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 eceptor -p

G ene expres s ion

FKH R -pC R EB-p

Ak t

P I3K

IR Ss

.

.

.

MAPK

R af

R as

sos

G rb2

shc

L igand(Ins ulin/IG 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

Result

Insulin 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.