Chapter.10
Cellular and molecular
processes regulating glucose
transporter 4 tranlocation
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
membrane
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
glucose clearance from the circulation
No insulin : 95%, sequestered in
intracellular compartment
Insulin 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
exocytosis
Insulin stimuli condi.: exocytosis
endocytosis
GLUT4 trafficking : controls whole body
glucose homeostasis

Muscle, adipose tissue: express GLUT4
isoforms. These tissue, glucose uptake is
rate-limiting step for glucose metablism
The translocation hypothesis
T2DM & insulin resistance




Glut4 translocation 과 glucose transport의
감소.
Directly corrleated in severity of Insulin
resistance
Glut4 expression 의 감소
Glut4 overexpression: glucose tolerance와
diabetic phenotype 개선(db/db)
GLUT4 intracellular storage
compartments
2-D e-microscope 관찰(adipocyte,
cardiac, Skmuscle)


GLUT4 protein: small vesicle내 관찰
그외: tubulovsicular structures beneath the
cell 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
associated membrnae protein2(VAMP-2)에
많이 존재
Endosomal v-SNARE, endosomal VAMP3/cellubrevin 에는 존재치 않음(recycling
endosome population)
GLUT4 intracellular storage
compartments
Cellugyrin




4-transmembrane protein
Marker for a distinct population of glut4containing vesicle.
인슐린 감수성 없는glut4 positive
compartment에 존재
 insulin reponsive glut4-containing vescle
do not contain this protein
GLUT4 endocytosis
GLUT4


Undergoes continuous recycling through
multiple rounds of endocytosis and
exocytosis both presence and absence of
insulin.
Insulin removed,

glut4=> internalized, recruited back to
intracellular storage site in preparation for the
next round of insulin stimulated translocation
GLUT4 endocytosis
Basal state,

Small amount of GLUT4 at the plasma
membrane => localized to coated pit.
Insulin 자극,

Induces the distribution of the translocated
GLUT4 in noncoated regions of plasma
membrane
 새로이 translocated 된 GLUT4의
incorporation 은 clathrin-coated pits 으로
diffusion 보다 빠르다.
GLUT4 endocytosis
Primarily occurs through clathrin-coated
pit, dynamin-dependent mechanism.
Dymamins =>endocytosis 초기 단계 작용하
는 GTPase의 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
several plasma membrane protein that undergo
endocytosis
Mutation=> aberrant localization to membrane.
Phenylalanine(F)is necessary for binding of the
glut4 amino-terminus to the chlathrin adaptor
protein(adptin)
Clathrin coated vesicle로의 targeting
Mechanism of GLUT4 sorting
Di-leucine domain



Carboxy terminal에 존재
여러 recycling protiein의 endocytic signal 에
서의 중요한 역할
Trans-golgi와 glut4 containing vesicle 사이
의 sorting 에서 중요한 역할
Plasma membrane fusion of glucose
transporter 4 vesicles
t-SANRE 와 v-SNARE사이의 fusion
SNAP:soluble NSF attachment protein
SNARE: 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, VAMP2가 많이 존재
t-SNARE
 Munc와 결합
 Mucn는 VAMP2와 t-SNARE반응에 필요함
Insulin induces the interactin of VAMP2 with syn4/SANP23
complex
Fusion hypothesis by munc protein
 Basal에서 munc18c가 VAMP2와의 결합억제 하다가 인슐린 자
극하에서 conforamtional change를 하여 vesicle fusion 일어 나
게끔 함
Plasma membrane fusion of glucose
transporter 4 vesicles

Syn4 or SNAP23 inhibition



Blocking Ab, dominant interfering mutant, peptide inhibitor
introduction
Spcifically inhibit insulin-stimulated Glut4 translocation
Syn4 KO


-/- embryonic lethality
+/-  IGT, insulin Resitance
Overexpression of Munc18c
 Blocks insulin stimulated glut4 translocation
Genetic 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 IRS
Association and activiation of the SH2domain containing protein PI3K
Production of PIP3
PIP3 interacts with PDK1
Phosphorylation cascade initiation
PKB/Akt or atypical PKC phosphorylation
Insulin signaling pathways regulating
glut4 translocation
PKB/Akt KO(animal study)

Insulin resistance and IGT
In vitro study

Overexpression of dominant-interfering
PKB mutant, 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
translocation
Active 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 translocation
PIP3 kinase: widely accepted necessary for glut4
translocation


Inhibition study: glut4 translocation, glucose
transport inhibited
PTEN overexpression(PIP3 lipid lipase)



Glut4 translocaiton blocked
 PIP3-K activity and PIP3 lipid product required
for glut4 translocation
PIP3-K is necessary (no sufficient)

Maybe PIP3-independent path way exist
Insulin signaling pathways regulating
glut4 translocation
IR phosphorylate the substrates Cbl & APS
Cbl interacts with Cbl associated protein(CAP)
CAP can bind to the lipid raft protein
flotillin(found in caveolin)
This binding recruits phosphorylated Cbl
Recruitment of the SH2/SH3 adaptor protein CrkII
CrkII catalyze GTP to GDP of TC10
Glut4 translocaiton
TC10 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 protein
Each isoforms of GLUTs has specific
role(tissue)
GLUT4 is key regulator of glucose
homeostasis
Cardiac/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/fusion 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 가능성
Futher studies


Identify the downstream targets of insulinmediated glut4 translocation
The specific mechnism of
budding/priming/docking/fusion of Glut4
vesicles with the p/membrane