Signaling of the p21-activated kinase (PAK1) coordinates insulin-stimulated actin remodeling and glucose uptake in skeletal muscle cells

Abstract

Skeletal muscle accounts for ∼ 80% of postprandial glucose clearance, and skeletal muscle glucose clearance is crucial for maintaining insulin sensitivity and euglycemia. Insulin-stimulated glucose clearance/uptake entails recruitment of glucose transporter 4 (GLUT4) to the plasma membrane (PM) in a process that requires cortical F-actin remodeling; this process is dysregulated in Type 2 Diabetes. Recent studies have implicated PAK1 as a required element in GLUT4 recruitment in mouse skeletal muscle in vivo, although its underlying mechanism of action and requirement in glucose uptake remains undetermined. Toward this, we have employed the PAK1 inhibitor, IPA3, in studies using L6-GLUT4-myc muscle cells. IPA3 fully ablated insulin-stimulated GLUT4 translocation to the PM, corroborating the observation of ablated insulin-stimulated GLUT4 accumulation in the PM of skeletal muscle from PAK1(-/-) knockout mice. IPA3-treatment also abolished insulin-stimulated glucose uptake into skeletal myotubes. Mechanistically, live-cell imaging of myoblasts expressing the F-actin biosensor LifeAct-GFP treated with IPA3 showed blunting of the normal insulin-induced cortical actin remodeling. This blunting was underpinned by a loss of normal insulin-stimulated cofilin dephosphorylation in IPA3-treated myoblasts. These findings expand upon the existing model of actin remodeling in glucose uptake, by placing insulin-stimulated PAK1 signaling as a required upstream step to facilitate actin remodeling and subsequent cofilin dephosphorylation. Active, dephosphorylated cofilin then provides the G-actin substrate for continued F-actin remodeling to facilitate GLUT4 vesicle translocation for glucose uptake into the skeletal muscle cell.

Keywords: Diabetes; F-actin remodeling; GLUT4 vesicle exocytosis; L6-GLUT4myc muscle cells; PAK1; Rac1; Skeletal muscle; Small Rho family GTPase.

Fig. 1

Group I PAK isoforms in skeletal muscle and L6-GLUT4-myc cells. Hindlimb muscle from wild-type (WT) mice was homogenized and analyzed for the three Group I PAK members PAK1, 2 and 3 for (A) mRNA content using qRT-PCR (normalized to GAPDH) from three sets of tissues, and (B) protein expression. L6-GLUT4-myc myoblasts were assessed similarly for protein expression of all three Group I PAKs; mouse brain lysates served as control (CTRL) as they co-express all three isoforms. Vertical black lines denote splicing of lanes from the PAK1, PAK2, PAK3 and actin immunoblots. (C) Lysate proteins from L6-GLUT4-myc myoblast cells left unstimulated or insulin-stimulated (100 nM, 10 min) were resolved by 12% SDS-PAGE for immunoblot detection of phosphorylated- and total- PAK1 and PAK2 proteins and (D) specifically for phosphorylated PAK1. Data are representative of three independent experiments, with average insulin-stimulated p-PAK1/total PAK1 ratios shown below the blots, normalized to basal = 1.0 for each experiment.

Fig. 2

IPA3 inhibits insulin-stimulated PAK1 phosphorylation in L6-GLUT4-myc myoblasts. (A) Dose-optimization: L6 myoblasts were pretreated with IPA3 at 20, 25 or 30 μM for 10 min followed by insulin stimulation for an additional 10 min. Detergent cleared cell lysates were prepared and proteins resolved by SDS-PAGE for immunoblot and optical density scanning quantitation for p-PAK1 relative to total PAK1 (in arbitrary units). (B) Time course: L6 myoblasts were treated with 25 μM IPA3 for 0, 20, 40 or 60 min, with insulin added in the final 10 min (100 nM). Cell lysates were immunoblotted for p-PAK1 and PAK1 and quantified as in (A) above. (C) p-AKT and total AKT were immunoblotted and quantified from cells treated with vehicle (−) or 25 μM IPA3 for 60 min as described in (A) above. Bar graphs represent the average ± SE of three independent cell passages; *p < 0.05. The vertical black line denotes splicing of lanes from within the same gel exposure.

Fig. 3

PAK1 activity is essential for insulin stimulated GLUT4 vesicle translocation and glucose uptake in skeletal muscle cells. (A) Surface GLUT4: L6-GLUT4-myc myoblasts treated with IPA3 or vehicle (DMSO) were stimulated with insulin (100 nM, 20 min) for LiCor analyses of surface GLUT4 levels. Immunofluorescent intensity of cell surface GLUT4 was normalized to nucleic acid staining dye, Syto 60, and data displayed as the fold-stimulation of insulin-stimulated surface GLUT4, relative to unstimulated/basal level. Bars represent the average ± SE of four independent cell passages; *p < 0.05, vs vehicle (−) treated cells. (B) Glucose uptake: L6-GLUT4-myc myotubes were used for 2-deoxyglucose uptake assays as described in Section 2. Data represent the average (±SE) fold stimulation in response to insulin relative to basal level glucose uptake, in at least three independent passages of cells; *p < 0.05, vs vehicle (−) treated cells. (C) L6-GLUT4-myc myoblasts were transfected to express GFP-tagged PAK1 inhibitory domain (PID) or non-inhibitory control (PID-L107F) and assessed for insulin-stimulated GLUT4 as described in (A) above. Bars represent the average ± SE of three independent cell passages; *p < 0.05, vs PID-L107F-expressing cells. (D) Myoblasts were transfected with control (siCon) or PAK2-selective (siPAK2) siRNA oligonucleotides, normalized to actin (arbitrary units). Bars represent the average ± SE of three independent cell passages; *p < 0.05, vs siCon. (E) GLUT4 vesicle translocation assays from siPAK2 or control (siCon) transfected cells, as described in (A) above. No significant differences were detected in four independent experiments.

Fig. 4

Insulin-induced PAK1 signaling is important for insulin-induced F-actin remodeling in L6-GLUT4-myc myoblasts. L6-GLUT4-myc myoblasts were transfected to express the LifeAct-GFP biosensor for live-cell imaging of F-actin remodeling. Myoblasts treated with vehicle or IPA3 were imaged using an custom spinning-disk confocal microscope and captured every min from 1 min prior insulin addition and on through to 10 min after insulin stimulation (100 nM). Arrows denote regions of ruffling. Images represent still images taken from at least four cell movies of each treatment condition conducted using four independent passages of cells. Movies are included as Supplemental movies 1–4, two movies for each condition shown here.

Fig. 5

PAK1 signaling is required selectively for insulin-stimulated cofilin dephosphorylation in L6-GLUT4-myc myoblasts. L6-GLUT4-myc myoblasts treated with vehicle or IPA3 were stimulated with insulin (100 nM, 10 min) and resulting detergent cleared cell lysates resolved on 12% SDS-PAGE for simultaneous immunoblot detection of (A) p-PAK1 and total PAK1, (B) p-LIMK and total LIMK, (C) p-cofilin and total cofilin, and (D) p-ERK and total ERK. Optical density scanning was used for quantification, with bar graphs representing the average ± SE of fold changes in activations evaluated in four independent experiments; *p < 0.05. Vertical black lines denote splicing of lanes from within the same gel exposure within each figure panel.