How does insulin work?
(~1984)
Pierre De Meyts = Chuck;
head of research at Novo
Nordsk
1Aug
How does insulin work: functional pathways?
Insulin
Insulin Receptor
Activates a series of downstream signaling
cascades, leading to pleiotropic effects on:
LIVER, MUSCLE, FAT
Glucose
Transport
Cell
Growth
Fat, Muscle
Muscle,
Liver
+ Glycogen Synthesis
- Gluconeogenesis
Lipid
Metabolism
Gene
Expression
Protein
Synthesis
Size
Survival
Mitogenesis
How can all of this occur mechanistically?
How does insulin work: mechanistically ~’95
Insulin Receptor
Inactive
Ras
Step 1 Binding of insulin
causes phosphorylation of
receptor cytosolic domain and
of IRS-1
Step 3 Sos promotes
GDP
GTP
dissociation of GDP from Ras;
GTP binds and Sos dissociates
from Ras
Active Ras
Step 2 Binding of GRB2 to
IRS1 and Sos to GRB2 couples
insulin signal to inactive Ras
MAP kinase
cascade
Q: why then doesn't EGF mimic insulin actions in cells that have both receptors?
Redrawn from Lodish ‘95
Model for Insulin Mechanism of Action
~1996
a
a
THM: both
pathways are
real and
important
ATP
IRS-1/2
YP
Grb-2/SOS
Ras/Raf
- Mitogenesis
- Protein synthesis
- Amino acid uptake
- Fatty acid synthesis
- DNA synthesis
- Lipolysis
- Glycogen
metabolism
YP
PI3-kinase
PI-3,4-P2
MEK
X
X MAPK
PKB kinase(s)
(PDKs)
Akt/PKB(s)
Glucose
Transport
Structural features of IRS-1 that Mediate
Pathway(s)
Insulin
Receptor?
Membrane
Association
Binds to NPEpY
motif in insulin
receptor
Grb2
PH
PTB
Y608MPM
P85 of PI3K
Y895VNI
Y939MNM
Y1172IDL
Y1222ASI
SHP-2 Ptase
Redrawn from M. White
Multiple Phosphotyrosines in IRS1 & 2
Comparison of IRS1 and IRS2 protein sequences, including the relative location of the amino-terminal
pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains, and numerous known phosphorylation
sites revealed by MS/MS or expected tyrosine phosphorylation sites. The amino acid sequences surrounding
tyrosine sites are shown, and motifs conserved between IRS1 and IRS2 are coded with a similar background
color. The kinase regulatory loop-binding (KRLB) domain in IRS2 is indicated by a yellow box, which includes
6
the tyrosine residue that binds in the ATP binding pocket (Y621).
From Morris White Review 2010
The insulin signaling network ~’06
Insulin signaling
impacts many cellular
processes, including
the metabolism of
glucose, protein, and
lipids, as well as cell
growth and
differentiation.
GAB-1
P85
p110
Thus, the insulin
signaling network is
broad and complex.
7
Biddinger & Kahn Ann Rev Physiol '06
Insulin Receptor tyrosine phosphorylation
initiates several regulatory cascades
Insulin
pY
Insulin Receptor (Tyr kinase)
pY
Shc pY
Grb2
pY APS CAP
CG3
TC10
Cbl
Crk-II
• MAP kinase pathway Grb2/So
pY
pY
IRS-1
Cell proliferation
Muscle/Fat
Physiological
Effects
IRS-2
s/Ras
Glucose transport
Liver/Brain
MAJOR INSULIN ACTION ROUTE
PI3K pathway
Physiological
Effects
Molecular mechanisms of
IR/IGFR Responses,
current
9
Simplified representation of Phosphitidly Inositols and
the point of action of PI3K
How does insulin
influence this
pathway?
ATP
P
PI3K
ADP
From Alessi et al
IRS can bring PI3K to membrane;
but how does this lead to activation
of Akt/PKB?
Activation
mechanism for
PKB by 3,4,5phosphatidylinositol lipids
Note AKT = PKB
THM: Both have to be
brought to membrane
bound PIs
Note: nearly all ser/thr
kinases have to be
PO4 on their
“activation loop”
What are some of the important
pathways that are regulated by
Insulin?
Example of glucose transport in a cell
type specific manner
How does ( a ) Resting cell
Insulin
Regulate
Glucose
Transport?
Glucose
Glut-1
Transporter
Insulin
Insulin
Receptor
Intracellular vesicles
Glut-4
Transporter
Insulin
binds
( b ) Insulin- stimulated
Exocytosis
PI3K/PKB
activation
required, but
not sufficient
for this
process
Redrawn from Lodish ‘95
Exocytosis of glucose
transporter molecules
(mechanism unknown))
( c ) Insulin removed
Endocytosis
On removal of insulin, endocytosis
removes glucose transporter
molecules from plasma membrane
and deactivates remaining
transporters
Insulin signaling regulates GLUT4 exocytosis by
engaging 2 pathways in the “trafficking machinery”
The APS (adaptor protein with pleckstrin homology (PH) and Src homology 2 (SH2) domains)–insulin signaling cascade is initiated when the
activated IR recruits the adaptor proteins APS, c-CBL and c-CBL-associated protein (CAP) (a). IR-catalyzed Tyr phosphorylation of c-CBL
triggers recruitment of the adaptor protein CRK and the guanine nucleotide exchange factor (GEF) C3G to the plasma membrane (PM),
where C3G activates the small GTPase TC10. GTP-bound TC10 interacts with the exocyst complex, thereby creating targeting sites for the
glucose transporter type 4 (GLUT4) storage vesicle (GSV) at the plasma membrane. TC10 also interacts with CDC42-interacting protein 4
(CIP4), which is associated with the RAB5 and RAB31 GEF GAPEX5. Translocation of GAPEX5 to the cell surface modulates the activation
state of its target small GTPases, which are involved in GSV retention and translocation. The insulin receptor simultaneously initiates the
phosphoinositide 3-kinase (PI3K)-dependent signaling cascade by phosphorylating IR substrate (IRS) proteins, thus producing docking sites
for the recruitment and activation of PI3K (b). This kinase converts phosphatidylinositol-4,5 -diphosphate (PIP2) to
phosphatidylinositol- 3,4,5-trisphosphate (PIP3), which serves as a platform for recruitment of phosphoinositide-dependent kinase 1 (PDK1)
and AKT. When at the PM, AKT is phosphorylated by PDK1 and mammalian target of rapamycin complex 2 (mTORC2), which results in AKT
activation. AKT promotes GSV exocytosis by phosphorylating and inactivating two GTPase-activating proteins (GAPs), AS160 and
15the RAL–
GAP complex (RGC, consisting of a regulatory subunit (RGC1) and a catalytic subunit (RGC2)), which regulate small GTPases that are
involved in GSV retention and targeting, respectively.
Leito & Saltiel Nature Reviews Molecular Cell Biology 13, 383-396 (June 2012)
Pathway of Glycogen formation from
glucose - effects of insulin
Glucose
Glut-4
Glucose
G-6-P
glycolysis
Skeletal
muscle only
Insulin
PKB
glycogenin/branching
enzymes)
UDPGlucose
Kinase
(GSK-3)
THM: PKB inhibits PO4 of GSK3,
ie "inhibits the inhibitor"
Glycogen
Glycogen
synthase
(active)
Glycogen
synthase-P
(inactive)
Phosphatase(s
)
Unique substrate specificity of GSK3
THM: often a priming phosphorylation – Why is GSK a substrate?
Nature Reviews Molecular Cell Biology 2; 769-776
Does this also give us a hint how
phosphorylation of GSK3 by PKB
inhibits activity?
Yes, PO4 interferes with substrate binding
The molecular mechanism by which PKB
phosphorylation inhibits GSK3
In the absence of insulin, GSK3 is fully active. In this state, substrates that already have a 'priming phosphate' bind to a specific pocket, aligning
it such that GSK3 can phosphorylate a serine located four residues amino-terminal to the priming phosphate. After agonist stimulation, GSK3
becomes phosphorylated at a serine residue near its amino terminus, (Ser21) in GSK3a and Ser9 in GSK3b. This transforms the amino
terminus into a 'pseudosubstrate' inhibitor, the phosphoserine occupying the same binding site as the priming phosphate of the substrate, and
blocking access to the active site. GSK3 does not phosphorylate its own amino terminus, because residue 17 in GSK3a and residue 5 in GSK3b
are not serine or threonine. As a result of phosphorylation by PKB/AKT, GSK3 activity is inhibited, and its substrates are then dephosphorylated
by protein phosphatases. By contrast, the priming phosphate tends to be resistant to dephosphorylation, and the protein kinases that
19
phosphorylate these sites are often not regulated by extracellular signals. So, substrates are already primed and available for phosphorylation
by
GSK3 when it becomes reactivated after agonist stimulation has ceased. Arginine 96 (R96), R180 and lysine 205 (K205) are the key residues
involved in binding the priming phosphate and the phosphorylated amino terminus.
How do we determine the
relative importance of the
various pathway components?
Conditional and Tissue-specific
Mammalian gene disruption
Cre-loxP tissue-specific gene targeting
Cre
gene
X
IRS
loxP site: ATAACTTCGTATAATGTATGCTATACGAAGTTAT
Cre
gene
IRS
No Cre gene
Expression
No Cre protein made
Make transgenic animal with
gene of interest flanked by
specific loxP sequences
The Cre enzyme recognizes a
sequence motif of 34 bp, called
loxP. If the target gene is flanked
by two loxP sites in the same
orientation, Cre protein excises
the intervening target gene.
Cre
gene
Cre protein
Cre-mediated
recombination
IRS
IRS
IRS
Tissue-specific deletion of the
target gene is generated by
crossing the mutant mice
harboring the target gene
flanked by two loxP sites to
various strains expressing Cre
protein in tissue-specific manner
No IRS made
Wildtype +/+
KO or tissue specific KO -/depending on Cre expression
Now additional options, eg.
21
Tamoxifen or tet on/off systems
Serum Glucose/Insulin Effects of IRS-1 Disruption
Glucose Tolerance Curve
Blood Glucose/Insulin
500-
100-
-20
80-
60-
-10
4020-
Blood glucose (mg%)
120-
Plasma Insulihn(uU/ml)
Blood glucose (mg%)
30
-0
0Control
KO
Glucose
Control
400-
KO
300200-
0-
KO
Control
100-
Insulin
|
0
30-
Control
20-
10-
0-
|
|
|
|
|
30
60
90
Time after glucose injection (min)
Glucose Uptake in Adipocytes
Blood glucose (%fasting)
Glucose uptake(nMol/cell/min)
NOT Diabetic !
|
KO
|
0
|
|
|
|
|
|
30
60
90
Time after glucose injection (min)
|
120
|
120
Glucose response to IGF-1
100-
KO
50Control
0- |
0
|
|
|
|
|
|
|
30
60
90
120
Time after glucose injection (min)
Redrawn from Araki & Kahn Nature 372:Nov 10
Irs1-/-/Irs3-/- double
knockout mice are
hyperglycemic and
hyperinsulinemic
Why does it take 2 KOs?
(Top) Blood glucose in 2-month-old
mice after overnight fast (n = 6-14).
**, P < 0.001 for Irs1-/-/Irs3-/- versus
WT, Irs1-/-, and Irs3-/- mice. *, P < 0.05
for Irs1-/-/Irs3-/- versus WT, Irs3-/- mice.
#, P < 0.05 for Irs1-/- versus WT and
Irs3-/- mice.
(Bottom) Plasma insulin levels in 2month-old mice after overnight fast (n
= 6-14). *, P < 0.05 for Irs1-/-/Irs3-/versus WT and Irs3-/- mice; for male
mice, also versus Irs1-/- mice.
THM: Probable compensation
Genes & Development 16: 3213-3222
23
Disruption of IRS-2 causes type 2 diabetes in mice
After fast
Fasting blood
glucose and glucosetolerance test, fasting
insulin levels and
insulin-tolerance test,
and in vivo glucose
disposal and hepatic
glucose production.
a, After a 15 h
overnight fast, blood
glucose levels were
determined. WT, wild
type. b, Glucosetolerance tests after
Plasma insulin
intraperitoneal
loading with 2 g Dglucose per kg were
performed on 6week-old animals of
the indicated
genotype. c, Serum
insulin levels were
measured after a 15
h overnight fast.
d, Insulin-tolerance tests are expressed as
percentage of initial blood glucose concentration.
Glucose tolerance
test
Insulin tolerance
test
24
Nature 391, 900 – 904
Effect of IRS2 disruption depends on genetic
background
Why does one only see the phenotype in some strains?
Proportion of animals with diabetes among offspring from N2 intercrosses with either the 129/Sv or the C57Bl/6J genetic
25
background. **, p 0.01 compared with IRS-2-/- mice derived from N2 intercrosses with the 129/Sv background; ##, p 0.01
compared with those having the C57Bl/6 background.
J Biol Chem. 278:14284-90
What are phenotypes for tissue specific
IR and IRS Knockouts?
Are major roles of IRS proteins tissue specific?
Why do tissue specific knockouts?
Differences in responses with muscle or
adipose-specific KO of GLUT4 or IR
Q: Why
differences?
27
Kahn Review 03
Advantages of tissue specific
knockouts
Tissue specific knockouts allow one to begin to assess in
a quantitative manner the relative contribution of various
parts of complex pathway to overall phenotype.
Also allows many but not all developmental issues to be
addressed
Pathogenesis of obesity-associated insulin resistance
N.S. Kalupahana et al. / Molecular Aspects of Medicine (2011)
Molecular mechanisms of insulin resistance
N.S. Kalupahana et al. / Molecular Aspects of Medicine (2011)
How do other pathways talk to IRS-1?
Functional Interactions that Can
Modify IRS-1 by Phosphorylation
31
M. White Can J P Jul 06
Beavo Take Home Question 2013
Please read the posted review by Kahn and the research paper by James. 1)
For the James’ paper briefly outline your opinion about at least two strengths
of the approach that they are using to help further refine the well-studied
insulin signaling pathway. 2) Identify what you feel are at least two limitations
of the approach that they use and briefly explain your reasoning for saying so.
Your answer therefore should have two parts and each part will be graded
with approximately equal weight. (One page total please).
‘nuff for now
The influence of genetic background
on mouse life span
Inbred strains include F344 (Fischer), C57Bl/6, DBA/2, and BN (Brown Norway); hybrid strains include B6D2 (F1) and F344BN
(F1). Data are adapted from
34
http://www.nia.nih.gov/ResearchInformation/ScientificResources/AgedRodentColoniesHandbook/StrainSurvivalInformation.htm
Feedback Phosphorylation Can
target IRS-1 to Proteosome
35
Mechanisms that can produce
insulin resistance
Fig 8 - Multiple
mechanisms exist
for downregulating insulin
signaling, such as
decreased
synthesis,
increased
degradation,
inhibitory serine
phosphorylation,
interaction with
inhibitory proteins,
and alteration of
the ratios of
different signaling
molecules.
Biddinger & Kahn Ann Rev Physiol
Extranuclear signaling of the estrogen receptor
Figure 4 Upon binding to its ligands, like 17b-estradiol (E2), the estrogen receptor (ER) not only activates the classic signaling
37
pathway resulting in modulating gene transcription in the nucleus but also triggers PI3K activation in the cytoplasm, by directly
binding to the p85 subunit. Listed are some of the biological effects of ER-dependent PI3K activation. Hirsch et al 07
Signaling pathways downstream of PI3K
Figure 3. The product of PI3K activity, PIP3, serves as a docking site for a large number of PH domain (in brown) containing proteins
38 (listed
in circle around PIP3). These effector proteins represent the front line of PIP3-dependent signaling. Further signal amplification is achieved by
their activation and inhibition of multiple more specialized downstream targets, eventually producing specific biological responses.
The effects of genetic ablations on
glucose homeostasis
Fig. The PI 3-kinase
portion of the insulin
signaling network illustrates
the complexity of insulin’s
metabolic signaling. There
are four IRS proteins, five
regulatory subunits of PI 3kinase (with three more
produced by alternative
splicing), three catalytic
units of PI 3-kinase, and
three isoforms of Akt. Loss
of a particular signaling
molecule or isoform can
have positive (green),
slightly negative (orange),
negative (red), or neutral
(white) effects on glucose
homeostasis. Gray denotes
that the effects of deletion
on glucose homeostasis are
unknown.
Biddinger & Kahn Ann Rev Physiol '06
39
Rab Proteins
mediate vesicle
fusion in response
to insulin
Schematic of the PPIn 3-kinase arm of the insulinsignaling cascade.
Negative regulators of insulin signaling leading to
GLUT4-vesicle translocation are depicted as stop
signs (red octagons); positive regulators are shown
as green boxes. PTEN (phosphatase and tensin
homolog deleted on chromosome ten) is a 3’phosphoinositol phosphatase that converts
PtdIns(3,4,5)P3 to PtdIns(4,5)P2.
SHIP2 (type-II SH2-domain-containing inositol 5phosphatase) converts PtdIns(3,4,5)P3 to
PtdIns(3,4,)P2. The mTOR/RICTOR complex
participates, together with PDK1 and
PtdIns(3,4,5)P3, in activating the serine/threonine
kinase Akt. mTOR, when complexed with RAPTOR,
also functions downstream of Akt in the pathway
leading to protein synthesis. Akt seems to
phosphorylate and inactivate the Rab GTPaseactivating protein AS160. This permits the
conversion of an as yet unidentified Rab protein to
the active GTP-bound form, which is proposed to
regulate one or more steps of GLUT4 vesicle
translocation to the cell surface.
40
From Pessin, TiBS Apr., 06
Class IA PI3K function in leptin- and insulininduced signaling
Fig 6. Though a wealth of evidence points to a major role of PI3Ka in insulin signaling, it is still unclear whether
41
PI3Kb has indeed no importance in the process.
Hirsch et al J Endocrinol. 2007
Insulin and
Metformin
Signaling to
a PKC in
Liver
2009
Elements of the IRS2 branch of the insulin signaling cascade are shaded gray, whereas elements of the IRS1 branch of the cascade are
not shaded. IRS1 and IRS2 play a common role in the activation of the PI3 kinase/[PDK1/AKT]/FOXO cascade. The IRS2/PI3
kinase/[PDK1/aPKCi/l]/CBP cascade inactivates the CREB:CBP:CRTC2 transcription complex. Metformin also activates aPKCi/l
through a different mechanism, which circumvents hepatic insulin resistance to phosphorylate CBP and inactivate the
CREB:CBP:CRTC2 transcription complex, especially during hepatic insulin resistance associated with obesity and type 2 diabetes.
Phosphorylation sites (pS or pY) highlighted in green indicate activation steps, whereas sites highlighted in red indicate inhibitory
steps.
Morris F White Cell Metabolism 2009
Molecular mechanisms of IR/IGFR Responses,
‘07
43
Morris White Review 2007
Legend for previous figure - The mammalian insulin-like signaling
cascade, ‘07/’08
In mammals, the insulin/insulin-like growth factor (IGF) family consists of three hormones: insulin, insulin-like growth factor 1
(IGF1), and insulin-like growth factor 2 (IGF2). These peptide ligands bind as indicated to five distinct receptor isoforms that
generate cytoplasmic signals: two insulin receptor isoforms, IRa and IRb (red ); the insulin like growth factor receptor, IGF1R
(blue); and two hybrid receptors, IRa::IGF1R and IRb::IGF1R. IGF2 also binds to the mannose-6-phosphate receptor, which
mediates the endocytosis and degradation of IGF2 (not shown). Activation of the receptors promotes tyrosine phosphorylation of
cellular proteins—IRS1 and IRS2 occur in nearly all cells, but other substrates exist in specific cells and tissues. The SOS/GRB2
branch activates the ras→RAF→MEK→ERK1/2 cascade. Activated ERK stimulates transcriptional activity by direct
phosphorylation of ELK1 and by phosphorylation of FOS through p90rsk (RSK). Activation of PI 3-kinase during recruitment to
IRS1/2 produces PI(3,4)P2 and PI(3,4,5)P3 (antagonized by the action of PTEN or SHIP2), which recruit PDK1 and AKT to the
plasma membrane, where AKT is activated by PDK-mediated phosphorylation. AKT phosphorylates many substrates, including
the cyclin-dependent kinase inhibitor p21kip, GSK3β, BAD, eNOS, and FOXO1; phosphorylation of FOXO1 inactivates this
transcription factor and causes its sequestration in the cytosol, which alters the expression of many genes, including those
encoding IGFBP1, PGC1α, PEPCK, and SOD2. The mTOR kinase is activated by RHEBGTP, which accumulates upon
inhibition of the GAP activity of the TSC1::TSC2 complex by AKT-mediated phosphorylation. The p70s6k is primed for
activation by PDK1-mediated phosphorylation and ultimately activated by mTOR-mediated phosphorylation. Insulin stimulates
protein synthesis by altering the intrinsic activity or binding properties of key translation initiation and elongation factors (eIFs
and eEFs, respectively), including eIF4A and eIF4G. In particular, phosphorylation of 4E-BP1 releases eIF4E to form an active
complex promoting translation initiation. In β-cells, GLP-1 and glucose promote Ca2+ and cAMP-mediated signaling that
stimulates IRS2 expression through CREB and TORC2. Regarding phosphorylation depicted, green indicates stimulatory
phosphorylation, and purple represents inhibitory phosphorylation.
Abbreviations: AKT, protein kinase B; BAD, Bcl-2-associated death promoter; cAMP, cyclic-3 -5 -adenosine monophosphate;
CREB, cAMP response element (CRE) binding protein; cyclase, adenylylcyclase; eNOS, endothelial nitric oxide synthase; EPAC,
exchange protein activated by cAMP; GAP, guanosine triphosphatase–associated protein; GLP1, glucagon-like peptide 1; GRB2,
factor receptor binding protein 2; GSK3β, glycogen synthase kinase 3β; IGFBP1, IGF binding protein-1; INS, insulinoma; IRS1,
insulin receptor substrate 1; JNK, Jun NH2-terminal kinase; MAP2K, MAPK kinase; MAP3K, MAPK kinase kinase; mTOR,
mammalian target of rapamycin; PDK, PI-dependent protein kinase; PDX1: pancreatic and duodenal homeobox factor 1;
PEPCK, phosphoenolpyruvate carboxykinase; PGC1α, peroxisome proliferator–activated receptor (PPAR) gamma coactivator 1
alpha; PH, pleckstrin homology domain; PKA, protein kinase A; PTB, phosphotyrosine-binding domain; PTEN and SHIP2,
phospholipid phosphatases; RIP1, Ser/Thr protein kinase receptor–interacting protein; SOD2, superoxide dismutase 2; SOS, sonof-sevenless; TORC2, transducer of regulated CREB activity 2; TRADD: tumor necrosis factor receptor (TNFR)-associated44death
domain; TSC, tuberous sclerosis complex
At least six receptors can transduce Insulin
or IGF-1 signals in mammals
45