Insulin-induced cortical actin remodeling promotes GLUT4 insertion at muscle cell membrane ruffles
Peter Tong, … , Atsunori Ueyama, Amira Klip
Peter Tong, … , Atsunori Ueyama, Amira Klip
Published August 1, 2001
Citation Information: J Clin Invest. 2001;108(3):371-381. https://doi.org/10.1172/JCI12348.
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Article

Insulin-induced cortical actin remodeling promotes GLUT4 insertion at muscle cell membrane ruffles

Abstract

Insulin stimulates glucose uptake by recruiting glucose transporter 4 (GLUT4) from an intracellular compartment to the cell surface; this phenomenon is defective in type 2 diabetes. Here we examine the involvement of actin filaments in GLUT4 translocation and their possible defects in insulin resistance, using L6 myotubes expressing myc-tagged GLUT4. Insulin caused membrane ruffling, a dynamic distortion of the myotube dorsal surface. Fluorescence microscopy and immunogold staining of surface GLUT4myc coupled to backscatter electron microscopy revealed a high density of this protein in membrane ruffles. The t-SNAREs syntaxin4 and SNAP-23 were also abundant in these regions. Below the membrane, GLUT4 and the vesicular protein VAMP2, but not VAMP3, colocalized with the actin structures supporting the membrane ruffles. GLUT4myc externalization and membrane ruffles were reduced by jasplakinolide and by swinholide-A, drugs that affect actin filament stability and prevent actin branching, respectively. Insulin resistance generated by prolonged (24 hours) exposure of myotubes to high glucose and insulin diminished the acute insulin-dependent remodeling of cortical actin and GLUT4myc translocation, reminiscent of the effect of swinholide-A. We propose that GLUT4 vesicle incorporation into the plasma membrane involves insulin-dependent cortical actin remodeling and that defective actin remodeling contributes to insulin resistance.

Authors

Peter Tong, Zayna A. Khayat, Carol Huang, Nish Patel, Atsunori Ueyama, Amira Klip

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Figure 2

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Dorsal actin-rich structures cause the membrane ruffling in L6 myotubes....
Dorsal actin-rich structures cause the membrane ruffling in L6 myotubes. Serum-deprived L6 GLUT4myc myotubes were left untreated (a and c) or stimulated with 100 nM insulin for 10 minutes (b and d) at 37°C. The plasma membrane surface was then stained using FITC-conjugated Con-A (25 μg/mL for 30 minutes at 4°C), followed by permeabilization and staining for F-actin using rhodamine-phalloidin, as described in Methods. Scale bar = 10 μm. Arrows indicate regions of membrane ruffling. The images are representative of three experiments.