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Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice

Lizunov, Vladimir A.; Stenkula, Karin LU ; Lisinski, Ivonne; Gavrilova, Oksana; Yver, Dena R.; Chadt, Alexandra; Al-Hasani, Hadi; Zimmerberg, Joshua and Cushman, Samuel W. (2012) In American Journal of Physiology: Endocrinology and Metabolism 302(8). p.950-960
Abstract
Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice. Am J Physiol Endocrinol Metab 302: E950-E960, 2012. First published January 31, 2012; doi:10.1152/ajpendo.00466.2011.-Insulin regulates glucose uptake into fat and muscle by modulating the subcellular distribution of GLUT4 between the cell surface and intracellular compartments. However, quantification of these translocation processes in muscle by classical subcellular fractionation techniques is confounded by contaminating microfibrillar protein; dynamic studies at the molecular level are almost impossible. In this study, we introduce a muscle-specific transgenic mouse model in which HA-GLUT4-GFP is expressed under the... (More)
Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice. Am J Physiol Endocrinol Metab 302: E950-E960, 2012. First published January 31, 2012; doi:10.1152/ajpendo.00466.2011.-Insulin regulates glucose uptake into fat and muscle by modulating the subcellular distribution of GLUT4 between the cell surface and intracellular compartments. However, quantification of these translocation processes in muscle by classical subcellular fractionation techniques is confounded by contaminating microfibrillar protein; dynamic studies at the molecular level are almost impossible. In this study, we introduce a muscle-specific transgenic mouse model in which HA-GLUT4-GFP is expressed under the control of the MCK promoter. HA-GLUT4-GFP was found to translocate to the plasma membrane and T-tubules after insulin stimulation, thus mimicking endogenous GLUT4. To investigate the dynamics of GLUT4 trafficking in skeletal muscle, we quantified vesicles containing HA-GLUT4-GFP near the sarcolemma and T-tubules and analyzed insulin-stimulated exocytosis at the single vesicle level by total internal reflection fluorescence and confocal microscopy. We found that only 10% of the intracellular GLUT4 pool comprised mobile vesicles, whereas most of the GLUT4 structures remained stationary or tethered at the sarcolemma or T-tubules. In fact, most of the insulin-stimulated exocytosis emanated from pretethered vesicles, whereas the small pool of mobile GLUT4 vesicles was not significantly affected by insulin. Our data strongly suggest that the mobile pool of GLUT4 vesicles is not a major site of insulin action but rather locally distributed. Most likely, pretethered GLUT4 structures are responsible for the initial phase of insulin-stimulated exocytosis. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
hemagglutinin, glucose transporter 4, green fluorescent protein, insulin, fusion
in
American Journal of Physiology: Endocrinology and Metabolism
volume
302
issue
8
pages
950 - 960
publisher
American Physiological Society
external identifiers
  • wos:000302730600006
  • scopus:84859476275
ISSN
1522-1555
DOI
10.1152/ajpendo.00466.2011
language
English
LU publication?
yes
id
173b9b9c-fb76-4893-9ff1-6a13f9fac1f2 (old id 2571050)
date added to LUP
2012-06-01 08:53:27
date last changed
2017-11-19 03:52:26
@article{173b9b9c-fb76-4893-9ff1-6a13f9fac1f2,
  abstract     = {Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice. Am J Physiol Endocrinol Metab 302: E950-E960, 2012. First published January 31, 2012; doi:10.1152/ajpendo.00466.2011.-Insulin regulates glucose uptake into fat and muscle by modulating the subcellular distribution of GLUT4 between the cell surface and intracellular compartments. However, quantification of these translocation processes in muscle by classical subcellular fractionation techniques is confounded by contaminating microfibrillar protein; dynamic studies at the molecular level are almost impossible. In this study, we introduce a muscle-specific transgenic mouse model in which HA-GLUT4-GFP is expressed under the control of the MCK promoter. HA-GLUT4-GFP was found to translocate to the plasma membrane and T-tubules after insulin stimulation, thus mimicking endogenous GLUT4. To investigate the dynamics of GLUT4 trafficking in skeletal muscle, we quantified vesicles containing HA-GLUT4-GFP near the sarcolemma and T-tubules and analyzed insulin-stimulated exocytosis at the single vesicle level by total internal reflection fluorescence and confocal microscopy. We found that only 10% of the intracellular GLUT4 pool comprised mobile vesicles, whereas most of the GLUT4 structures remained stationary or tethered at the sarcolemma or T-tubules. In fact, most of the insulin-stimulated exocytosis emanated from pretethered vesicles, whereas the small pool of mobile GLUT4 vesicles was not significantly affected by insulin. Our data strongly suggest that the mobile pool of GLUT4 vesicles is not a major site of insulin action but rather locally distributed. Most likely, pretethered GLUT4 structures are responsible for the initial phase of insulin-stimulated exocytosis.},
  author       = {Lizunov, Vladimir A. and Stenkula, Karin and Lisinski, Ivonne and Gavrilova, Oksana and Yver, Dena R. and Chadt, Alexandra and Al-Hasani, Hadi and Zimmerberg, Joshua and Cushman, Samuel W.},
  issn         = {1522-1555},
  keyword      = {hemagglutinin,glucose transporter 4,green fluorescent protein,insulin,fusion},
  language     = {eng},
  number       = {8},
  pages        = {950--960},
  publisher    = {American Physiological Society},
  series       = {American Journal of Physiology: Endocrinology and Metabolism},
  title        = {Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice},
  url          = {http://dx.doi.org/10.1152/ajpendo.00466.2011},
  volume       = {302},
  year         = {2012},
}