scholarly journals Actin filaments play a critical role in insulin-induced exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes

2000 ◽  
Vol 346 (2) ◽  
pp. 321-328 ◽  
Author(s):  
Waka OMATA ◽  
Hiroshi SHIBATA ◽  
Lu LI ◽  
Kuniaki TAKATA ◽  
Itaru KOJIMA
Keyword(s):  
Plasma Membrane ◽  
Glucose Transport ◽  
Actin Filaments ◽  
Insulin Action ◽  
Cytochalasin D ◽  
Glut4 Translocation ◽  
Intracellular Pool ◽  
Rat Adipocytes ◽  
Latrunculin A ◽  
Isolated Rat

Actin-based cytoskeletons have been implicated in insulin-stimulated glucose transport and translocation of the insulin-regulated glucose transporter, GLUT4, from the intracellular pool to the plasma membrane. However, most previous studies were done using adherent cell systems such as L6 myotubes and 3T3-L1 adipocytes, and very little information is available on the significance of the actin filaments to the insulin action in isolated adipocytes, a widely used experimental system. In the present study, we investigated the physiological role of actin filaments in the subcellular trafficking of GLUT4 in isolated rat adipocytes. We first compared the effects of two actin-disrupting reagents, latrunculin A and cytochalasin D, on the organization of the actin filaments as well as on the insulin action on glucose transport by laser confocal microscopy combined with biochemical analysis of the insulin action. Treatment of the cells with latrunculin A induced dose- and time-dependent disappearance of the filamentous actin, which correlated very well with inhibition of the insulin effect on glucose transport. Although cytochalasin D at 50 μM significantly inhibited insulin-stimulated glucose transport, it was not effective in disassembly of the actin filaments; rather, many intense punctate signals were observed in cytochalasin D-treated cells. In the actin-disrupted adipocytes treated with latrunculin A, insulin-induced GLUT4 translocation was inhibited completely. In addition, latrunculin A remarkably inhibited both insulin-induced glucose transport and GLUT4 translocation in the presense of Dk-(62-85), a potent inhibitor of GLUT4 endocytosis, suggesting that intactness of the actin filaments was necessary for insulin-induced exocytosis of the GLUT4-containing vesicles. On the other hand, latrunculin A showed little inhibitory effect on either endocytosis of the trypsin-cleaved 35-kDa fragment of GLUT4 or decay of the glucose transport activity after addition of wortmannin in insulin-stimulated cells. The results of our experiment show clearly that, in rat adipocytes, (i) latrunculin A may be a more suitable tool than cytochalasin D for disruption of actin filaments, and (ii) actin filaments play a crucial role in exocytotic recruitment of GLUT4 to the plasma membrane from the intracellular pool, but not in its endocytosis.

scholarly journals Proteolytic cleavage of cellubrevin and vesicle-associated membrane protein (VAMP) by tetanus toxin does not impair insulin-stimulated glucose transport or GLUT4 translocation in rat adipocytes

1997 ◽  
Vol 321 (1) ◽  
pp. 233-238 ◽  
Author(s):  
Eric HAJDUCH ◽  
J. Carlos ALEDO ◽  
Colin WATTS ◽  
Harinder S. HUNDAL
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Tetanus Toxin ◽  
Glucose Transporter ◽  
Detectable Effect ◽  
Glut4 Translocation ◽  
Rat Adipocytes ◽  
Isolated Rat

Acute insulin stimulation of glucose transport in fat and skeletal muscle occurs principally as a result of the hormonal induced translocation of the GLUT4 glucose transporter from intracellular vesicular stores to the plasma membrane. The precise mechanisms governing the fusion of GLUT4 vesicles with the plasma membrane are very poorly understood at present but may share some similarities with synaptic vesicle fusion, as vesicle-associated membrane protein (VAMP) and cellubrevin, two proteins implicated in the process of membrane fusion, are resident in GLUT4-containing vesicles isolated from rat and murine 3T3-L1 adipocytes respectively. In this study we show that proteolysis of both cellubrevin and VAMP, induced by electroporation of isolated rat adipocytes with tetanus toxin, does not impair insulin-stimulated glucose transport or GLUT4 translocation. The hormone was found to stimulate glucose uptake by approx. 16-fold in freshly isolated rat adipocytes. After a single electroporating pulse, the ability of insulin to activate glucose uptake was lowered, but the observed stimulation was nevertheless nearly 5-fold higher than the basal rate of glucose uptake. Electroporation of adipocytes with 600 nM tetanus toxin resulted in a complete loss of both cellubrevin and VAMP expression within 60 min. However, toxin-mediated proteolysis of both these proteins had no effect on the ability of insulin to stimulate glucose transport which was elevated approx. 5-fold, an activation of comparable magnitude to that observed in cells electroporated without tetanus toxin. The lack of any significant change in insulin-stimulated glucose transport was consistent with the finding that toxin-mediated proteolysis of both cellubrevin and VAMP had no detectable effect on insulin-induced translocation of GLUT4 in adipocytes. Our findings indicate that, although cellubrevin and VAMP are resident proteins in adipocyte GLUT4-containing vesicles, they are not required for the acute insulin-induced delivery of GLUT4 to the plasma membrane.

scholarly journals Role of Insulin-dependent Cortical Fodrin/Spectrin Remodeling in Glucose Transporter 4 Translocation in Rat Adipocytes

2006 ◽  
Vol 17 (10) ◽  
pp. 4249-4256 ◽  
Author(s):  
Libin Liu ◽  
Mark P. Jedrychowski ◽  
Steven P. Gygi ◽  
Paul F. Pilch
Keyword(s):  
Plasma Membrane ◽  
Glucose Transporter ◽  
Cytochalasin D ◽  
Resting Cells ◽  
Glut4 Translocation ◽  
Glucose Transporter 4 ◽  
Rat Adipocytes ◽  
Protein Receptor ◽  
Latrunculin A ◽  
Syntaxin 4

Fodrin or nonerythroid spectrin is an abundant component of the cortical cytoskeletal network in rat adipocytes. Fodrin has a highly punctate distribution in resting cells, and insulin causes a dramatic remodeling of fodrin to a more diffuse pattern. Insulin-mediated remodeling of actin occurs to a lesser extent than does that of fodrin. We show that fodrin interacts with the t-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 4, and this interaction is increased by insulin stimulation and decreased by prior latrunculin A treatment. Latrunculin A disrupts all actin filaments, inhibits glucose transporter 4 (GLUT4) translocation, and causes fodrin to partially redistribute from the plasma membrane to the cytosol. In contrast, cytochalasin D disrupts only the short actin filament signal, and cytochalasin D neither inhibits GLUT4 translocation nor fodrin redistribution in adipocytes. Together, our data suggest that insulin induces remodeling of the fodrin–actin network, which is required for the fusion of GLUT4 storage vesicles with the plasma membrane by permitting their access to the t-SNARE syntaxin 4.

Effect of purified phospholipases on glucose transport, insulin binding, and insulin action in isolated rat adipocytes

1982 ◽  
Vol 4 (4) ◽  
pp. 261-271 ◽  
Author(s):  
Tj. Wieringa ◽  
G. Bruin ◽  
W. P. M. Meerwijk ◽  
H. M. J. Krans
Keyword(s):  
Glucose Transport ◽  
Insulin Action ◽  
Insulin Binding ◽  
Rat Adipocytes ◽  
Isolated Rat

Effect of purified phospholipases on glucose transport, insulin binding, and insulin action in isolated rat adipocytes

1982 ◽  
Vol 14 (4) ◽  
pp. 261-271
Author(s):  
Tj. Wieringa ◽  
G. de Bruin ◽  
W. P. M. van Meerwijk ◽  
H. M. J. Krans
Keyword(s):  
Glucose Transport ◽  
Insulin Action ◽  
Insulin Binding ◽  
Rat Adipocytes ◽  
Isolated Rat

Immunoelectron microscopic evidence that GLUT4 translocation explains the stimulation of glucose transport in isolated rat white adipose cells

2000 ◽  
Vol 113 (23) ◽  
pp. 4203-4210 ◽  
Author(s):  
D. Malide ◽  
G. Ramm ◽  
S.W. Cushman ◽  
J.W. Slot
Keyword(s):  
Adipose Tissue ◽  
Glucose Transport ◽  
Morphological Evidence ◽  
Glut4 Translocation ◽  
Transport Activity ◽  
Brown Adipose ◽  
Quantitative Immunoelectron Microscopy ◽  
Adipose Cells ◽  
Isolated Rat ◽  
Stimulation Of

We used an improved cryosectioning technique in combination with quantitative immunoelectron microscopy to study GLUT4 compartments in isolated rat white adipose cells. We provide clear evidence that in unstimulated cells most of the GLUT4 localizes intracellularly to tubulovesicular structures clustered near small stacks of Golgi and endosomes, or scattered throughout the cytoplasm. This localization is entirely consistent with that originally described in brown adipose tissue, strongly suggesting that the GLUT4 compartments in white and brown adipose cells are morphologically similar. Furthermore, insulin induces parallel increases (with similar magnitudes) in glucose transport activity, approximately 16-fold, and cell-surface GLUT4, approximately 12-fold. Concomitantly, insulin decreases GLUT4 equally from all intracellular locations, in agreement with the concept that the entire cellular GLUT4 pool contributes to insulin-stimulated exocytosis. In the insulin-stimulated state, GLUT4 molecules are not randomly distributed on the plasma membrane, but neither are they enriched in caveolae. Importantly, the total number of GLUT4 C-terminal epitopes detected by the immuno-gold method is not significantly different between basal and insulin-stimulated cells, thus arguing directly against a reported insulin-induced unmasking effect. These results provide strong morphological evidence (1) that GLUT4 compartments are similar in all insulin-sensitive cells and (2) for the concept that GLUT4 translocation almost fully accounts for the increase in glucose transport in response to insulin.

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