Format

Send to

Choose Destination
Biochem Biophys Res Commun. 2001 Sep 21;287(2):445-54.

Insulin-regulated trafficking of dual-labeled glucose transporter 4 in primary rat adipose cells.

Author information

1
Experimental Diabetes, Metabolism, and Nutrition Section, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Bethesda, Maryland 20892-0842, USA.

Abstract

In isolated rat adipose cells, physiologically relevant insulin target cells, glucose transporter 4 (GLUT4) subcellular trafficking can be assessed by transfection of exofacially HA-tagged GLUT4. To simultaneously visualize the transfected GLUT4, we fused GFP with HA-GLUT4. With the resulting chimeras, GFP-HA-GLUT4 and HA-GLUT4-GFP, we were able to visualize for the first time the cell-surface localization, total expression, and intracellular distribution of GLUT4 in a single cell. Confocal microscopy reveals that the intracellular proportions of both GFP-HA-GLUT4 and HA-GLUT4-GFP are properly targeted to the insulin-responsive aminopeptidase-positive vesicles. Dynamic studies demonstrate close similarities in the trafficking kinetics between the two constructs and with native GLUT4. However, while the basal subcellular distribution of HA-GLUT4-GFP and the response to insulin are indistinguishable from those of HA-GLUT4 and endogenous GLUT4, most of the GFP-HA-GLUT4 is targeted to the plasma membrane with little further insulin response. Thus, HA-GLUT4-GFP will be useful to study GLUT4 trafficking in vivo while GFP on the N-terminus interferes with intracellular retention.

PMID:
11554749
DOI:
10.1006/bbrc.2001.5620
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center