Chapter.10 Cellular and molecular processes regulating glucose transporter 4 tranlocation.

Chapter.10

Cellular and molecular processes regulating glucose transporter 4 t

ranlocation

Overview

Glucose: energy sorce

Glucose level: interrelationship between intestinal glucose absorbtion, hepatic glucose output, uptake of CNS, pheripheral tissue

Blood glucose clearance By glucose transporter family in plasma membr

ane

overview

Glucose transporter Sodium-dependent transporter(SGLT)

Intestine/kidney

Facilitative glucose transporter family(GLUT) All mammalian cell type GLUT4 => adipocyte, striated muscle

GLUT4 overview

Mainly expressed in adipocyte, striated muscle Responsible for the majority of postprandial glu

cose clearance from the circulationNo insulin : 95%, sequestered in intracellular compartmentInsulin stimuli : cell surface Insulin stimulated glucose uptake

GLUT4 main interests

Intracellular storage site

Their distribution

The trafficking path way of GLUT4

Signaling mechanisms regulating these event

The translocation hypothesis

=> recruitment model of insulin stimulated glucose uptake

The translocation hypothesis

under basal condition: slow rate of exocytosisInsulin stimuli condi.: exocytosis endocytosis GLUT4 trafficking : controls whole body glucose homeostasis Muscle, adipose tissue: express GLUT4 isofor

ms. These tissue, glucose uptake is rate-limiting step for glucose metablism

The translocation hypothesis

T2DM & insulin resistance Glut4 translocation 과 glucose transport 의

감소 . Directly corrleated in severity of Insulin resistan

ce Glut4 expression 의 감소 Glut4 overexpression: glucose tolerance 와 dia

betic phenotype 개선 (db/db)

GLUT4 intracellular storage compartments

2-D e-microscope 관찰 (adipocyte, cardiac, Skmuscle)

GLUT4 protein: small vesicle 내 관찰 그외 : tubulovsicular structures beneath the cel

l surface membrane. Trans-golgi, chlathrin-vesicle, plasma membrane, endosones, secretory granule.

GLUT4 intracellular storage compartments

Specific GLUT4-containg vesicle 존재 Vesicle SNAP recpetor(v-SNARE), vesicle assoc

iated membrnae protein2(VAMP-2) 에 많이 존재

Endosomal v-SNARE, endosomal VAMP-3/cellubrevin 에는 존재치 않음 (recycling endosome population)

GLUT4 intracellular storage compartments

Cellugyrin 4-transmembrane protein Marker for a distinct population of glut4-contai

ning vesicle. 인슐린 감수성 없는 glut4 positive compartmen

t 에 존재 insulin reponsive glut4-containing vescle do

not contain this protein

GLUT4 endocytosis

GLUT4 Undergoes continuous recycling through multipl

e rounds of endocytosis and exocytosis both presence and absence of insulin.

Insulin removed, glut4=> internalized, recruited back to intracellular s

torage site in preparation for the next round of insulin stimulated translocation

GLUT4 endocytosisBasal state, Small amount of GLUT4 at the plasma membra

ne => localized to coated pit.Insulin 자극 , Induces the distribution of the translocated GL

UT4 in noncoated regions of plasma membrane

새로이 translocated 된 GLUT4 의 incorporation 은 clathrin-coated pits 으로 diffusion 보다 빠르다 .

GLUT4 endocytosis

Primarily occurs through clathrin-coated pit, dynamin-dependent mechanism.

Dymamins =>endocytosis 초기 단계 작용하는 GTPase 의 일종

GTPase-defective dynamin mutant overexpression => prevents glut4 endocytosis

Mechanism of GLUT4 sorting

There are two regulation domains

FQQI

Di-leucine

Mechanism of GLUT4 sorting

FQQI motif Cytoplasmic domain of glut4(amino terminal) Plasma endocytosis 에서의 중요한 역할 Tyrosine based internalization motifs identified in sever

al plasma membrane protein that undergo endocytosis Mutation=> aberrant localization to membrane. Phenylalanine(F)is necessary for binding of the glut4 a

mino-terminus to the chlathrin adaptor protein(adptin) Clathrin coated vesicle 로의 targeting

Mechanism of GLUT4 sorting

Di-leucine domain Carboxy terminal 에 존재 여러 recycling protiein 의 endocytic signal

에서의 중요한 역할 Trans-golgi 와 glut4 containing vesicle 사이의

sorting 에서 중요한 역할

Plasma membrane fusion of glucose transporter 4 vesicles

t-SANRE 와 v-SNARE 사이의 fusion

SNAP:soluble NSF attachment proteinSNARE: SNAP receptor

t-SNARE Target membrane SNAP receptor Syntaxin4 + SNAP23 Syntaxin : 35kd, a-terminal:cytoplasm oriented

Plasma membrane fusion of glucose transporter 4 vesicles

Insulin responsive glut4 vesicle v-SNARE, VAMP2 가 많이 존재t-SNARE Munc 와 결합 Mucn 는 VAMP2 와 t-SNARE 반응에 필요함

Insulin induces the interactin of VAMP2 with syn4/SANP23 complex

Fusion hypothesis by munc protein Basal 에서 munc18c 가 VAMP2 와의 결합억제 하다가 인슐린

자극하에서 conforamtional change 를 하여 vesicle fusion 일어 나게끔 함

Plasma membrane fusion of glucose transporter 4 vesicles

Syn4 or SNAP23 inhibition Blocking Ab, dominant interfering mutant, peptide inhibitor introductio

n Spcifically inhibit insulin-stimulated Glut4 translocation

Syn4 KO -/- embryonic lethality +/- IGT, insulin Resitance

Overexpression of Munc18c Blocks insulin stimulated glut4 translocationGenetic loss of Munc18c Prevents vesicle fusion

regulatory functions of Munc18 are significantly more complex

Insulin signaling pathways regulating glut4 translocation

Insulin stimulated tyrosine phosphorylation of IRSAssociation and activiation of the SH2-domain containing protein PI3KProduction of PIP3PIP3 interacts with PDK1Phosphorylation cascade initiationPKB/Akt or atypical PKC phosphorylation

Insulin signaling pathways regulating glut4 translocation

PKB/Akt KO(animal study) Insulin resistance and IGTIn vitro study Overexpression of dominant-interfering PKB mu

tant, blocking Ab Blocks insulin responsive GLUT4 translocation

Expressionn of PKB activity to GLUT4 containing compartment glut4 translocation enhancing

Insulin signaling pathways regulating glut4 translocation

Atypical PKC peptide inhibitor and expression of dominant-interfering mutant or PKC bloking Ab. Prevents insulin-stiumulated GLUT4 translocati

onActive atypical PCK mutants induces GLUT4 translocation

PKC associated with GLUT4 compartment

Insulin signaling pathways regulating glut4 translocation

PKB/PKC controversial these are necessary for insulin-response glut4 translocationPIP3 kinase: widely accepted necessary for glut4 translocation Inhibition study: glut4 translocation, glucose transport i

nhibited PTEN overexpression(PIP3 lipid lipase)

Glut4 translocaiton blocked PIP3-K activity and PIP3 lipid product required for gl

ut4 translocation PIP3-K is necessary (no sufficient)

Maybe PIP3-independent path way exist

Insulin signaling pathways regulating glut4 translocation

IR phosphorylate the substrates Cbl & APSCbl interacts with Cbl associated protein(CAP) CAP can bind to the lipid raft protein flotillin(found in caveolin)This binding recruits phosphorylated CblRecruitment of the SH2/SH3 adaptor protein CrkIICrkII catalyze GTP to GDP of TC10Glut4 translocaitonTC10 inhibition Glut4 translocation 억제

Glucose transporter 4 activation

Newly traslocated GLUT4 Low transport activity Must undergo activation step

Insulin signaling generally needed for glut4 activation. Second signaling event has not been elucidated

Exact signaling to activates GLUT4 is not clear

summary

Facilitative glucose transporters are 12 membrane spanning proteinEach isoforms of GLUTs has specific role(tissue)GLUT4 is key regulator of glucose homeostasisCardiac/skeletal muscle and adipose tissue provides glut4 localization store site and rapidly redistributed to cell surface by insulin

summary

Glut4-regulated vesicle populations are enriched in the v-SNARE protein (VAMP2)VAMP2 => combination with t-SANRE protein syntaxin4 and SNAP23 Core compllex necessary for the docking/fusio

n of the glut4 vesicle PI3-K pathway induced by insulin essential for glut4 translocation (but sufficient)

summary

CAP/Cbl/TC10 pathway PI3-K pathway 와의 연관 가능성Futher studies Identify the downstream targets of insulin-medi

ated glut4 translocation The specific mechnism of budding/priming/doc

king/fusion of Glut4 vesicles with the p/membrane