Kinetic Evidence for Unique Regulation of GLUT4 Trafficking by Insulin and AMP-activated Protein Kinase Activators in L6 Myotubes

Daniel J Fazakerley,Geoffrey D Holman,Anna Marley,David E James,Jacqueline Stöckli,Adelle C.F Coster

doi:10.1074/jbc.m109.051185

Abstract

In L6 myotubes, redistribution of a hemagglutinin (HA) epitope-tagged GLUT4 (HA-GLUT4) to the cell surface occurs rapidly in response to insulin stimulation and AMP-activated protein kinase (AMPK) activation. We have examined whether these separate signaling pathways have a convergent mechanism that leads to GLUT4 mobilization and to changes in GLUT4 recycling. HA antibody uptake on GLUT4 in the basal steady state reached a final equilibrium level that was only 81% of the insulin-stimulated level. AMPK activators (5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and A-769662) led to a similar level of antibody uptake to that found in insulin-stimulated cells. However, the combined responses to insulin stimulation and AMPK activation led to an antibody uptake level of ∼20% above the insulin level. Increases in antibody uptake due to insulin, but not AICAR or A-769662, treatment were reduced by both wortmannin and Akt inhibitor. The GLUT4 internalization rate constant in the basal steady state was very rapid (0.43 min−1) and was decreased during the steady-state responses to insulin (0.18 min−1), AICAR (0.16 min−1), and A-769662 (0.24 min−1). This study has revealed a nonconvergent mobilization of GLUT4 in response to activation of Akt and AMPK signaling. Furthermore, GLUT4 trafficking in L6 muscle cells is very reliant on regulated endocytosis for control of cell surface GLUT4 levels.

Highlights

Insulin stimulates glucose uptake into muscle and fat cells by triggering the translocation of the facilitative glucose transporter GLUT4 from intracellular storage vesicles to the plasma membrane
GLUT4 trafficking has been extensively studied in adipocytes where it has been found that the major effect of insulin is to stimulate exocytosis [1,2,3,4,5], it has been reported that insulin inhibits GLUT4 endocytosis [2, 6]
We have found that GLUT4 recycling is considerably faster in the muscle cell line L6 than in adipocytes [1,2,3,4,5]

Summary

Introduction

Insulin stimulates glucose uptake into muscle and fat cells by triggering the translocation of the facilitative glucose transporter GLUT4 from intracellular storage vesicles to the plasma membrane. We report that AMPK agonists and insulin added simultaneously to L6 myotubes resulted in additive effects on GLUT4 levels at the cell surface and in the recycling pathway, indicating the presence of distinct pools of GLUT4 in muscle cells. For measurement of steady-state trafficking of HA-GLUT4 under insulin and AICAR- and A-769662-stimulated conditions, 200 nM insulin and/or 2 mM AICAR or 100 ␮M A-769662 was added to cells 30 min before anti-HA antibody.

Results

Conclusion