Immunocytochemical Evidence that GLUT4 Resides in a Specialized Translocation Post-endosomal VAMP2-positive Compartment in Rat Adipose Cells in the Absence of Insulin

Malide, Daniela; Nk, Dwyer; Blanchette‐Mackie, E. Joan; Sw, Cushman

doi:10.1177/002215549704500806

Cited by 91 publications

(84 citation statements)

References 67 publications

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“…Randhawa et al 1996;Martin et al, 1996Martin et al, , 1998Malide et al, 1997), whereas VAMP3 has been detected in the recycling endosome and was in part responsible for TfR recycling (Galli et al, 1994;Martin et al, 1996).…”

Section: Vamp2 and Vamp3 Segregate Into Distinct Intracellular Comparmentioning

confidence: 99%

VAMP2, but Not VAMP3/Cellubrevin, Mediates Insulin-dependent Incorporation of GLUT4 into the Plasma Membrane of L6 Myoblasts

Randhawa

Bilan

Khayat

et al. 2000

MBoC

102

117

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Like neuronal synaptic vesicles, intracellular GLUT4-containing vesicles must dock and fuse with the plasma membrane, thereby facilitating insulin-regulated glucose uptake into muscle and fat cells. GLUT4 colocalizes in part with the vesicle SNAREs VAMP2 and VAMP3. In this study, we used a single-cell fluorescence-based assay to compare the functional involvement of VAMP2 and VAMP3 in GLUT4 translocation. Transient transfection of proteolytically active tetanus toxin light chain cleaved both VAMP2 and VAMP3 proteins in L6 myoblasts stably expressing exofacially myc-tagged GLUT4 protein and inhibited insulin-stimulated GLUT4 translocation. Tetanus toxin also caused accumulation of the remaining C-terminal VAMP2 and VAMP3 portions in Golgi elements. This behavior was exclusive to these proteins, because the localization of intracellular myc-tagged GLUT4 protein was not affected by the toxin. Upon cotransfection of tetanus toxin with individual vesicle SNARE constructs, only toxin-resistant VAMP2 rescued the inhibition of insulin-dependent GLUT4 translocation by tetanus toxin. Moreover, insulin caused a cortical actin filament reorganization in which GLUT4 and VAMP2, but not VAMP3, were clustered. We propose that VAMP2 is a resident protein of the insulin-sensitive GLUT4 compartment and that the integrity of this protein is required for GLUT4 vesicle incorporation into the cell surface in response to insulin.

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Section: Vamp2 and Vamp3 Segregate Into Distinct Intracellular Comparmentioning

confidence: 99%

VAMP2, but Not VAMP3/Cellubrevin, Mediates Insulin-dependent Incorporation of GLUT4 into the Plasma Membrane of L6 Myoblasts

Randhawa

Bilan

Khayat

et al. 2000

MBoC

102

117

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“…Third, there are biochemical (Holman et al, 1994;Livingstone et al, 1996;Martin et al, 1996;Wei et al, 1998;Bogan and Lodish, 1999;Lee et al, 1999;Millar et al, 1999;Hashiramoto and James, 2000) and morphological (Slot et al, 1991a;Slot et al, 1991b;Martin et al, 1996;Malide et al, 1997a;Malide et al, 1997b;Martin et al, 1997) experiments indicating that GLUT4 localizes to specialized compartments. Although, most studies agree on the presence of a major GLUT4 compartment distinct from endosomes and the TGN, the degree of overlap is variable dependent on the experimental method or cell type used, e.g., isolated rat adipocytes versus 3T3-L1 adipocytes Martin et al 1996;Malide et al, 1997a;Bogan and Lodish, 1999;Lee et al, 1999;Millar et al, 1999;Hashiramoto and James, 2000). An insulin-responsive aminopeptidase, IRAP (Ross et al, 1996;Malide et al 1997b;Martin et al, 1997;Ross et al, 1997;Elmendorf et al, 1999;Garza and Birnbaum, 2000), and a V-SNARE, VAMP2, are colocalized with GLUT4 in this discrete intracellular compartment.…”

mentioning

confidence: 99%

Insulin Recruits GLUT4 from Specialized VAMP2-carrying Vesicles as well as from the Dynamic Endosomal/Trans-Golgi Network in Rat Adipocytes.

Ramm

Slot

James

et al. 2000

MBoC

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Insulin treatment of fat cells results in the translocation of the insulin-responsive glucose transporter type 4, GLUT4, from intracellular compartments to the plasma membrane. However, the precise nature of these intracellular GLUT4-carrying compartments is debated. To resolve the nature of these compartments, we have performed an extensive morphological analysis of GLUT4-containing compartments, using a novel immunocytochemical technique enabling high labeling efficiency and 3-d resolution of cytoplasmic rims isolated from rat epididymal adipocytes. In basal cells, GLUT4 was localized to three morphologically distinct intracellular structures: small vesicles, tubules, and vacuoles. In response to insulin the increase of GLUT4 at the cell surface was compensated by a decrease in small vesicles, whereas the amount in tubules and vacuoles was unchanged. Under basal conditions, many small GLUT4 positive vesicles also contained IRAP (88%) and the v-SNARE, VAMP2 (57%) but not markers of sorting endosomes (EEA1), late endosomes, or lysosomes (lgp120). A largely distinct population of GLUT4 vesicles (56%) contained the cation-dependent mannose 6-phosphate receptor (CD-MPR), a marker protein that shuttles between endosomes and the trans-Golgi network (TGN). In response to insulin, GLUT4 was recruited both from VAMP2 and CD-MPR positive vesicles. However, while the concentration of GLUT4 in the remaining VAMP2-positive vesicles was unchanged, the concentration of GLUT4 in CD-MPR-positive vesicles decreased. Taken together, we provide morphological evidence indicating that, in response to insulin, GLUT4 is recruited to the plasma membrane by fusion of preexisting VAMP2-carrying vesicles as well as by sorting from the dynamic endosomal-TGN system. INTRODUCTIONInsulin stimulates glucose transport in adipocytes and myocytes by triggering the translocation of the glucose transporter GLUT4 (Birnbaum, 1989;Charron et al., 1989;James et al., 1989) from intracellular locations to the cell surface (Cushman and Wardzala, 1980;Suzuki and Kono, 1980). Establishing the precise nature of the intracellular compartment(s) from which GLUT4 is recruited to the cell surface will provide major insight into the molecular basis for this effect. Insulin regulates both endosomal and trans-Golgi network (TGN) trafficking in a variety of cell types. For example, in adipocytes, insulin triggers translocation of transmembrane proteins other than GLUT4 to the cell surface, such as the ubiquitously expressed glucose transporter GLUT1 (Piper et al., 1991), the cation-independent mannose-6-phosphate receptor (MPR;Appell et al., 1988) and the transferrin-receptor (TfR; Tanner and Lienhard, 1987). However, several observations suggest that the effects of insulin on GLUT4 trafficking may be unique compared with its effects on endosomal and/or TGN trafficking. First, insulin effects on the endosomal/TGN system are modest compared with GLUT4 trafficking. The surface expression of molecules that are thought to transit the TGN/endosomes, such as GLUT1, MPR, a...

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“…These and other morphological and biochemical data [14][15][16] suggest that the interaction of GLUT4 with endosomal, endosome-like or post-endosomal compartments may be part of the mechanism that maintains the unique distribution of GLUT4, resulting in a principally intracellular localization in the steady state. A typical characteristic of eukaryotic endosomes, along with other organelles (i.e.…”

Section: Introductionmentioning

confidence: 65%

“…Thus GLUT4 vesicles derived from insulin-stimulated fat-cell intracellular membranes display changes in density [41], consistent with insulin-induced changes in membrane protein\lipid composition of these vesicles. Although the exact nature of intracellular GLUT4 vesicles and the different membrane pools that contain GLUT4 have not yet been fully elucidated, morphological studies visualize GLUT4 membranes essentially as populations of endosomes, endosome-like or post-endosomal structures sequestered intracellularly in resting insulin-sensitive cells [12][13][14][15][16].…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Arrest of endosome acidification by bafilomycin A1 mimics insulin action on GLUT4 translocation in 3T3-L1 adipocytes

Chinni

Shisheva²

1999

Biochemical Journal

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In insulin-sensitive fat and muscle cells, the major glucose transporter GLUT4 is constitutively sequestered in endosomal tubulovesicular membranes, and moves to the cell surface in response to insulin. While sequence information within GLUT4 appears to be responsible for its constitutive intracellular sequestration, the regulatory elements and mechanisms that enable this protein to achieve its unique sorting pattern under basal and insulin-stimulated conditions are poorly understood. We show here that arrest of endosome acidification in insulin-sensitive 3T3-L1 adipocytes by bafilomycin A " , a specific inhibitor of the vacuolar proton pump, results in the rapid and dose-dependent translocation of GLUT4 from the cell interior to the membrane surface ; the effects of maximally stimulatory concentrations of bafilomycin A " (400-800 nM) were equivalent to 50-65 % of the effects of acute insulin treatment. Like insulin, bafilomycin A "

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Immunocytochemical Evidence that GLUT4 Resides in a Specialized Translocation Post-endosomal VAMP2-positive Compartment in Rat Adipose Cells in the Absence of Insulin

Cited by 91 publications

References 67 publications

VAMP2, but Not VAMP3/Cellubrevin, Mediates Insulin-dependent Incorporation of GLUT4 into the Plasma Membrane of L6 Myoblasts

VAMP2, but Not VAMP3/Cellubrevin, Mediates Insulin-dependent Incorporation of GLUT4 into the Plasma Membrane of L6 Myoblasts

Insulin Recruits GLUT4 from Specialized VAMP2-carrying Vesicles as well as from the Dynamic Endosomal/Trans-Golgi Network in Rat Adipocytes.

Arrest of endosome acidification by bafilomycin A1 mimics insulin action on GLUT4 translocation in 3T3-L1 adipocytes

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