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.

Modulation of the caveolin-3 and Akt status in caveolae by insulin resistance in H9c2 cardiomyoblasts

“…These results are consistent with recent studies reporting that high glucose treatment alone did not impair insulin-induced glucose uptake in adipocytes and skeletal muscle (Nelson et al, 2000;Kawanaka et al, 2001). However, an experimental condition of high glucose (25 mM) plus high insulin (100 nM) for 24 h exposure, demonstrated to produce insulin resistance in L6 myoblasts and 3T3-L1 adipocytes (Thomson et al, 1997;Tong et al, 2001), significantly decreased the insulinstimulated glucose uptake in H9c2 cells (Figure 2). It has been shown that insulin promotes membrane ruffling by a rapid remodeling of actin filaments and that the disruption of the actin cytoskeleton inhibited insulin-mediated GLUT4 translocation in myoblasts, fibroblasts and adipocytes (Tsakiridis et al, 1994;Clodi et al, 1998;Wang et al, 1998;Olson et al, 2001;Tong et al, 2001).…”

“…To test an experimental hyperglycemic condition, cells were incubated in DMEM containing 25 mM glucose supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml) for 0.5 or 24 h. Cells were serum starved for 24 h in DMEM containing 5 mM (control) or 25 mM (hyperglycemic condition) glucose, supplemented with 0.5% bovine serum albumin (BSA) (Fraction V; Sigma) and then compared to each other by incubating with or without 100 nM insulin (Porcine, Sigma I0259) for 10 min. To investigate an experimental condition of insulin resistance, cells were incubated for 24 h in DMEM supplemented with 10% FBS containing 5 mM glucose (control) or 25 mM glucose plus 100 nM insulin (insulin resistance condition) (Thomson et al, 1997;Tong et al, 2001) prior to serum starvation for 4 h in DMEM supplemented with 0.5% BSA, and treated with or without 100 nM insulin for 5-15 min.…”

“…We then tested the effect of the exposure of H9c2 cells to high glucose (25 mM) plus high insulin (100 nM) condition for 24 h, an experimental strategy reported to cause insulin resistance in L6 myotubes and 3T3-L1 adipocytes (Thomson et al, 1997;Tong et al, 2001), to insulin-induced glucose uptake. When cells under the insulin resistance condition were treated with insulin, there was no marked change in basal uptake but the insulin-stimulated uptake of 2-deoxy[ 3 H]glucose was significantly reduced (approximately 46% stimulation vs 16% stimulation in insulin resistance, thus a 65% decrease in the uptake) as compared to control cells grown in 5 mM glucose alone (Figure 2).…”

“…To induce an experimental condition of insulin resistance, H9c2 cells were grown in high glucose (25 mM) plus high insulin (100 nM)-containing media for 24 h (Thomson et al, 1997;Tong et al, 2001). Akt activation and its translocation to caveolae in response to insulin was then investigated by subcellular fractionation, sucrose gradient caveolar enriched membrane fractionation, glucose uptake measurements and immunofluorescence microscopy for analysis of actin remodeling.…”

Adverse effect of obesity on red cell membrane arachidonic and docosahexaenoic acids in gestational diabetes

“…If the membrane protein structure changes by extracellular forces such as peroxidation and glycosylation, its folding property will alter, thereby its lipid composition may have to change to match. Abnormal glucose transporters [41], decreased insulin receptor binding protein [42], Ca ++ transmembrane movement [43], alteration of uncoupling and remodelling of actin [44,45] are examples of altered membrane protein function depressed in diabetes and obesity. The red cell membrane is essentially an example of plasma membrane.…”