The actin-related p41ARC subunit contributes to p21-activated kinase-1 (PAK1)-mediated glucose uptake into skeletal muscle cells

Abstract

Defects in translocation of the glucose transporter GLUT4 are associated with peripheral insulin resistance, preclinical diabetes, and progression to type 2 diabetes. GLUT4 recruitment to the plasma membrane of skeletal muscle cells requires F-actin remodeling. Insulin signaling in muscle requires p21-activated kinase-1 (PAK1), whose downstream signaling triggers actin remodeling, which promotes GLUT4 vesicle translocation and glucose uptake into skeletal muscle cells. Actin remodeling is a cyclic process, and although PAK1 is known to initiate changes to the cortical actin-binding protein cofilin to stimulate the depolymerizing arm of the cycle, how PAK1 might trigger the polymerizing arm of the cycle remains unresolved. Toward this, we investigated whether PAK1 contributes to the mechanisms involving the actin-binding and -polymerizing proteins neural Wiskott-Aldrich syndrome protein (N-WASP), cortactin, and ARP2/3 subunits. We found that the actin-polymerizing ARP2/3 subunit p41ARC is a PAK1 substrate in skeletal muscle cells. Moreover, co-immunoprecipitation experiments revealed that insulin stimulates p41ARC phosphorylation and increases its association with N-WASP coordinately with the associations of N-WASP with cortactin and actin. Importantly, all of these associations were ablated by the PAK inhibitor IPA3, suggesting that PAK1 activation lies upstream of these actin-polymerizing complexes. Using the N-WASP inhibitor wiskostatin, we further demonstrated that N-WASP is required for localized F-actin polymerization, GLUT4 vesicle translocation, and glucose uptake. These results expand the model of insulin-stimulated glucose uptake in skeletal muscle cells by implicating p41ARC as a new component of the insulin-signaling cascade and connecting PAK1 signaling to N-WASP-cortactin-mediated actin polymerization and GLUT4 vesicle translocation.

Keywords: actin; glucose transporter type 4 (GLUT4); insulin resistance; insulin signaling; serine/threonine protein kinase PAK 1 (PAK1); skeletal muscle.

Figure 1.

An insulin-dependent interaction of p41ARC with N-WASP is regulated by PAK1. A and B, L6-GLUT4myc myoblasts were pretreated with vehicle (DMSO) or 25 μm IPA3 for 50 min and stimulated with 100 nm insulin for an additional 10 min. Whole-cell lysates of L6-GLUT4myc myoblasts were analyzed for phosphorylated (p)-p41ARC^Thr-21, p41-ARC, PAK1, and p-PAK1/2^Thr-423/402 (the p-PAK1 band migrates at 68 kDa) levels. The means ± S.D. from at least three independent sets of cell lysates are shown in the bar graph. *, p < 0.05; **, p < 0.01. Black vertical lines between lanes indicate splicing of lanes from within the same gel exposures. C, the whole-cell lysates from above were then immunoprecipitated (IP) with N-WASP antibody, and coimmunoprecipitated p41ARC was determined by immunoblotting (IB). The ratio of p41ARC/Ponceau S staining of the same blot was quantified, and the means ± S.D. from three independent sets of cell lysates were determined. *, p < 0.05.

Figure 2.

N-WASP inhibition blunts insulin-stimulated glucose uptake and GLUT4 translocation. A, L6-GLUT4myc myoblasts were treated with vehicle (DMSO) or 10 μm WISK and stimulated with 100 nm insulin for 20 min simultaneously. Cells were fixed and left unpermeabilized for labeling with anti-myc antibody. The immunofluorescent intensity of cell surface GLUT4 was normalized to the nucleic acid staining dye Syto 60. Values are means ± S.D. from three independent sets of cells. **, p < 0.01. B, L6-GLUT4myc myoblasts were treated with 0, 5, 10, 20, or 25 μm WISK together with 100 nm insulin for a total of 20 min. Cells were fixed and left unpermeabilized for labeling with anti-myc antibody. The immunofluorescent intensity of cell surface GLUT4 was normalized to the nucleic acid staining dye Syto 60. Values are means ± S.D. from three independent sets of cells. ***, p < 0.001. C, L6-GLUT4myc myotubes were used for 2-deoxyglucose uptake assays. Values are means ± S.D. from three independent sets of cells. *, p < 0.05. D and E, L6-GLUT4myc myoblasts were treated with DMSO or WISK, and the cellular ATP levels and percentage of viable cells were determined, respectively. Values are means ± S.D. from three independent sets of cells. **, p < 0.01; ***, p < 0.001.

Figure 3.

Insulin-stimulated F-actin remodeling requires N-WASP activation. A, L6-GLUT4myc myoblasts transfected to express the F-actin binding LifeAct-GFP biosensor were pretreated with vehicle (Veh, DMSO) or 10 μm WISK for 10 min, and live-cell confocal imaging was initiated. F-actin remodeling was monitored every minute from 1 min prior to insulin addition to 10 min after insulin addition. Arrows indicate sites of F-actin remodeling. At least 20 GFP-positive cells were live-imaged, with >10 treated with WISK, from three independent passages of L6 cells. Scale bar = 100 μm. B, F- and G-actin from L6-GLUT4myc myoblasts treated with vehicle (DMSO) or 10 μm WISK, left unstimulated (Basal), or stimulated with 100 nm insulin for 20 min were resolved by SDS-PAGE for immunoblotting using anti-actin. A representative blot showing the F:G-actin ratio is calculated as a percent of total per sample. C, quantitative bar graph representation of the F:G-actin ratio from three independent passages of L6 cells. p > 0.05 for all comparisons.

Figure 4.

Inhibition of PAK1 activation decreases the insulin-stimulated association of Actin with N-WASP. A, L6-GLUT4myc myoblasts were pretreated with vehicle (DMSO) or 25 μm IPA3 for 50 min and stimulated with 100 nm insulin for an additional 10 min. Whole-cell lysates were subjected to immunoprecipitation (IP) with anti-N-WASP antibody, and precipitated proteins were resolved using SDS-PAGE for immunoblot analyses. Actin levels were normalized for loading using Ponceau S staining of the same blot section in each of three independent sets of cell lysates. Values represent the means ± S.D. ***, p < 0.001. B, whole-cell lysates from the experiments in A were analyzed for p-PAK1/2^{Thr-423,Thr-402} (p-PAK1 band at 68 kDa shown) and total PAK1 protein levels. The pPAK1 band (68 kDa) was quantified as a fraction of corresponding total PAK1 in each of three independent co-immunoprecipitations. *, p < 0.05. C, L6-GLUT4myc myoblasts pretreated with vehicle (DMSO) or 25 μm IPA3 were stimulated with 100 nm insulin for 10 min. F- and G-actin were separated by ultracentrifugation and analyzed on SDS-PAGE. Data represent the means ± S.D. of three sets of cell F:G-actin ratios. p > 0.05. D, L6-GLUT4myc myoblasts pretreated with vehicle, 25 μm IPA3, or 10 μm Latrunculin B (LatB) were stimulated with 100 nm insulin for 5 min and harvested for use in STX4 activation assays, as described under “Experimental Procedures.” Values are the means ± S.D. of three independent experiments using at least two independent batches of recombinant GST-VAMP2 protein. **, p < 0.01.

Figure 5.

A PAK1-regulated, insulin-dependent N-WASP–cortactin interaction. A, L6-GLUT4myc myoblasts were transfected to express GFP-tagged N-WASP and, after 48 h, pretreated with vehicle (DMSO) or 25 μm IPA3 for 50 min and stimulated with 100 nm insulin for an additional 10 min. Whole-cell lysates were immunoprecipitated (IP) with anti-GFP antibody, and coimmunoprecipitated cortactin was determined by IB. Bars represent the means ± S.D. of four independent sets of L6 cells. **, p < 0.01. B, AKT activation in cells treated with or without WISK, representative of three independent sets of cells. p > 0.05/not significant (NS). C, L6-GLUT4myc myoblasts treated with vehicle or 10 μm WISK were stimulated with 100 nm insulin for 5 min and harvested for use in STX4 activation assays, as described under “Experimental Procedures.” Values are the means ± S.D. of four independent experiments using at least two independent batches of recombinant GST-VAMP2 protein. **, p < 0.01.