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.

Septin 7 reduces nonmuscle myosin IIA activity in the SNAP23 complex and hinders GLUT4 storage vesicle docking and fusion

“…We can not rule out the possibility that NM-IIA regulates the final steps of GSV trafficking also via modulating actin dynamics, as the activity of NM-IIA is necessary for F-actin localization at the plasma membrane in adipocytes [36], and knockdown of NMHC-IIA leads to loss of actin stress fiber organization in podocytes [37]. Defective actin remodeling impairs the fusion of GSVs with the plasma membrane in adipocytes [38] and muscle cells [39], and glucose uptake depends on an intact actin cytoskeleton also in podocytes [8]. NM-IIA also regulates the intrinsic activity of GLUT4 at the plasma membrane after insulin stimulation [14], and also other proteins may, either positively or negatively, regulate GLUT4 activity [40], indicating the complexity of GLUT4 regulation.…”

Arp2/3- and Cofilin-coordinated Actin Dynamics Is Required for Insulin-mediated GLUT4 Translocation to the Surface of Muscle Cells

“…Indeed, in insulin-stimulated muscle cells, GLUT4 vesicles accumulate within the cortical actin mesh visualized by electron microscopy (Tong et al , 2001; Randhawa et al , 2008). GLUT4 itself may tether to actin filaments via α-actinin-4 (Foster et al , 2006; Talior-Volodarsky et al , 2008), and indeed upon α-actinin-4 silencing, GLUT4 vesicles do not accumulate at the muscle cell periphery, and there is no insulin-dependent gain in surface GLUT4 (Talior-Volodarsky et al , 2008).…”

Cortactin, an actin binding protein, regulates GLUT4 translocation via actin filament remodeling

“…In adipocytes, both microtubules and actin stress fibers contribute to GLUT4 translocation as disruption of either myosin Myo1c, an actin motor, or kinesins KIF3 or KIF5b, which are microtubule motors, impairs insulin-stimulated GLUT4 translocation [13–17]. In contrast, disruption of actin fibers in L6 muscle cells with jasplakinolide or inhibition of actin branching with swinholide A blocks GLUT4 translocation and actin ruffling, but disruption of microtubules with colchicine has no effect suggesting that actin fibers represent the major route for GLUT4 vesicle trafficking in these cells [18, 19]. GLUT4 vesicles isolated from 3T3-L1 cells contain actin-polymerizing activity and demonstrate actin comet tails in vitro [20].…”