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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
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
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)