Rac1 is a novel regulator of contraction-stimulated glucose uptake in skeletal muscle

Abstract

In skeletal muscle, the actin cytoskeleton-regulating GTPase, Rac1, is necessary for insulin-dependent GLUT4 translocation. Muscle contraction increases glucose transport and represents an alternative signaling pathway to insulin. Whether Rac1 is activated by muscle contraction and regulates contraction-induced glucose uptake is unknown. Therefore, we studied the effects of in vivo exercise and ex vivo muscle contractions on Rac1 signaling and its regulatory role in glucose uptake in mice and humans. Muscle Rac1-GTP binding was increased after exercise in mice (~60-100%) and humans (~40%), and this activation was AMP-activated protein kinase independent. Rac1 inhibition reduced contraction-stimulated glucose uptake in mouse muscle by 55% in soleus and by 20-58% in extensor digitorum longus (EDL; P < 0.01). In agreement, the contraction-stimulated increment in glucose uptake was decreased by 27% (P = 0.1) and 40% (P < 0.05) in soleus and EDL muscles, respectively, of muscle-specific inducible Rac1 knockout mice. Furthermore, depolymerization of the actin cytoskeleton decreased contraction-stimulated glucose uptake by 100% and 62% (P < 0.01) in soleus and EDL muscles, respectively. These are the first data to show that Rac1 is activated during muscle contraction in murine and human skeletal muscle and suggest that Rac1 and possibly the actin cytoskeleton are novel regulators of contraction-stimulated glucose uptake.

FIG. 1.

A: Rac1 protein expression in mouse soleus (SOL), extensor digitorum longus (EDL), and gastrocnemius (Gast) (n = 9). B: Rac1 expression in human soleus and gastrocnemius (n = 8). C: Intracellular localization of Rac1 and VAMP3 in mouse hind limb (gastrocnemius and quadriceps) muscle. L, lysate; P1 and 2, plasma membrane fractions; Cyt, cytosol; F1-8 intracellular fractions (n = 7). Statistical significance is indicated by *P < 0.05; ** P < 0.01. Values represent mean ± SEM.

FIG. 2.

A: Rac1-GTP binding in incubated mouse soleus muscle stimulated with (black bar) or without (white bar) electrically induced contraction (100 Hz 15-s intervals, 2-s train, 0.2-ms impulses) (n = 7). B: Rac1-GTP binding in incubated mouse soleus muscle kept at resting tension (2–5 mN; basal) or stretched (150 mN, 15 min) (n = 7). C: Mouse quadriceps muscle, basal and after 30 min running at speed of 10 m/min (exercise 10 m/min = 40% of max running speed) (n = 7). D: Rac1-GTP binding (top) and p-PAK^Thr423 (middle), and representative blots (bottom) in mouse quadriceps muscle basal or in response to 30-min treadmill running at 50 or 70% (Ex 50/70%) of maximum running speed (n = 8). E: Representative images and bar graph (FWHM) showing Rac1 staining of single fibers isolated from mouse EDL muscle in the basal state or after 30 min of treadmill running at 70% of maximum running speed (n = 5). Statistical significance compared with basal is indicated by *P < 0.05; **P < 0.01; ***P < 0.001. Values represent mean ± SEM.

FIG. 3.

A: Rac1-GTP binding in human soleus (SOL) and gastrocnemius (Gast) in the basal state and after 45 min of inclined walking at ∼69% Vo_{2 peak} (n = 5–9). B: Exercise-stimulated fold changes from basal in soleus (SOL) and gastrocnemius (Gast). C: Bar graph and representative Western blot analysis of p-Rac1^Ser71 in response to exercise in human soleus and gastrocnemius muscle (n = 5–9). D: Bar graph and Western blot analysis of p-PAK^Thr423 in response to exercise in human soleus and gastrocnemius muscle (n = 5–9). Statistical significance compared with basal is indicated by *P < 0.05; **P < 0.01. Values represent mean ± SEM.

FIG. 4.

A: Rac1-GTP binding after ex vivo AICAR (2 mmol/L) stimulation of mouse soleus and EDL muscles (n = 5–6). B: Bar graphs and representative blots showing p-AMPK^Thr172 and p-ACC^Ser212 in response to AICAR (n = 5–6). C: Representative blots and bar graphs of the effect of insulin (100 nmol/L; 10 min), DNP (0.5 mmol/L; 20 min), and AICAR (2 mmol/L; 30 min) on Rac1-GTP binding in C2C12 myotubes (n = 5). D: Rac1-GTP binding in WT and AMPK KD mouse quadriceps muscle basal or in response to 30 min of treadmill running at 70% (Exe 70%) of the maximum running speed (n = 6–9). Statistical significance compared with basal is indicated by *P < 0.05; **P < 0.01; ***P < 0.001. Values represent mean ± SEM.

FIG. 5.

A: Contraction-stimulated 2DG uptake in soleus and EDL without or with 200 μmol/L NSC23766, 1-h preincubation (n = 12). B: Contraction-stimulated 2DG uptake in soleus and EDL without or with 10 μmol/L Rac1 Inhibitor II (Inhib II), 1-h preincubation (n = 10). C: Representative blots and bar graphs show contraction-stimulated p-AMPK^Thr172 and p-ACC^Ser212 in soleus and EDL muscle without or with 200 μmol/L NSC23766. D: Representative blots and bar graphs show contraction-stimulated p-AMPK^Thr172, p-ACC^Ser212, and p-PAK^Thr423 signaling and Rac1 and GLUT4 total protein in soleus and EDL muscle without or with 10 μmol/L Rac1 Inhibitor II (Inhib II). E: Insulin-stimulated (60 nmol/L) 2DG uptake in mouse white adipose (gonadal) tissue without or with 10 μmol/L Rac1 Inhibitor II (Inhib II), 1-h preincubation (n = 8). Statistical significance between basal and contraction is indicated by *P < 0.05; **P < 0.01; ***P < 0.001. Effect of inhibitor on contraction-stimulated 2DG or p-PAK^Thr423 is indicated by #P < 0.05; ##P < 0.01; ###P < 0.001. Main effect of inhibitor is indicated by †P < 0.05. Values represent mean ± SEM.

FIG. 6.

A: Bar graph shows mean ± SEM of Rac1 knockout efficiency in soleus and EDL muscle of muscle-specific inducible Rac1 KO mice and WT controls (n = 11–13). B: Representative Western blots showing the effect of doxycycline (1 g/L) treatment on Rac1 protein in WT vs. Floxed Rac1 KO mice (Rac1 fl/fl*Cre) (gastrocnemius muscle). C: Contraction-stimulated 2DG uptake in incubated isolated soleus and EDL muscles from WT and Rac1 KO mice (n = 11–13). D: Contraction-stimulated fold changes from basal in 2DG uptake in soleus and EDL from WT and Rac1 KO mice. E: Representative Western blots of p-AMPK^Thr172, p-ACC^Ser212, PAS, p-PAK^Thr423, p-TBC1D4^Thr642 (Soleus), p-TBC1D1^Ser237 (EDL), and actin, GLUT4, and Rac1 total proteins in response to contraction. F: Bar graph shows mean ± SEM of contraction-induced p-AMPK ^Thr172, p-ACC ^Ser212, PAS, p-PAK^Thr423/PAK1, p-TBC1D4^Thr542, and p-TBC1D1^Ser237 in soleus and EDL from Rac1 KO mice and WT controls. G: Bar graph shows mean ± SEM of total PAK1 and GLUT4 protein in soleus and EDL of WT and Rac1 KO mice. Statistical significances between basal and contraction are indicated by *P < 0.05; **P < 0.01; ***P < 0.001. Effect of genotype on contraction-stimulated 2-DG and p-PAK^Thr423 is indicated by #P < 0.05. Main effect of genotype is indicated by †P < 0.05; †††P < 0.001. Values represent mean ± SEM.

FIG. 7.

A: AICAR-stimulated (2 mmol/L) 2DG uptake in incubated isolated soleus and EDL without or with 10 μmol/L Rac1 Inhibitor II (Inhib II), 1-h preincubation (n = 8). B: Bar graphs show mean ± SEM of AICAR-induced p-AMPK ^Thr172, p-ACC ^Ser212, p-PAK^Thr423, in soleus and EDL without or with 10 μmol/L Rac1 Inhibitor II (Inhib II) (n = 8). C: Representative Western blots of p-AMPK^Thr172, p-ACC^Ser212, p-PAK^Thr423, and GLUT4 and Rac1 total proteins in response to AICAR without or with 10 μmol/L Rac1 Inhibitor II (Inhib II). D: AICAR-stimulated 2DG uptake in mouse soleus and EDL muscles from WT and muscle-specific inducible Rac1 KO (n = 8) mice. E: Bar graphs show mean ± SEM of AICAR-induced p-AMPK ^Thr172, p-ACC ^Ser212, p-PAK^Thr423, and total Rac1 and PAK1 protein in soleus and EDL muscles from WT or Rac1 KO mice (n = 8). F: Representative Western blots of p-AMPK^Thr172, p-ACC^Ser212, and p-PAK^Thr423/PAK1, and GLUT4, Rac1, and PAK1 total proteins in response to AICAR in WT and Rac1 KO soleus and EDL muscles. G: AICAR-stimulated GLUT4 translocation in C2C12-GLUT4myc myotubes in response to DNP (0.5 mmol/L) or AICAR (2 mmol/L) without or with 200 μmol/L NSC23766, 1-h preincubation (n = 6). Statistical significances between basal and AICAR are indicated by *P < 0.05; **P < 0.01; ***P < 0.001. Significant differences in AICAR-stimulated signaling are indicated by #P < 0.05. Main effect of genotype is indicated by ††P < 0.01 and †††P <0.0001. Values represent mean ± SEM.

FIG. 8.

A: Contraction-stimulated 2DG uptake in mouse soleus and EDL without or with 10 μmol/L Latrunculin B, 1-h preincubation (n = 13–18). B: Contraction-stimulated 2DG uptake in mouse soleus and EDL without or with 1 μmol/L Latrunculin B, 1-h preincubation (n = 6–12). Statistical significances between basal and contraction are indicated by **P < 0.01; *** P < 0.001. Significant differences in contraction-stimulated glucose uptake are indicated by #P < 0.05; ## P < 0.01. Values represent mean ± SEM.