α-Actinin-4 Is Selectively Required for Insulin-induced GLUT4 Translocation

Ilana Talior-Volodarsky,Varinder K Randhawa,Hilal Zaid,Amira Klip

doi:10.1074/jbc.m801750200

Ilana Talior-Volodarsky, Varinder K Randhawa + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m801750200

Copy DOI

Abstract

Insulin induces GLUT4 translocation to the muscle cell surface. Using differential amino acid labeling and mass spectrometry, we observed insulin-dependent co-precipitation of actinin-4 (ACTN4) with GLUT4 (Foster, L. J., Rudich, A., Talior, I., Patel, N., Huang, X., Furtado, L. M., Bilan, P. J., Mann, M., and Klip, A. (2006) J. Proteome Res. 5, 64-75). ACTN4 links F-actin to membrane proteins, and actin dynamics are essential for GLUT4 translocation. We hypothesized that ACTN4 may contribute to insulin-regulated GLUT4 traffic. In L6 muscle cells insulin, but not platelet-derived growth factor, increased co-precipitation of ACTN4 with GLUT4. Small interfering RNA-mediated ACTN4 knockdown abolished the gain in surface-exposed GLUT4 elicited by insulin but not by platelet-derived growth factor, membrane depolarization, or mitochondrial uncoupling. In contrast, knockdown of alpha-actinin-1 (ACTN1) did not prevent GLUT4 translocation by insulin. GLUT4 colocalized with ACTN4 along the insulin-induced cortical actin mesh and ACTN4 knockdown prevented GLUT4-actin colocalization without impeding actin remodeling or Akt phosphorylation, maintaining GLUT4 in a tight perinuclear location. We propose that ACTN4 contributes to GLUT4 traffic, likely by tethering GLUT4 vesicles to the cortical actin cytoskeleton.

Highlights

Insulin-dependent GLUT4 translocation requires dynamic remodeling of filamentous actin (20 –23), the gain in surface GLUT4 elicited by plateletderived growth factor (PDGF), depolarization, or mitochondrial uncouplers is independent of actin dynamics (24 –26)
PDGF did not increase the binding of ACTN4 to GLUT4myc (Fig. 1B), highlighting the specificity of insulin on this association
These experiments were tested in myoblasts, as the PDGF receptor is only expressed at this stage of the myogenic process

Summary

Introduction

This treatment prevented the insulin-stimulated association of GLUT4myc with ACTN4 (Fig. 1C), offering additional support for the specificity of this interaction in response to insulin. ACTN4 Knockdown Reduces Insulin-stimulated Glucose Transport and Surface GLUT4 Gain—To assess the functional relevance of ACTN4 to GLUT4 traffic, we explored the effects of silencing ACTN4 expression using a specific small interfering RNA oligonucleotide (siACTN4). Significant knockdown (60 Ϯ 2%) of ACTN4 protein was achieved in siACTN4 (Fig. 2A) relative to NT or non-related control siRNA (siNR)-treated cells.

Results

Conclusion