Insulin stimulates glucose transport in adipose and muscle tissue by stimulating the movement (‘translocation’) of an intracellular pool of glucose transporters (the Glut4 isoform) to the plasma membrane. We have engineered a series of chimaeras between Glut4 and green fluorescent protein (GFP) from Aequoria victoria and expressed these proteins in 3T3-L1 adipocytes by microinjection of plasmid cDNA. In the absence of insulin, GFP-Glut4 is localized intracellularly within a perinuclear compartment and multiple intracellular punctate structures. In response to insulin, chimaeric GFP-Glut4 species exhibit a profound redistribution to the cell surface with kinetics comparable with the endogenous protein. The intracellular localization of GFP-Glut4 overlaps partially with compartments labelled with Texas Red transferrin, but is largely distinct from intracellular structures identified using Lysotracker-Red®. K+-depletion resulted in the accumulation of GFP-Glut4 at the cell surface, but to an lesser extent than that observed in response to insulin. In contrast with native Glut4, removal of the insulin stimulus or treatment of insulin-stimulated cells with phosphatidylinositol 3′-kinase inhibitors did not result in re-internalization of the chimaeric GFP-Glut4 from the plasma membrane, suggesting that the recycling properties of this species differ from the native Glut4 molecule. We suggest that the recycling pathway utilized by GFP-Glut4 in the absence of insulin is distinct from that used to internalize GFP-Glut4 from the plasma membrane after withdrawal of the insulin stimulus, which may reflect distinct pathways for internalization of endogenous Glut4 in the presence or absence of insulin.
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December 1999
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Research Article|
November 24 1999
Trafficking of Glut4–Green Fluorescent Protein chimaeras in 3T3-L1 adipocytes suggests distinct internalization mechanisms regulating cell surface Glut4 levels
Kate A. POWELL;
*Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
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Lachlan C. CAMPBELL;
Lachlan C. CAMPBELL
2
*Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
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Jeremy M. TAVARÉ;
Jeremy M. TAVARÉ
†Department of Biochemistry, University of Bristol, University Walk, Bristol BS8, U.K.
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David P. LEADER;
David P. LEADER
*Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
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Jill A. WAKEFIELD;
Jill A. WAKEFIELD
*Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
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Gwyn W. GOULD
Gwyn W. GOULD
1
*Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
1To whom correspondence should be addressed (e-mail G.Gould@bio.gla.ac.uk).
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Publisher: Portland Press Ltd
Received:
June 30 1999
Revision Received:
September 01 1999
Accepted:
September 21 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 344 (2): 535–543.
Article history
Received:
June 30 1999
Revision Received:
September 01 1999
Accepted:
September 21 1999
Citation
Kate A. POWELL, Lachlan C. CAMPBELL, Jeremy M. TAVARÉ, David P. LEADER, Jill A. WAKEFIELD, Gwyn W. GOULD; Trafficking of Glut4–Green Fluorescent Protein chimaeras in 3T3-L1 adipocytes suggests distinct internalization mechanisms regulating cell surface Glut4 levels. Biochem J 1 December 1999; 344 (2): 535–543. doi: https://doi.org/10.1042/bj3440535
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