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Insulin regulates fusion of GLUT4 vesicles independent of Exo70-mediated tethering

Lizunov, Vladimir A ; Lisinski, Ivonne ; Stenkula, Karin LU ; Zimmerberg, Joshua and Cushman, Samuel W (2009) In Journal of Biological Chemistry 284(12). p.9-7914
Abstract

Insulin regulates cellular glucose uptake by changing the amount of glucose transporter-4 (GLUT4) in the plasma membrane through stimulation of GLUT4 exocytosis. However, how the particular trafficking, tethering, and fusion steps are regulated by insulin is still debated. In a 3T3-L1 adipocyte cell line, the Exocyst complex and its Exo70 subunit were shown to critically affect GLUT4 exocytosis. Here we investigated the effects of Exo70 on tethering and fusion of GLUT4 vesicles in primary isolated rat adipose cells. We found that Exo70 wild type was sequestered away from the plasma membrane in non-stimulated cells, and its overexpression had no effect on GLUT4 trafficking. The addition of insulin increased the amount of Exo70 in the... (More)

Insulin regulates cellular glucose uptake by changing the amount of glucose transporter-4 (GLUT4) in the plasma membrane through stimulation of GLUT4 exocytosis. However, how the particular trafficking, tethering, and fusion steps are regulated by insulin is still debated. In a 3T3-L1 adipocyte cell line, the Exocyst complex and its Exo70 subunit were shown to critically affect GLUT4 exocytosis. Here we investigated the effects of Exo70 on tethering and fusion of GLUT4 vesicles in primary isolated rat adipose cells. We found that Exo70 wild type was sequestered away from the plasma membrane in non-stimulated cells, and its overexpression had no effect on GLUT4 trafficking. The addition of insulin increased the amount of Exo70 in the vicinity of the plasma membrane and stimulated the tethering and fusion of GLUT4 vesicles, but the rates of fusion and GLUT4 exposure were not affected by overexpression of Exo70. Surprisingly, the Exo70-N mutant induced insulin-independent tethering of GLUT4 vesicles, which, however, did not lead to fusion and exposure of GLUT4 at the plasma membrane. Upon insulin stimulation, the stationary pretethered GLUT4 vesicles in Exo70-N mutant cells underwent fusion without relocation. Taken together, our data suggest that fusion of GLUT4 vesicles is the rate-limiting step regulated by insulin downstream of Exo70-mediated tethering.

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author
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publishing date
type
Contribution to journal
publication status
published
keywords
3T3-L1 Cells, Adipocytes/metabolism, Animals, Biological Transport/drug effects, Cell Membrane/genetics, Cells, Cultured, Exocytosis/drug effects, Glucose Transporter Type 4/genetics, Hypoglycemic Agents/metabolism, Insulin/metabolism, Male, Membrane Fusion/drug effects, Mice, Mutation, Rats, Secretory Vesicles/genetics, Vesicular Transport Proteins/genetics
in
Journal of Biological Chemistry
volume
284
issue
12
pages
9 - 7914
publisher
American Society for Biochemistry and Molecular Biology
external identifiers
  • scopus:65549096355
  • pmid:19155211
ISSN
0021-9258
DOI
10.1074/jbc.M806460200
language
English
LU publication?
no
id
de2a511f-aff2-4e42-b691-b32e2e3b6726
date added to LUP
2021-05-20 14:35:14
date last changed
2024-03-08 13:46:15
@article{de2a511f-aff2-4e42-b691-b32e2e3b6726,
  abstract     = {{<p>Insulin regulates cellular glucose uptake by changing the amount of glucose transporter-4 (GLUT4) in the plasma membrane through stimulation of GLUT4 exocytosis. However, how the particular trafficking, tethering, and fusion steps are regulated by insulin is still debated. In a 3T3-L1 adipocyte cell line, the Exocyst complex and its Exo70 subunit were shown to critically affect GLUT4 exocytosis. Here we investigated the effects of Exo70 on tethering and fusion of GLUT4 vesicles in primary isolated rat adipose cells. We found that Exo70 wild type was sequestered away from the plasma membrane in non-stimulated cells, and its overexpression had no effect on GLUT4 trafficking. The addition of insulin increased the amount of Exo70 in the vicinity of the plasma membrane and stimulated the tethering and fusion of GLUT4 vesicles, but the rates of fusion and GLUT4 exposure were not affected by overexpression of Exo70. Surprisingly, the Exo70-N mutant induced insulin-independent tethering of GLUT4 vesicles, which, however, did not lead to fusion and exposure of GLUT4 at the plasma membrane. Upon insulin stimulation, the stationary pretethered GLUT4 vesicles in Exo70-N mutant cells underwent fusion without relocation. Taken together, our data suggest that fusion of GLUT4 vesicles is the rate-limiting step regulated by insulin downstream of Exo70-mediated tethering.</p>}},
  author       = {{Lizunov, Vladimir A and Lisinski, Ivonne and Stenkula, Karin and Zimmerberg, Joshua and Cushman, Samuel W}},
  issn         = {{0021-9258}},
  keywords     = {{3T3-L1 Cells; Adipocytes/metabolism; Animals; Biological Transport/drug effects; Cell Membrane/genetics; Cells, Cultured; Exocytosis/drug effects; Glucose Transporter Type 4/genetics; Hypoglycemic Agents/metabolism; Insulin/metabolism; Male; Membrane Fusion/drug effects; Mice; Mutation; Rats; Secretory Vesicles/genetics; Vesicular Transport Proteins/genetics}},
  language     = {{eng}},
  month        = {{03}},
  number       = {{12}},
  pages        = {{9--7914}},
  publisher    = {{American Society for Biochemistry and Molecular Biology}},
  series       = {{Journal of Biological Chemistry}},
  title        = {{Insulin regulates fusion of GLUT4 vesicles independent of Exo70-mediated tethering}},
  url          = {{http://dx.doi.org/10.1074/jbc.M806460200}},
  doi          = {{10.1074/jbc.M806460200}},
  volume       = {{284}},
  year         = {{2009}},
}