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Regulation of protein phosphorylation by
insulin/IGF-1
Yang,Yu-Ying Tseng, Yu-Hua C.Ronald Kahn
Insulin
Insulin an anabolic hormone with strong metabolic
effects. If concentration of glucose is too high, then insulin will be released to metabolize the glucose. When it binds to its receptor, the receptor will autophosphorylation, activate a family of IRS proteins, and lead the transmitting the signal downstream.
IGF-1
IGF-1 insulin-like growth factor 1,has it’s own
receptor.It has a similar signal transduction pathways to insulin’s. IGF-I mediates many actions of growth hormone, and it stimulates cell replication, cell differentiation and the synthesis of cellular products. As for their biological effects, in general, IGF-1 is mainly responsible for mitogenic effects
Insulin/IGF-1 pathway
r e c e p t o r - p
G e n e e x p r e s s i o n
F K H R - pC R E B - p
A k t
P I 3 K
I R S s
.
.
.
M A P K
R a f
R a s
s o s
G r b 2
s h c
L i g a n d ( I n s u l i n / I G F - 1 )
IRS(insulin receptor substrate)proteins
IRS-1 its deficiency causes growth retardation and
insulin resistance, which will lead to type 2 diabetes.
IRS-2 insulin resistance, lead to serious diabetes
when it absents
IRS-3 no glucose metabolism or growth problems
when it is absent
IRS-4 slight defects in growth, reproduction, and
glucose homoeostasis
Methodbrown pre-adipocytes from different IRS KO miceWestern blotting
-- sample preparation cell + <insulin/IGF-1> for <5mins/30mins> scrap cell normalize cell -- loading gel-- transfer-- immunoblotting-- ECL
0 5 30 0 5 30 0 5 30 0 5 30 0 5 30 0 5 30
Wt IRS-1 KOIRS-3 KO
m2IRS-1,3 KO IRS-2 KO
IRS-3 KOm1
ST5
(min)
CREB-pATF-p
0 5 30 0 5 30 0 5 30 0 5 30 0 5 30
Wt IRS-1 KOIRS-1 KO+ IRS-1
IRS-1 KO+ IRS-2
IRS-1 KO + IRS-4
IRS-1 KO + IRS-3
0 5 30
CREB-pATF-p
ST6
(min)
Creb-p Insulin (100 nm)
0 5 30 0 5 30 0 5 30 0 5 30 0 5 30 0 5 30
Wt IRS-1 KOIRS-3 KO
m1IRS-1,3 KO IRS-2 KO
IRS-3 KOm2ST5
(min)
CREB-pATF-p
Creb-p (IGF-1 100nm)
0 5 30 0 5 30
Wt IRS-1 KOST6
(min)0 5 30 0 5 30
IRS-1 KO+ IRS-1
0 5 30 0 5 30
IRS-1 KO+ IRS-2
IRS-1 KO+ IRS-3
IRS-1 KO+ IRS-4
CREB-pATF-p
Akt-Juk
(min)
IGF-1 (100 nm)
ST 4
0 5 30 0 5 30 0 5 30 0 5 30 0 5 300 5 30 0 5 300 5 30
Wt IRS-1 KO IRS-2 KO IRS-1,3 KO P85a KOP85a KO+ P85a
IRS-1 KO+ IRS-1 IRS-3 KO
Akt
Jnk
(min)
Insulin (100 nm)
ST 4
0 5 30 0 5 30 0 5 30 0 5 30 0 5 300 5 30 0 5 300 5 30
Wt IRS-1 KO IRS-2 KO IRS-1,3 KO P85a KOP85a KO+ P85a
IRS-1 KO+ IRS-1 IRS-3 KO
Akt
Jnk
ResultInsulin and IGF-1 almost have the same stimulative efficiency.
Compare with Wt cell, the KO cells did have lower efficiency in signaling.
IRS1+3KO cells didn’t lower much efficiency in signaling.
Akt-Juk shows stronger bands than Creb-p in the same time period stimulating.
Conclusion
both insulin and IGF-1 can cross-react with each other's receptor when used at high dosages.Using differentiation protocols, both with and without insulin, preadipocyte cell lines derived from IRS-1 KO mice exhibited a marked decrease in differentiation and protein
accumulation (10 to 40%) compared to wild-type cells (90 to 100%).
IRS-1KO gene maybe complementary to IRS-2, IRS-3 gene, but not IRS-4 gene.
IRS-1KO and IRS-3 KO cell didn’t result in much deficiency in signaling, maybe there’s other pathways for the insulin/IGF-1 signaling.