h
aspirated centrifuged measured APN
(ELISA kits) 13

Effect of ET-1 on adiponectin secretion
serum free-treated adipocyte
preincubated 1 h
Inhibitor
/
Receptor blocker
Inhibitor /
Receptor blocker
wash 3 times
incubated with 10 -8 M ET-1 4 h
(presence/absence these inhibitor or receptor blocker)
determined adiponectin secretion
14

Adiponectin mRNA extraction
Tri Reagent® kit
1% agarose gel electrophoresis
UV light absorbance at 260 nm
30 min
37°c
incubated with RNase-free DNase I
inactivated DNase
10 min
100°c
15

RT-PCR
RNA template Heated, 5 min, 70°c
RT ----> total RNA, RTase, poly (dT) 12-18 primer, dNTP,
human placental ribonuclease inhibitor
100°c, 10 min
PCR ----> RT template solution, Vent DNA polymerase,
dNTP, primers adipoQ-5’ & adipoQ-3’
1.5% agarose gel electrophoresis 0.83 kb cDNA of adiponectin gene
16

Northern blot analysis
Adiponectin PCR products
cDNAs probe ( 32 P)
Total RNA
Northern blot analysis
GAPDH mRNA normalized values
17

Statistical analysis
‣ mean ± SD
‣ one way ANOVA, P

Results
19

Time-Effect of ET-1 on adiponectin mRNA levels
10 -7 M ET-1
20

Dose-Effect of ET-1 on adiponectin mRNA levels
21

ET-1 acts through ET A R to decrease
adiponectin mRNA levels
22

ET-1 decreases adiponectin mRNA levels
through ERK-dependent pathway
23

Role of ERK activation on ET-1-downregulated
adiponectin mRNA levels
24

ET-1-mediated decrease in adiponectin
mRNA levels is not due to degradation of
adiponectin mRNA
With ET-1
Without ET-1
25

Effect of ET-1 on adiponectin secretion
without ET-1
with ET-1
26

Effect of ET-1 on adiponectin secretion
27

Acute ET-1 treatment stimulates adiponectin
secretion via IP 3 /Ca 2+ pathway
28

Acute ET-1 treatment stimulates adiponectin
secretion via IP 3 /Ca 2+ pathway
29

ERK activation was not involved in the
ET-1 stimulated adiponectin release
30

presence
absence
2-APB
2-APB
presence
absence
presence
absence
ET-1
ET-1
ET-1
ET-1
31

2-APB
ET-1
ET-1
2-APB
ET-1 significantly decreased adiponectin mRNA levels
but 2-APB cannot prevent ET-1 downregulated
adiponectin mRNA expression
IP 3 /Ca 2+ pathway was not involved in the ET-1
downregulated adiponectin mRNA expression
32

Discussion
33

Major finding of this study
ET-1 has regulatory effects on
adiponectin expression
and secretion
via two different signaling pathways
34

Adiponectin gene expression
PD98059
ERK-dependent
pathway
ET-1 + ET A R
adiponectin mRNA
35

Adiponectin secretion
Short-term
2-APB
ET-1
IP 3 /Ca 2+ pathway
adiponectin secretion
36

Adiponectin secretion
Long term
ET-1
Downregulation adiponectin mRNA
synthesis of adiponectin and
intracellular adiponectin content
levels of adiponectin secretion
37

Bedi et al.
ET-1 did not regulate adiponectin gene expression
chronic ET-1 treatment significantly decreased
adiponectin secretion through PIP 2 modulation
of actin cytoskeleton.
38

Bedi’s study,
ET-1
did not effect adiponectin mRNA levels
in 3T3-L1 adipocytes.
Real-time RT-PCR
This study,
ET-1
adiponectin mRNA levels
in 3T3-L1 adipocytes.
Northern blot analysis
39

Bedi et al.
Chronic ET-1 treatment significantly decreased adiponectin
secretion through post-transcriptional regulation
via
interfering adiponectin packaging
and/or vesicular trafficking
decreased intracellular adiponectin content partially
explain why prolonged ET-1 treatment inhibited
adiponectin secretion in 3T3-L1 adipocytes
40

ET-1 interferes with several metabolic activities
hepatic glucose
production
(Roden M et al)
glucose uptake in skeletal
muscle and adipocytes
(Yang XY et al, Usui I et al)
41

Lee YC et al,
ET-1
ET A R
impairs insulin-stimulated
glucose uptake in rat adipocytes
In vivo studies,
ET-1 causes systemic insulin
resistance
in rats and human
(Juan CC et al, Ottosson-Seeberger A et al, Wilkes JJ et al)
ET-1 stimulates lipolysis in 3T3-L1
adipocyte
(Juan CC et al)
42

In obesity and diabetes, circulating ET-1 levels
are significantly increased
(Ferri C et al, Takahashi K et al)
lipolysis and free fatty acids release
FFA-induced insulin resistance
in humans
43

Adiponectin
insulin-sensitizing adipocytokine
insulin sensitivity
(Satoh H et al, Fu Y et al)
44

In the present study,
ET-1
adiponectin mRNA levels
in 3T3-L1 adipocytes
ET-1 may suppress adiponectin and
aggravate insulin-resistance
in obesity and type2 DM
45

Adipocyte-derived adiponectin
• paracrine effect on the cardiovascular system
• suppressing the development of cardiac
hypertrophy and atherosclerosis
(Shibata R et al, Okamoto Y et al)
46

In obesity and diabetes
plasma ET-1
+
cardiovascular system is highly
infiltrated with adipose tissue
suppresses adiponectin expression
protective effects on the cardiovascular system
indirectly accelerates the progression
of cardiovascular diseases
47

ET-1
adipocyte differentiation
(Hauner H et al, Shinohara O et al)
insulin-stimulated glucose uptake
(Usui I et al, Lee YC et al)
lipoprotein lipase activity in adipocytes
(Ishida F et al)
stimulate lipolysis in adipocytes
(Juan CC et al)
48

ET-1
stimulates leptin production
(Xiong Y et al)
in 3T3-L1
resistin release
(Zhong Q et al)
Thus, ET-1 can directly or indirectly
modulate adipocyte functions
49

Dysregulation of the action of ET-1 on adipocytes
disrupt body energy homeostasis
metabolic disorders
50

In the present study,
ET-1
ET-1
ERK-dependent
pathway
suppressed adiponectin gene expression
+
Juan CC et al, previously report
stimulated ERK activation
lipolysis in adipocytes
signaling pathways leading to ERK activation are
important in adipocyte physiology
51

Bost et al.
essential and specific role of the ERK
pathway at each step of adipogenesis from
embryonic stem cells to adipocytes
52

Abnormal modulation of the
ERK pathway
affects adipogenesis
obesity
53

Conclusion
ET-1 has distinct regulatory effects
on adiponectin expression and
secretion via two different signaling
pathways
Adiponectin gene expression
ERK dependent
pathway
Adiponectin secretion
IP 3 /Ca 2+ pathway
54

Conclusion(cont.)
‣ Support the pathophysiological role
of ET-1 in adipocyte function
‣ Dysregulation of the effect of ET-1 on
adipose tissue may contribute to the
pathogenesis of metabolic disorders
55

Conclusion(cont.)
Cardiovascular system
regulates expression of adipocytokines
and energy homeostasis
via
vasoactive factors e.g. ET-1
56

THANK YOU
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