Insulin Signalling

1
Insulin Signalling BCH 2021, November
2006 Amira Klip The Hospital for Sick Children,
Toronto Amira_at_sickkids.ca
2
Our brain is the main glucose consumerbetween
meals
Blood glucose
Liver (glucose store)
brain
Fat cells
pancreas
All other organs
Skeletal muscle
3
Dietary glucose is stored in the liver, muscle
and fat cells
Blood glucose
liver
brain
Fat cells
pancreas
insulin
Skeletal muscle
4
Pancreas, liver, fat, muscle and brain determine
glucose homeostasis
Blood glucose
insulin
Adapted from Nature 414, 2001
Skeletal muscle
5
Insulin directly acts on liver, muscle and fat
Liver
Adipose tissue
Muscle
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The insulin receptor dimer
From Chang et al (2004) Mol Med 1065
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Insulin receptor conformation
From Yip and Ottensmeyer (2003) JBC 278 27329
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Insulin and Insulin-like Receptors
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Structure relationships of IRS family
IRS2 human Irs2 mouse DroIRS drosophila (chico)
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Levels of insulin action
Acute glucose and lipid metabolism
Intermediate Proteins synthesis
Long-acting Gene expression
11
Insulin receptor constitutive dimer
IRS-1,2,3,4
PM
NPXY
PH
PTB
IIIIIIII
adaptor
PHIP
(pY)
(pY)n
Grb
GEF
shc
Sos
Small G
Ras
Raf
MEK
Ser/thr kinases
Tyr/thr kinase
ERK
P90 rsk
gene expression
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Insulin receptor
PM
IRS-1,2,3,4
p85
(pY)
Grb
shc
PI kinase
Sos
p110
Ras
Ser/thr kinases
PDK1,2
PKB/ Akt1,2,3
-
Raf
Gsk-3
mTOR
MEK
-
ERK
eIF-2B
eIF-4E
4E-BP1
P90 rsk
-


mRNA translation and protein synthesis
13
Regulation of metabolism by insulin
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Insulin stimulates glucose uptake into muscle and
fat tissues, via GLUT4
GLUT4 is the major insulin-responsive glucose
transporter
Pancreas
15
Glucose Transporter (GLUT) Family (SLC2)
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Glucose enters muscles through GLUT4
PM
PM
cytosol
cytosol
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GLUT4 continuously recycles, insulin increases
GLUT4 at the surface
exocytosis
What are the signals involved?
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Insulin receptor
PM
IRS-1,2
p85
(pY)
Grb
shc
AS160 is a Rab-GAP
PI kinase
Sos
p110
Ras
Ser/thr kinases
PDK1,2
PKB/Akt1,2,3
Raf
AS160
MEK
eIF-4E
mTOR
ERK
eIF-2B

4E-BP1
P90 rsk

-
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Signal transduction versatility, ups and downs

• isoforms
• cellular location
• intensity (amplitude)
• duration
• frequency (transient or sustained)
• feedback regulation ( or - )

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siRNA-mediated knock-down of IRS-1 or IRS-2
Huang, Thirone et al (2005) J Biol Chem 280 1226
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IRS-1, but not IRS-2, is required for
insulin-induced GLUT4 translocation
siRNA to IRS-1
siRNA to IRS-2

100
100
80
80

of maximal response
60
of maximal response
60
40
40
20
20
0
0
Insulin (nM)
Insulin, nM
5
100
100
5
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IRS-1, but not IRS-2, is required for
insulin-stimulation of glucose uptake
Glucose uptake
siRNA IRS-1
siRNA IRS-2

100
100
80
80

60
60
of maximal response
of maximal response
40
40
20
20
0
0
Insulin (nM)
100
5
Insulin (nM)
100
5
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IRS-1 and IRS-2 have complementary but not
redundant functions in muscle cells
IRS-1
IRS-2
PI 3-kinase
PI 3- kinase
p38
ERK
Akt1
Akt2
actin
Mitogenic pathway
Lipid metab, liver muscle
GLUT4 translocation
Glc metab, liver
Huang, Thirone et al (2005) J Biol Chem 280 1226
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Insulin signallingan integrated circuit
Thong et al, (2005) Physiology 20271
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The circuit has built-in feedback inhibition
P
P
Pser
Pser
IRS-1
Pser
Pser
Rho Kinase
Pser
pY pY
PI 3-kinase
p85
p110
Grb2 SOS Ras Raf MEK ERKs
MKKK MKK JNK
AMPK
PDKs
mTOR
S6Kinase
PKCl/z
Akt/PKB
GSK3
IL-1 R
mPLK
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All roads lead to IRS-1
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Additional negative regulation
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AS-160 a Rab-GAP
S588
S341
T642
S318
S751
S570
R973
119
189
357
439
1
917
1136
1299
4P mutant S318A, S588A, S751A, T642A cannot be
phosphorylated Rab-GDP prevails
GAP domain
PTB
PTB
As per Lienhard et al
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Insulin-induced GLUT4-vesicle translocation and
fusion in rounded-up myoblasts
Basal
Insulin
Intact cells
surface Myc
Permeabilized cells
Myc
C-terminus
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AS160 mutant 4P prevents the insulin and
PI-3,4,5-P3-induced GLUT4 translocation
3.0
untransfected
2.5
4P
2.0
Cell surface GLUT4myc (fold over basal
untransfected)
1.5
1.0
0.5
0.0
Basal
100 nM Insulin
10 ?M PIP3Carrier
Hypertonic Sucrose
Thong et al, unpublished
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AS160-4P allows significant GLUT4 arrival, but
eliminates GLUT4 fusion
Varinder Randhawa, Alex Cheng
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Insulin signals leading to GLUT4
Insulin Receptor
P
P
IRS-1
P P P P
PI 3-Kinase
PDK
P P
P P
GLUT4 translocation
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Recurrent themes in insulin signalling
ATP expender
ATP provider
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Is that all it takes? If not complicated enough
a new signalling pathway involving
APS-Cap-Cbl-C3G-TC10 was proposed (Saltiel and
Pessin)
Actin dynamics and GLUT4 translocation
35
A second signalling pathway?
Insulin Receptor
P
P
IRS-1
P P P P
p85
p110
PI 3,4,5-P3
PDK
Akt
AS160
GLUT4 translocation
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CAP-Cbl-TC10 do not participate in actin
remodelling in myoblasts
IR
CAP Cbl TC10

• CAP is expressed only in myotubes (not
  myoblasts)

• Cbl is phosphorylated in response to insulin
  only in myotubes

• Endogenous TC10 is not detectable with available
  antibodies

• Transfected TC10 is GTP-loaded in myoblasts and
  myotubes
• stimulated with insulin

• TC10 mutants do not prevent insulin-dependent
  actin
• remodelling into a mesh, nor GLUT4
  translocation

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Insulin signalling pathway
Insulin Receptor
P
P
IRS-1
P P P P
p85
p110
PI 3,4,5-P3
PDK
Akt
AS160
GLUT4 translocation
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Insulin-dependent actin filament remodelling
Basal
Insulin
Myotubes
Myoblasts
Rounded-up myoblasts
Acta Physiol Scand 178 297-308 (2003)
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Disrupting actin polymerization prevents
insulin-dependent GLUT4myc externalization
2.5
Basal

• Insulin effect also prevented by
• Jasplakinolide
• Swinholide A

Insulin
2
1.5
Cell surface GLUT4myc
1
0.5
0
Control
Cyto D
Latrunculin B
Adapted from J. Clin. Invest. 108 371-381
(2001) And Mol. Biol. Cell,
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Insulin signalling pathway
Insulin Receptor
P
P
IRS-1
P P P P
p85
p110
PI 3,4,5-P3
PDK
Akt
AS160
GLUT4 translocation
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Rho GTPases

• General Biological Roles
• Cell cycle regulation
• Vesicle traffic
• Morphogenesis
• Cell migration
• Actin remodelling
• GTPases in insulin signalling
• RhoA (fibroblasts)
• Cdc 42 (adipocytes)
• TC10 (adipocytes)
• Rac (muscle)

TC10
Takai et al 2002
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Rac activation in response to insulin
Serum depletion
Stimulation (100 nM Insulin) lysis
hPAK1B CRIB aa56-272

GST
Lellean JeBailey
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DN Rac inhibits insulin-induced actin
remodelling and GLUT4 translocation
DN-Rac
Actin
Basal
Fold GLUT4myc Translocation
Insulin
Lellean JeBailey
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Rac1 knockdown Reduces GLUT4 translocation
2.5
2.0
1.5
Fold GLUT4 Translocation
1.0
0.5
0
Adapted from JeBailey et al (2006) Diabetes in
press
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siRac 1 Inhibits PAK, LIMK but not Akt Activation
Insulin - - -
siUR - - -
- siRac 1 - - - -

ib Rac
ib p-Pak Thr 423
ib p-Pak Ser 199
ib p-LIMK
ib p-Akt Ser 473
ib Actin
Lellean JeBailey
46
Insulin signalling pathway
Insulin Receptor
P
P
IRS-1
P P P P
p85
p110
PI 3,4,5-P3
PDK
Akt
AS160
GLUT4 translocation
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Insulin signalling pathway
Insulin Receptor
p85
p110
PI 3,4,5-P3
PDK
AS160
GLUT4 translocation
48
Insulin signalling pathway
Insulin Receptor
p85
p110
PI 3,4,5-P3
PDK
AS160
GLUT4 translocation
49
Thank you!