Ch. 15 The Insulin Signaling Network and Insulin Action

2004. 6. 22

Insulin signaling at target tissue Normal growth and development Normal homeostasis of glucose, fat, and protein metabolism

Improved understanding of signaling pathways involving insulin action Better understanding of pathophysiology of insulin resistance assc

ociated with obesity and type 2 diabetes Newer and more effective therapeutic agents

This chapter Current understanding of insuin action

Strucutre of IR, elements that constitute insuling signaling pathways

Impairments in signaling pathways and new paradigms regarding the molecular basis of insulin resistance

Insulin receptor and its substrates

STRUCTURE OF STRUCTURE OF INSULIN RECEPTORINSULIN RECEPTOR

Family of homologous receptor tyrosine kinases (RTKs)- IR / IGF-1R(insulin-like growth factor-1) - IRR (insulin receptor related receptor) : orphan receptor

Proteolytic cleavage of a single polypeptide chain precursor → α & β chains linked by disulfide bonds

→ biologically active α2β2 receptor heterotetramer

Product of a single gene containing 22 exons - 1,355 amino acids, M.Wt of 153,917

- Exon 1 – 11 : α subunit / Exon 12-22 : β subunit- Alternative splicing of exon 11 in hematopoietic tissues- Longer transcript predominates in liver, muscle, and adipose tissues

Two IR isoformsTwo IR isoforms

IR type A (IR-A or Ex11-) (-) Sequence coding for 12 a.a. in C-terminus of α chain of Rc. Preferentiallly activates type Ia of PI3-kinase and p70S6K Promotes insulin gene transcription in pancreatic β cells

IR type B (IR-B or Ex11+) (+) Sequence coding for 12 a.a. in C-terminus of α chain of Rc. Stimulates class II of PI3-kinase and PKB (Akt) Promotes glucose/insulin stimulated β-glucokinase transcription

Different localization of two IR isoforms in plasma memb. of β cells and different sensitivity for insulin

The Extracellular Domain

Entire α-subunit and about one third of β-subunit

Ligand-binding domains, with α-subunit being the primary lignand-binding site

Analogous region of IR (residues 1-468) Cannot bind ligand on its own, high-affinity insulin binding Restored by addition of residues 704 to 719 of IR

⇒ carboxyl-terminal region of α-subunit

: part of ligand- binding site

Three repeats of fibronectin type III domain (FnIII) Deletion of residues 450 to 601

: blunted Tyr autophosphorylation response

⇒ involved in transmission of insulin-binding signal to tyrosine kinase

N-linked and O-linked sugar moieties Mutations of a.a. of potential sugar binding sites

: alter IR kinase acitivity Improper glycosylation : accelerates IR degradation

⇒ required to preserve proper structure and function of IR

The Transmembrane Domain

Part of β-subunits

α-helix structure with seven turns

Function primarily as passive lipid anchor

23 hydrophobic a.a., flanked at C-terminal end by a short sequence of basic residues (Arg-Lys-Arg) Potential interactions with head group of negatively cha

rged phospholipids May represent a stop-transfer signal, anchoring IR in me

mbrane during biosynthesis

The Intracellular Region

C-termianal 403 a.a. of β-subunits, 3 subdomains

1. Juxtamembrane (JM) domain

At least one autophosphorylation site (Tyr 972) in NPXY motif

- Binding site for IR substrates such as Shc and IRS proteins

Regulation of IR internalization (NPXY, GPLY, di-Leu motifs)

Replacement of Tyr972 with Phe or Ala

- Inability of mutant Rc to interact with substrate proteins (IRS-I & Crk-II)

→ Impairs receptor signal transmission and both metabolic and growth-

promoting effects of insulin

- Rescued by overexpression of IRS-1

⇒ Involved in interactions of Rc with substrate proteins

2. Tyrosine kinase domain of IR : hallmark of RTK family 84% homology with IGF-1R Two lobes with a single connection

N-terminal lobe : ATP-binding site C-terminal lobe

: active site (catalytic loop), three autophosphorylation sites (activation loop), and kinase-insert region

3. C-terminus domain of IR Two autophosphorylation sites, still unresolved role Mutations of these Tyr residues

Augments insulin-dependent activation of MAPK and PI3-K Impaired metabolic effects and impaired induction of c-fos bu

t augmented mitogenic signaling Negatively regulate growth-promoting effects of insulin

INSULIN ACTION AT THE INSULIN ACTION AT THE CELLULAR LEVELCELLULAR LEVEL

Diverse biologic responses by binding of insulin to IR Glucose transport Glycogen and protien synthesis Mitogenensis Cell survival

Ligand Binding and Receptor AuLigand Binding and Receptor Autophosphorylationtophosphorylation

Binding of insulin to specific regions of α-subunit

→ rapid conformational change in Rc

→ activation of tyrosine kinase domain

(transautophosphorylation)

: kinase domain in one half of the receptor-dimer

phosphorylates cytoplasmic tyrosine residues in

activation loop of the other half of the receptor-dimer

→ activation loop swings out of the catalytic site to give

unrestricted access to ATP and substrates

Insulin Receptor SubstratesInsulin Receptor Substrates

Insulin receptor substrates Three isoforms of Shc, IRS proteins (IRS-1 to -4), p60dok, Cbl,

APS, and Gab-1 Tyr-phosphorylated IR substrates by activated IRK

: Function as signaling scaffolds, providing a docking interface for proteins having SH2 domains

SH2 proteins P-Tyr phosphatase SHP2 (SH-PTP2) and cytoplasmic Tyr kina

se Fyn : Enzymes p85 regulatory subunit of PI3-kinase, Grb2, or APS

Function as adaptor proteins for downstream effectors that further propagate the metabolic and growth promoting effects of insulin

IRS proteinsIRS proteins

Conserved pleckstrin homology (PH) domain1) Membrane - lipid interactions

Anchor IRS proteins to membrane phosphoinositides Helps to localize IRS proteins in close proximity to Rc - deletion of PH domain : ↓ tyrosine phosphorylation of IRS-1

2) Protein – protein interactions PH domain – interacting protien (PHIP) selectively binds Overexpression of PHIP : ↑ insulin-induced transcriptional responses Dominant-negative mutant of PHIP (DN-PHIP) : specifically blocks transcriptional & mitogenic signals by insulin : inhibits actin cytoskeletal reorganization and translocation to me

mbrane of GLUT-4⇒ PHIP : physiologic binding protein of IRS-1 PH domain, which pla

ys a role in insulin signaling

P-Tyr binding (PTB) domain of IRS proteins 75% sequence identitiy between IRS-1 and IRS-2 Function as a binding site to NPXY motif of JM region of IR

C-terminal region of IRS proteins Contains multiple Tyr-phosphorylation motifs that serve as doc

king sites for SH2 domain-containing proteins (p85α regulatory subunit of PI3-kinase, Grb2, Nck, Crk, Fyn, SHP-2…)

> 70 potential Ser/Thr phosphorylation sites Several kinases that can phosphorylate IRS-1

: casein kinase II, glycogen synthase kinase 3, MAPK, PI 3-kinase IRS-1

Insulin (-) : Ser (strong) and Tyr (weak) phosphorylation Insulin (+) : ↑ Tyr and Ser phosphorylation

Phosphorylation of IRS-1 on Ser/Thr residues reduces its ability to undergo Tyr phosphorylation by IRK serves to shut off insulin signaling

Relative role of IRS proteins in mediating insulin action 1) IRS-1 and IRS-2

IRS-1 knockout mice : generalized growth retardation, as well as insulin resistance and IGT, compensation of IRS-2 (PI3-K) IRS-2 null mice : insulin resistance but growth defects limited only to pancreatic β-cells → type 2 diabetes Complementary rather than redundant role in insulin signaling Attributed to selected structural differences, in addition to differences in their tissue distribution & subcellular localization

2) IRS-3 and IRS-4 Act as negative regulators of IRS-1 and IRS-2 in cultured cells IRS-3 and IRS-4 knoukout mice : mild defects in growth and metabolism

Shc family

Adapter proteins that serve as major substrates of IRKThree isoforms of 46, 52, and 66 kdInsulin-stimulated Tyr-phosphorylated Shc + SH2 domain of Grb2

→ activation of mSOS/Ras/MAPK signaling pathwayPTB & SH2 domain : Interaction with JM or C-terminal of IRPI 3-kinase as a necessary intermediary step facilitating insulin-stimulated Tyr-phosphorylation of Shc through PTB domainPP2A (Ser/Thr phosphatase)

: inhibit Tyr-phosphorylation of Shc independent of P-Tyr binding

Gab-1 (Grb2-associated binder-1)

Insulin receptor substrate

Tyr-phosphorylated Gab-1 recruits downstream signaling elements possessing SH2 domain (p85 PI 3-kinase, phospholipase C-γ, SHP-2 (protein tyrosine phosphatase), and Crk)

Substrate for other RTKs (EGF, FGF, HGF, NGF Rcs)

Physiologic roles of Gab-1 : little known May be part of signaling pathways leading to cell growth, t

ransformation, and apoptosis Mediator of osmotic shock signal transduction pathway

that induces glucose transport in adipocytes through Gab-1-associated PI 3-kinase acitivity

The Insulin Signaling NetworkThe Insulin Signaling Network

Three major pathways

: PI 3-kinase, MAPK, and Cbl/CAP pathways

MAPK pathway

: general signaling pathway leading to enhanced cell growth

PI 3-kinase and Cbl/CAP pathways

: biologic responses that are more unique to insulin action

Insulin signaling network

• Activated IRK

• Substrates

• 3 signaling pathways

• FunctionDNA / RNA / Protein Synthesis

Gluconeogenic Gene Transcription

Glycogen Synthesis

Glucose Transport

The PI 3-kinase pathway

Central role of metabolic & growth-promoting actions of insulin

p110 catalytic subunit and p85 regulatory subunit

Direct interactions of regulatory subunit with Tyr-phosphorylated YMXM and YXXM motifs of activated growth factor receptors, or with adapter proteins such as IRS proteins → activation of PI3K p110 catalytic subunit in close proximity to its lipid substrates in cell m

embrane May relieve an inhibitory effect of p85 on p110 kinase activity

p85α : predominant subunit that mediates most biologic responses to insulin (at least eight isoforms of regulatory subunits)

Inhibitors of class Ia PI 3-kinase, or transfections with dominant negative constructs of enzyme block most metabolic actions of insulin including stimulation of glucose transport, glycogen, and lipid synthesis.

Monomeric p85 inhibits IRS protein-mediated signal by competing with p85-p110 dimer. Partial depletion of p85 improves insulin signaling Complete depletion of p85 results in a significant decrease in PI 3-

kinase mediated biologic responses

⇒ optimal signaling depends on a critical molecular balance between regulatory and catalytic subunits

Association of p85-p110 complex with IRS molecules

→ production of PIP3

→ interaction with PH domain of PDK1, PKB, and other signaling molecules

→ their recruitment to plasma membrane

→ changes in their structure, function, and their substrate availability

Ser kinase activity independent of generation of PIP3

Atypical Protein Kinase C Isoforms : PAtypical Protein Kinase C Isoforms : PKCKCζζ and PKC and PKCλλ

Activated by PI 3-kinase and PDK1

Increase basal and insulin-stimulated translocation to the membrane of GLUT-4 in adipocytes and muscle cells

Play a critical role in physiologic negative feedback control mechanism, induced by insulin, that serves to terminate insulin action

: PKCζ-mediated phosphorylation of IRS proteins leads their dissociation from IR, thereby terminating insulin signaling

Protein Kinase B (Akt)Protein Kinase B (Akt)

Major substrates of PDK1

Mediating regulation of glucose transport, glycogen synthesi

s, protein synthesis, antilipolytic effects of insulin, as well as c

ell growth and cell survival induced by insulin

Phosphorylation at Thr308 by PDK1 & at Ser473 by PDK2 → maximal activation of PKB

Three PKB isoforms PKB1/PKBα : required for normal growth

Deletion of Aktβ(PKB2) : hepatic insulin resistance

Defect in a ability of insulin to activate PKB2 and -3 but not PKB1

: impaired insulin-stimulated glucose transport

Protein kinase B, Glucose Transport, Protein kinase B, Glucose Transport, and Glycogen Metabolismand Glycogen Metabolism

Regulates translocation of GLUT-4

: obscure mechanism

Important role in insulin-induced glycogen synthesis

: phosphorylation and inactivation by PKB of GSK3β

(phosphorylates and inactivates glycogen synthase (GS))

→ promotes GS activity and glycogen synthesis in response to insulin

GSK3β Inactivates by phosphorylation protein synthesis eukaryotic initiation factor (eIF)-2B

→ Insulin-mediated activation of PKB reverses, thereby enhancing protein synthesis

Glycogen Synthesis

DNA / RNA / Protein Synthesis

PI3K

PDK1

PKB

GSK3β

eIF-2B GS

Protein kinase B and Protein SynthesisProtein kinase B and Protein Synthesis

Mammalian target of rapamycin (mTOR)

- Ser/Thr kinase that serves as a molecular sensor that regulates protein synthesis on the basis of nutrients availability

Mechanisms of activation of insulin-induced protein synthesis at translational level PI3K

PDK1

P70 S6K PKB

mTOR

4E-BP1

eIF-4E

S6

DNA/RNA/Protein Syntheis

: translation repressorribosomal protein

Protein kinase B, Gene Expression, Protein kinase B, Gene Expression, and Cell survivaland Cell survival

Insulin inhibits nuclear translocations of transcription factors by phosphorylation these factors by PKB in an insulin-dependent mannerForkhead family (FH) FKHR (Foxo-1, forkhead box transcription factor O1), FKHRL1, AFX Insulin-mediated phosphorylation of Foxo-1 by PKB→ interaction of Foxo-1 with 14-3-3 family of proteins→ retention of Foxo-1 in cytoplasm and prevents Foxo-1 from translocati

ng to the nucleus→ Inhibition of expresssion of a number of Foxo-1-regulated genes : gain of function Foxo-1 mutation targeted to liver & pancreatic β-cells → increased hepatic glucose production and impaired β-cell compensat

ion due to decreased Pdx 1 expression → type 2 diabetes Foxo-1 functions in adipose cells to couple insulin signaling to adipog

enesis : switching preadipocytes from proliferation to terminal differntiation

Antiapoptotic functions of insulin and IGF-1 by PKB PKB phosphorylation of apoptosis-inducing protein Bad

→ creates binding sites for 14-3-3 proteins and

prevents Bad from binding to Bcl-2 family members,

Bcl-2, Bcl-XL, thus releasing them for a cell survival

response PRAS40

: novel substrate of PKB

: phosphoylation of PKB → binding of PRAS40 to 14-3-3

The Mitogen-Activated Protein KiThe Mitogen-Activated Protein Kinase Pathwaynase Pathway

MAPK kinase kinase (MKKKs) → MAPK kinase (MKK)↑ → MAPK↑

Three well-characterized subfamilies of MAPKs Extracellular signal-regulated kinases ERK1 and ERK2 c-Jun NH2-terminal kinases JNK1, JNK2, and JNK3 four p38 enzymes p38α, β, γ, δ

ERK1 and ERK2ERK1 and ERK2

Activated ERKs mediate growth-promoting effects of insulin by phosphorylating transcription factors such as Elk-1, leading to induction of gene expressionMagnitude and duration of ERK activation Critical determinants of final cell type-specific physiologic outcome Regulated by balance of both activating kinases and inactivating pho

sphatasesInsulin-induced adipocyte differentiation by ERK Activation of MEK/ERK pathway at late stages of adipogenesis : likely to block adipogenic gene expression due to MAPK-depende

nt phosphorylation of PPAR-γ Activation of pathway early during adipogenesis before PPAR expre

ssion might promote differentiation by activating transcription factors operating to initiate PPAR and C/EBP expression

- Insulin : principal regulator of MAPK pathway in preadipocytes and promotes their differentiation by enhancing expression of C/EBPα and PPAR-γ

C-Jun N-terminal KinaseC-Jun N-terminal Kinase

Stress-activated protein kinases on the basis of their activation in response to inhibition of protein synthesisBind and phosphorylate c-Jun (component of AP-1, important regulator of gene expression) and increase its transcriptional activityInsulin stimulation

→ binding of JNK to IRS-1 and phosphorylation on Ser307 → inhibition of insulin signalingDual function as a heterologous inhibitor of insulin action during acute and chronic inflammation and as a negative feedback regulator of insulin action by phosphorylating Ser307 in IRS-1

p38 Kinasesp38 Kinases

Activated by inflammatory cytokines, hormones, ligands for GPCRs, and stresses such as osmotic or heat shockUndergoes insulin- and IGF-1-dependent activation

PI 3-kinase-mediated activation of PKB and mTOR PI 3-kinase-mediated activation of Rac and p21-activated ki

nase (PAK) Insulin-stimulated activation of MKK3/6

Role of p38 Promoting differentiation of cultured muscle cell lines t

hrough phosphorylation of MAPK AP-2, degradation of IκBα, and induciton of NFκB

Mediates insulin-induced GLUT-4 transport activity, rather than GLUT-4 translocation

The CAP/TC10 pathway and Glucose The CAP/TC10 pathway and Glucose Transporter 4 TranslocationTransporter 4 Translocation

Cbl/Cbl-Associated Protein(CAP)/TC10 pathwayP-Tyr IR

→ APS

→ P-Tyr Cbl

→ Cbl-CAP complex

(3 SH3 domain)

→ lipid raft, flotillin

(SoHo domain)→ P-Tyr Cbl-CrkII→ CrkII-C3G complex→ activation of TC10

• Adapter protein• Expressed in insulin-sensitive tissue• Markedly induced during adipocyte diff.• Increased expression by PPARγ agonists

Constitutive activation of TC10 by overexpression of C3G : not mimic insulin action but potentiate action of insulinActive mutant of PI 3-kinase : fully insulin actionTwo pathways : synergistic signals in regulation of glucose transport

Activation of TC10 : specific for insulin

Disruption of its activation : blocks insulin-stimulated glucose transport and GLUT-4 translocation

Posttranslational modification (farnesylation and palmitoylation) of TC10 → secretory pathway → targeting to lipid raft domain

Downstream effectors that couple TC10 to GLUT-4 translocation Exo70 (a component of exocyst complex)

Targeting of GLUT-4 vesicle to plasma membrane, site of fusion Proximity with N-ethylmaleimide sensitive factor (NSF) attachmen

t protein (SNAP) receptor complex involved in docking and fusion Rab protein (Rab 4) : might mediate this tethering step Cdc42-interacting protein 4/2 (CIP4/2) : intracellular compartment

→ plasma membrane upon insulin stimulation

TC10 seems to stimulate GLUT-4 translocation in a PI 3-kinase-independent manner

TC10TC10

Cdc42 GTPase, 69% homology and 86% similarity to TC10 Downstream effector of Gαq/11 and upstream regulator of PI3

-kinase and PKCλ in insulin-stimulated pathway leading to GLUT-4 translocation

promote glucose transport by activating PI3-kinase pathway

Regulation of GLUT-4 translocation by TC10 & Cdc42 Reorganization of cytoskeleton cortical actin

Rho family GTPases : control formation of actin stress fibers lamellipodia and filopodia

Engagement of molecular motors, Myo1c Controls movement of intracellular GLUT-4-containing vesicles to

plasma membrane Proper structural organization of plasma membrane caveolin

and functional clathrin : regulate the rate of endocytosis of GLUT-4, thus affecting the overall rate of GLUT-4 recycling

- IRK directly catalyzes tyrosine phosphorylation of caveolin

INHIBITION OF INSULIN INHIBITION OF INSULIN RECEPTOR SIGNALINGRECEPTOR SIGNALING

Terminate insulin’s effect immediately following insulin stimulation Through action of lipid and protein phosphatases Through activation of Ser/Thr kinases that phosphorylate an

d uncouple various elements in insulin signaling pathways

Longer time scale Reduction in cellular component of IR, its substrates, and oth

er signaling elements

Insulin Receptor Internalization Insulin Receptor Internalization and Degradationand Degradation

Rapid internalization of IR following insulin binding

→ either to their degradation or recycling to cell surface

Surface redistribution

→ progressive concentration of receptor-insulin complex in clathrin-coated pits (internalization gates)

Stimulation of intrinsic Tyr kinase activity of IR following insulin binding is a prerequisite for surface redistribution

IR internalization independent of its ability to phosphorylate IRS proteins but impaired internalization of IR associated with impaired phosphorylation of Shc

Effect of ECM proteins on receptor internalization Interactions of different ECM proteins with cell surface integrins :

affect the rate of IR endocytosis Specific polymerized actin structures in the form of filamentous ac

tin tracks : required for maintain proper IR internalization Independent of effects of ECM proteins on IRK activity and its abil

ity to phosphorylate downstream effectors (IRS proteins)

Insulin signaling and insulin responsiveness are dually regulated by the adhesive properties of the cells- Ligation of ECM proteins by cell surface receptors (integrins) Generates signaling cascades that modulate the activity of IRK Dictate the rate and extent of IR internalization & degradation

Role of Protein & Lipid PhosphatasesRole of Protein & Lipid Phosphatases Lipid PhosphatasesLipid Phosphatases

Phosphatidyinositol-3-phosphatases Phosphatase and tensin homologue (PTEN) SH2 domain-containing inositol 5-phosphatase SHIP2

Overexpression of PTEN or SHIP2

→ decreased levels of PIP3

→ might terminate signal transduction or change the nature of phosphoinositides, altering binding specificity

Protein Tyr Phosphatases (PTPs)Protein Tyr Phosphatases (PTPs)

Prominent role in negative regulation of IR signaling - Dephosphorylate the IR and its substrates and thus serve to ter

minate IR signalingTransmembrane receptor-like PTPs LAR and PTPα LAR : dephosphorylating adapter proteins such as IRS-2 PTPα : not alter phosphorylation status of IR, IRS-1, or ShcEndoplasmic reticulum-associated PTP1B and its close homologue TCPTP Function as direct IR phosphatases, May act in concert PTP1B in complex with IRβ subunit activation loop, encompassing

pTyr residues at 1162 and 1163 Insulin stimulates Tyr phosphorylation & inactivation of PTP1B PTP1B may act as a point of counterregulation of insulin action by ca

techolamines (↑cAMP & activation of PKA & PTP1B)

Ser Phosphorylation as a Regulatory Ser Phosphorylation as a Regulatory Means to Terminate Insulin SignalingMeans to Terminate Insulin Signaling

Control mechanisms

Homologous desensitization (Autoregulation)

- Downstream enzyme inhibit upstream elements

Heterologous desensitization

- Signals from apparently unregulated receptor pathways can inhibit the signal

Ser/Thr Phosphorylation of the InsSer/Thr Phosphorylation of the Insulin Receptorulin Receptor

IR Basal state : both P-Ser and P-Thr but no P-Tyr residues Insulin (+) : abrupt increase in P-Tyr content of Rc

→ slower increase in its P-Ser and P-Thr content IRs with P-Ser residues in basal state

: Tyr autophosphorylation more slowly, or even not at all

Ser/Thr phosphorylation

: act as a physiologic feedback control mechanism to inhibit insulin-stimulated Tyr phosphorylation of Rc

Phosphorylation of Ser955/956, Ser1293/1294, Thr1336 Stimulated by insulin, but not major regulatoy sites

Ser/Thr kinases : largely unknown, phosphorylation of IR on Ser1078 without affecting its Tyr kinase activity

Phosphorylation of the Insulin Receptor IPhosphorylation of the Insulin Receptor Induced by Protein Kinases A and Cnduced by Protein Kinases A and C

Treatment of cells with inducers of PKA Ser/Thr phosphorylation of IR Impairs the ability of IR to function as a Tyr kinase Contribute to catecholamine-mediated insulin resistance

Several PKC isoforms Mediate Ser/Thr phosphorylation of IR in intact cells Inhibit receptor autophosphorylation Not inhibit the receptor tyrosine kinase Further classification for physiologic role of a direct phosph

orylation of IR by PKC

Ser/Thr Phosphorylation of the Insulin ReSer/Thr Phosphorylation of the Insulin Receptor and Insulin Resistanceceptor and Insulin Resistance

Insulin resistance associated with Type 2 diabetes, obesity, hypertension, chronic infection, and C-V dis.

Potential mechanism for induction of insulin resistance : Excessive Ser phosphorylation of IR

Polycystic ovary syndrome (PCOS) Decrease in IR autophosphorylation Significant increase in Ser phosphorylation of Rc β subunits and

decreased insulin-induced Tyr phosphorylation of IR (50%)

Increased Ser phosphorylation of IR decreases its Tyr kinase activity One mechanism for defect in insulin action in PCOS and oth

er forms of insulin resistance

Ser/Thr Phosphorylation of Insulin Ser/Thr Phosphorylation of Insulin Receptor Substrates ProteinsReceptor Substrates Proteins

Key negative feedback control mechanism uncouples IRS proteins from their upstream and downst

eam effectors terminates signal transduction in response to insulin

TNF-α, free fatty acids and cellular stress

→ Activation of Ser/Thr kinases

→ Ser/Thr phosphorylation of IRS protein

→ Inhibition of insulin signaling and

induction of insulin resistance

Insulin-Stimulated Ser/Thr Phosphoryation oInsulin-Stimulated Ser/Thr Phosphoryation of Insulin Receptor Substrate Proteins : f Insulin Receptor Substrate Proteins : DualDual

PKB : Protects IRS proteins from rapid action of PTPs

enables IRS proteins to maintain Tyr-phosphorylated acitive conformation

PKCζ

Mechanisms of inhibition of insulin-stimulated Tyr phosphorylation of IRS proteins by Ser/Thr phosphorylation of IRS proteins

(a)Releases IRS proteins from intracellular insoluble multiprotein complexes including cytoskeletal elements

(b)Dissociation of IRS proteins from JM domain of IR

(c) Inhibits the ability of downstream effectors such as PI3K to dock and bind to specific Tyr residues at C-terminal tail of IRS proteins

(d)Turns IRS proteins into inhibitors of IRK

(e) Induces degradation of IRS proteins

Downstream effectors of PI 3-kinase as negative regulator of IRS protein function mTOR :↑ phosphorylation of Ser residue at C-terminus of IRS

→ inhibits insulin-stimulated Tyr phosphorylation of IRS-1 and its ability to bind PI 3-kinase

PKCζ : phosphorylation of IRS proteins

→ dissociates IR-IRS complexes and inhibits the ability of IRS proteins to undergo insulin-stimulated Tyr phosphorylation

: function as an insulin-stimulated IRS kinase IKKβ : Ser/Thr kinase (degradation of IκB → activation of NFκB)

Serves as substrate for PKCζ and activated by PKCζ Insulin-stimulated IRS kinase

- ↓ insulin-stimulated Ser phosphorylation of IRS-1 by salicylates

- IKKβ stimulated by stress inducers : phosphorylation of IRS-1 on Ser307: adjacent to PTB domain, disruption

of interaction of between JM domain of IR and PTB domain of IRS-1

JNK : Direct interaction with IRS-1 and phosphorylation of IRS-1 at Ser307 in an insulin-dependent manner (similar to IKKβ)

Inducers of insulin resistance (phorbol esters, FFA, TNF-α)

: ↑ Ser/Thr phosphoryation and ↓insulin-stimulated Tyr phosphorylation of IRS Protein (pathologic condition)

TNF-α : activation of PKCζ & its downstream target IKKβ Induction of sphingomyelinase and production of ceramide

→ stimulation of PKCζ activity Induction of complex formation btw PKCζ, p62, and RIP proteins

→ link PKCζ to TNF-α signaling (adaptors of TNF-α Rc)

IKKβ : IRS kinase Stimulated by FFAs, proinflammatory cytokines, and other inducers of

insulin resistance Activation or overexpression of IKKβ : attenuates insulin signaling Inhibition of IKKβ by high doses of salicylates : ↑glucose metabolism

Ser/Thr Phosphoryation of Insulin Receptor Ser/Thr Phosphoryation of Insulin Receptor Substrate Proteins and Insulin ResisitanceSubstrate Proteins and Insulin Resisitance

Mechanism of activation of IKKβ by FFAs: FFA-derived metabolites (diacylglycerol and ceramide)

→ activation of PKCθ and PKCζ → activation of IKKβ Inhibition of IKKβ prevents Ser/Thr phosphorylation of IRS proteins by

high-fat diet, TNF-α, or phosphatase inhibitors

⇒ IKKβ as a point of convergence

Ser kinases downstream of insulin signaling and

Ser kinases activated by proinflammatory cytokines such as TNF-α

activate IKKβ to inhibit insulin signaling both under physiologic and pathologic conditions

JNK activated by proinflammatory cytokines, abnormally elevated in obesity IRS kinase that phosphorylate Ser307, to uncouple IRS-1 from IR

PKCθ : mediator of FFA-induced insulin resistance phosphorylation of IRS-1 at Ser307

PKCα : ↑ by phorbol esters or endothelin-1, phosphorylation at Ser612

GSK3, casein kinase II, novel kinase that phosphorylates IRS at Ser789

Phosphorylation-Mediated Degradation Phosphorylation-Mediated Degradation of Insulin Receptor Substrate Proteinof Insulin Receptor Substrate Protein

Ser phosphorylation of IRS proteins : mainly short-tem mechanism to inhibit insulin signaling

Ubiquitin/proteasome-mediated degradation of IRS proteins : long term insulin resistance

Ser/Thr phosphorylation via PI3-kinase/Akt/mTOR signaling pathway Phosphorylation of Ser307, in close proximity to PTB domain

(uncoupling interaction of IRS-1 with IR/ targeting IRS-1 to degradation) N-terminal region of IRS-1, including PH and PTB domain

Suppressors of cytokine signaling (SOCS) proteins ↑ by proinflammatory cytokines & inducers of insulin resistance SOCS1/3 bind to elongin BC-containing E3 ubiquitin-ligase compl

ex via conserved C-terminal SOCS box → promote ubiquitination and degradation of IRS1 & IRS2

Ser/Thr Phosphorylation of Shc ProteinsSer/Thr Phosphorylation of Shc ProteinsChronic stimulation with insulin

→ persisitent phosphorylation of mSOS by MAPK

→ dissociation mSOS from adaptor Grb2

→ allows GTPase RAS to return to inactive phase

Inhibitors that Uncouple the Insulin RInhibitors that Uncouple the Insulin Receptor from its Substrate Proteinseceptor from its Substrate Proteins

SOCS-3 : binds to phosphorylated Tyr960 of IR and prevents STAT-5B activation by insulin and insulin-induced IRS-1 tyrosine phosphorylation

Grb10 : interacts with regulatory kinase loop of IR and inhibition of insulin signaling