4997
J Physiol 594.17 (2016) pp 4997–5008
Rac1 governs exercise-stimulated glucose uptake in skeletal muscle through regulation of GLUT4 translocation in mice Lykke Sylow1 , Ida L. Nielsen1 , Maximilian Kleinert1 , Lisbeth L. V. Møller1 , Thorkil Ploug2 , Peter Schjerling3 , Philip J. Bilan4 , Amira Klip4 , Thomas E. Jensen1 and Erik A. Richter1 1
Molecular Physiology Group, Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, Denmark Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark 3 Institute of Sports Medicine, Department of Orthopedic Surgery, Bispebjerg Hospital and Center for Healthy Aging, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark 4 Cell Biology Program, The Hospital for Sick Children, Toronto, Ontario, Canada
The Journal of Physiology
2
Key point
r Exercise increases skeletal muscle energy turnover and one of the important substrates for the working muscle is glucose taken up from the blood.
r The GTPase Rac1 can be activated by muscle contraction and has been found to be necessary r r
for insulin-stimulated glucose uptake, although its role in exercise-stimulated glucose uptake is unknown. We show that Rac1 regulates the translocation of the glucose transporter GLUT4 to the plasma membrane in skeletal muscle during exercise. We find that Rac1 knockout mice display significantly reduced glucose uptake in skeletal muscle during exercise.
Abstract Exercise increases skeletal muscle energy turnover and one of the important substrates for the working muscle is glucose taken up from the blood. Despite extensive efforts, the signalling mechanisms vital for glucose uptake during exercise are not yet fully understood, although the GTPase Rac1 is a candidate molecule. The present study investigated the role of Rac1 in muscle glucose uptake and substrate utilization during treadmill exercise in mice in vivo. Exercise-induced uptake of radiolabelled 2-deoxyglucose at 65% of maximum running capacity was blocked in soleus muscle and decreased by 80% and 60% in gastrocnemius and tibialis anterior muscles, respectively, in muscle-specific inducible Rac1 knockout (mKO) mice compared to wild-type littermates. By developing an assay to quantify endogenous GLUT4 translocation, we observed that GLUT4 content at the sarcolemma in response to exercise was reduced in Rac1 mKO muscle. Our findings implicate Rac1 as a regulatory element critical for controlling glucose uptake during exercise via regulation of GLUT4 translocation. (Received 15 December 2015; accepted after revision 30 March 2016; first published online 9 April 2016) Corresponding author L. Sylow: Universitetsparken 13, 2100 Copenhagen O, Denmark. Email: [email protected] Abbreviations AMPK, AMP-activated protein kinase; 2-DG, 2-deoxyglucose; mKO, muscle-specific inducible Rac1 knockout; PBS, phosphate-buffered saline; Rac1, Ras-related C3 botulinum toxin substrate 1; TA, tibialis anterior; WT, wild-type.
Introduction Exercise promotes a large increase in skeletal muscle energy turnover, and one of the important substrates C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
for the working muscle is glucose taken up from the blood. Glucose uptake during exercise is regulated in three main steps: (i) increased glucose delivery; (ii) increased transport of glucose molecules across the muscle plasma DOI: 10.1113/JP272039
4998
L. Sylow and others
membrane; and (iii) augmented intracellular metabolism of glucose (Richter & Hargreaves, 2013). The increase in glucose transport across the plasma membrane is mediated by translocation of the glucose transporter, GLUT4, from intracellular storage sites to the sarcolemma and t-tubules, both in response to insulin (Marette et al. 1992; Kristiansen et al. 1996) or to exercise (Douen et al. 1990; Lauritzen et al. 2010). Despite intense research, the signalling mechanisms that mediate exercise-stimulated glucose uptake are still incompletely understood. Our group recently identified the Rho family GTPase, Rac1 (Ras-related C3 botulinum toxin substrate) as a previously unrealized signalling protein activated by muscle contractions (Sylow et al. 2013b). Rac1 regulates various cellular processes, including NADPH oxidase dependent reactive oxygen species production (Morel et al. 1991; Hordijk, 2006) and reorganization of the actin cytoskeleton (Ridley et al. 1992; Hall & Nobes, 2000; Chiu et al. 2011). A connection between Rac1 and glucose transport was originally observed in L6 muscle cells. Here Rac1 was found to be activated rapidly in response to insulin and contribute to insulin-induced GLUT4 translocation and glucose transport. This was brought about through the induction of cortical actin filament remodelling (JeBailey et al. 2007). Rac1 also regulates insulin-stimulated glucose uptake in mature skeletal mouse muscle. Notably, insulin-stimulated phosphorylation of PAK1/2Thr423/402 , a downstream target of Rac1, is impaired in skeletal muscle of insulin resistant human subjects (Sylow et al. 2013a) and in ob/ob mice (Sylow et al. 2014). Although the contribution of Rac1 to the regulation of glucose transport by in vivo exercise has not been examined, we recently showed that ex vivo electrically-induced contraction increases glucose transport to a lesser extent in muscles with decreased Rac1 signalling (Sylow et al. 2013b). However, electrical stimulation ex vivo induces intense supraphysiological isometric muscle contraction and recruits all fibres in the stimulated muscle, and is quite different from the conditions during in vivo exercise at a submaximal intensity. Hence, there is a need to examine the possible contribution of Rac1 to glucose uptake, as well as to GLUT4 in particular, in exercising skeletal muscles. Exercise-stimulated glucose uptake depends in part on the activation of AMP-activated protein kinase (AMPK) (Mu et al. 2001; O’Neill et al. 2011; Jensen et al. 2014), although this connection has been questioned in some studies (Maarbjerg et al. 2009; Fentz et al. 2015). In addition or on top of AMPK signalling, several other proteins and processes, including protein kinase C, LKB1, mechanical stretching, reactive oxygen species and nitric oxide, may participate in the regulation of exercise-stimulated glucose uptake in skeletal muscle (Richter & Hargreaves, 2013). Other as yet unidentified mechanisms probably also contribute to the regulation
J Physiol 594.17
of glucose uptake during muscle contraction (Jessen & Goodyear, 2005; Rose & Richter, 2005). Based on the arguments outlined above, Rac1 is one such candidate. The present study therefore aimed to investigate the involvement of Rac1 in in vivo exercise-induced glucose uptake, as well as the underlying mechanisms. Methods Tetracycline-inducible muscle-specific Rac1 knockout (mKO) mice
Inducible muscle-specific male and female Rac1 mKO mice were generated as described previously (Sylow et al. 2013b). In brief, Rac1 floxed mice (Chrostek et al. 2006) were crossed with mice containing a tetracycline-controlled transactivator coupled to the human skeletal muscle α-actin promoter, which drives the muscle-specific expression of the Cre recombinase (Rao & Monks, 2009). Control wild-type (WT) mice were littermates carrying the Cre recombinase or the floxed Rac1 gene on none, one or both alleles (excluding combined Cre and floxed Rac1). Rac1 mKO mice were homozygous for the floxed Rac1 gene and either homozygous or heterozygous for the Cre recombinase. Rac1 mKO was induced at 10–14 weeks of age by adding doxycycline in the drinking water (1 g l−1 ; Sigma-Aldrich, St Louis, MO, USA) for 3 weeks followed by a washout period of 3 weeks. All animals were maintained under a 12:12 h light/dark cycle and received standard rodent chow diet (Altromin no. 1324; Chr. Pedersen, Ringsted, Denmark) and water ad libitum. All experiments were approved by the Danish Animal Experimental Inspectorate and conform with the principles of UK regulations as described by Grundy (2015). Acclimatization to treadmill and maximal running capacity test
All mice were acclimatized to the treadmill for 3 × 5 min at 10 m min−1 and 2 × 5 min at 16 m min−1 at 0° incline during the week prior to the maximal running capacity test. The test was performed at 10° incline beginning with 5 min of warm up at 10 m min−1 , after which speed was increased by 1.2 m min−1 every 1 min until exhaustion. Testing was performed blinded. Glycogen-depleted maximal running capacity test
To investigate the effects of low muscle glycogen on maximal running capacity, WT and Rac1 mKO mice were exercised for 30 min at 75% of their maximal running capacity. This exercise bout almost depletes muscle glycogen stores to an equal extent in WT and C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
J Physiol 594.17
Rac1 in exercise-stimulated glucose uptake in muscle
Rac1 KO mice (data not shown). Following exercise, mice were left to recover for 2.5 h without access to food and a maximal running capacity test was performed as described. Tissue specific 2-deoxyglucose (DG) uptake measurements during treadmill running
Each mouse was exercised (16 m min−1 on average) at a relative work load corresponding to 65% of its maximum running capacity for 20 min at 10° incline. To determine 2-DG uptake in muscle and brain tissue, 2-[3 H]DG (Perkin Elmer, Boston, MA, USA) was injected I.P. in a bolus of saline containing 0.1 mM 2-DG and 60 μCi ml−1 2-[3 H]DG corresponding to 12 μCi/mouse (800 μl injected saline volume/100 g body weight) into fed WT or Rac1 mKO mice immediately before the onset of exercise. Control mice were placed on a still treadmill for the same amount of time. During exercise or rest, mice were removed from the treadmill (1 min) and blood samples were collected from the tail vein prior to and after 10 and 20 min and analysed for glucose concentration. At 10 and 20 min, blood samples were also analysed for specific 2-[3 H]DG tracer activity. After 20 min, mice were killed by cervical dislocation, and soleus, quadriceps, tibialis anterior and gastrocnemius muscles were excised and quickly frozen in liquid nitrogen and stored at −80°C until further processing. For the high-intensity experiment, mice were acclimatized as described above and exercised for 10 min at 20° incline at 85% of their maximal running capacity. Tissue specific 2-DG-6-phosphate accumulation was measured as described previously (Fueger et al. 2004) by precipitation of phosphorylated 2-DG, using 0.1 M Ba(OH)2 and 0.1 M ZnSO4 . The total tissue 2-[3 H]DG tracer activity found in 2-DG-6-phosphate was divided by the area under the curve of the mean specific activity at 10 min and 20 min and, for glucose, at 0, 10 and 20 min [for the high-intensity experiment, specific activity only at (end) 10 min was used for the calculations]; injected 3 H labelled 2-DG tracer or plasma glucose was identical between genotypes (data not shown). This was related to muscle weight and the time to obtain the tissue-specific 2-DG uptake (μmol g−1 h−1 ). Percentage increase by exercise was calculated by relating the exercise-stimulated values to the basal control values for the specific genotype group. Immunohistochemistry on cryosections for GLUT4 analysis
Staining of GLUT4 and α-sarcoglycan was carried out on 12 μm cryosections obtained from Tissue-Tek (Sakura Finetek USA, Inc., Torrance, CA, USA) embedded tibialis anterior muscle of basal or treadmill exercised WT and Rac1 mKO mice (from a subgroup of the mice C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
4999
that were used for 2-[3 H]DG uptake measurements). Cryosections were fixed for 30 min in ice-cold 4% Zamboni buffer (4% paraformaldehyde, 0.15% picric acid, 0.1 M Sorensen’s phosphate buffer, pH 7.3) and washed 3 × 10 min with phosphate-buffered saline (PBS) and incubated for 2 h with primary antibodies raised against GLUT4 (rabbit polyclonal; #PA523052; Thermo Scientific, Waltham, MA, USA) and α-sarcoglycan (mouse monoclonal, #IVD3(1)A9; Developmental Studies Hybridoma Bank, Iowa City, IA, USA). After primary antibody incubation, sections were washed 3 × 10 min in PBS and incubated with secondary antibody conjugated to Alexa 488 (anti-rabbit) and 568 (anti-mouse) for 1 h followed by another 3 × 10 min wash. Subsequently, the cryosections were mounted in Vectashield (Vector Laboratories, Inc., Burlingame, CA, USA). Negative controls were performed by incubating cryosections in the absence of primary antibodies. Microscope alignment was tested prior to image collection on each day using 5 μm fluorescent beads and images were acquired on the confocal microscope by recording a z-stack from the top to the bottom of a bead, images spaced 0.10 μm apart in the z-plane. Images were collected using a LSM710 microscope (Carl Zeiss, Oberkochen, Germany) through a 63×/1.40 oil DIC Plan-Apochromat objective at 20°C and analysed using Zen software (Carl Zeiss). The amount of GLUT4 protein at the plasma membrane was calculated at eight different locations across the plasma membrane of one muscle fibre by taking the ratio of the quantified mean intensity of GLUT4 to α-sarcoglycan staining in a 5 μm line drawn perpendicular through the plasma membrane. Care was taken not to quantify areas of the membrane containing unfused GLUT4 vesicles (visible as distinct green dots in close vicinity to the plasma membrane at the resolution of the confocal microscope) such that only the fraction of GLUT4 that was fused with the plasma membrane was quantified. The average of eight fibres from each mouse was used for data analysis. All image collections and quantifications were performed blinded. Following analysis, Tissue-Tek was mechanically removed from the tibialis anterior muscles and 2-DG uptake (n = 5 or 6 for each condition) was measured as described above. Immunohistochemistry on cryosections for capillary/fibre ratio analysis
Staining of capillaries was performed on 12 μm cryosections (cross-sections) on Tissue-Tek embedded tibialis anterior muscle of WT and Rac1 mKO mice using Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin 1 (Vector Laboratories) as primary label and Streptavidin/FITC (Dako, Glostrup, Denmark) as secondary label. Images were collected using an Axioplan 2 Universal microscope with Axiophot 2 Photo Module (Carl Zeiss) through a Plan-NEOFLUAR 20×/0.05 lens
5000
L. Sylow and others
J Physiol 594.17
(Carl Zeiss). The mean capillary-to-fibre ratio was calculated using ImageJ, version 1.47 (National Institutes of Health, Bethesda, MD, USA) as described previously (Baum et al. 2013). Image collections and quantifications were performed blinded.
the AMARA peptide (HAMARAASAAAIARRR; 100 μM) as substrate (Hayashi et al. 2000). α2 β2 γ 1 activity was measured by overnight α2 immunoprecipitation. The remaining lysates were then incubated with the α1 antibody to pull down the α1 β2 γ 1 complex.
Muscle analysis
L6 and C2C12 cell culture and Rac1 activity
Muscle tissue was pulverized in liquid nitrogen and homogenized 2 × 30 s at 30 Hz using a Tissuelyser II with stainless steel grinding balls (Qiagen, Valencia, CA, USA) in 50 mM Hepes (pH 7.5), 150 mM NaCl, 20 mM sodium pyrophosphate, 20 mM β-glycerophosphate, 10 mM NaF, 2 mM sodium orthovanadate, 2 mM EDTA, 1% NP-40, 10% glycerol, 2 mM phenylmethanesulfonyl fluoride, 1 mM MgCl2 , 1 mM CaCl2 , 10 μg ml−1 leupeptin, 10 μg ml−1 aprotinin and 3 mM benzamidine. After rotation end-over-end for 30 min, lysate supernatants were collected by centrifugation (13,000 g) for 20 min at 4°C.
L6 muscle cells were maintained and differentiated into multinucleated myotubes as described previously (JeBailey et al. 2004). C2C12 muscle cells were maintained and differentiated as reported previously (Miyatake et al. 2016). For all experiments, myotubes were incubated in serum-free α-minimal essential medium (Dulbecco’s modified Eagle’s medium for C2C12) containing 5 mmol l−1 glucose for 3 h prior to stimulation with insulin (100 nM, 10 min stimulation), isoproterenol (20 μM, 20 min stimulation), caffeine (5 mM, 20 min stimulation) or electrical pulse stimulation (24 ms pulse, 1 Hz repetition for 1 h). Following stimulation, cells were rinsed in PBS and the cells were lysed in lysis buffer (Cytoskeleton Inc., Denver, CO, USA) and centrifuged for 1 min at 10,000 g. Rac1 activities were measured in the supernatant using a commercially available Rac1 activation assay kit (BK 126; Cytoskeleton Inc.). In brief, immediately after centrifugation, the protein concentration in the lysates were determined (
J Physiol 594.17 (2016) pp 4997–5008
Rac1 governs exercise-stimulated glucose uptake in skeletal muscle through regulation of GLUT4 translocation in mice Lykke Sylow1 , Ida L. Nielsen1 , Maximilian Kleinert1 , Lisbeth L. V. Møller1 , Thorkil Ploug2 , Peter Schjerling3 , Philip J. Bilan4 , Amira Klip4 , Thomas E. Jensen1 and Erik A. Richter1 1
Molecular Physiology Group, Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, Denmark Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark 3 Institute of Sports Medicine, Department of Orthopedic Surgery, Bispebjerg Hospital and Center for Healthy Aging, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark 4 Cell Biology Program, The Hospital for Sick Children, Toronto, Ontario, Canada
The Journal of Physiology
2
Key point
r Exercise increases skeletal muscle energy turnover and one of the important substrates for the working muscle is glucose taken up from the blood.
r The GTPase Rac1 can be activated by muscle contraction and has been found to be necessary r r
for insulin-stimulated glucose uptake, although its role in exercise-stimulated glucose uptake is unknown. We show that Rac1 regulates the translocation of the glucose transporter GLUT4 to the plasma membrane in skeletal muscle during exercise. We find that Rac1 knockout mice display significantly reduced glucose uptake in skeletal muscle during exercise.
Abstract Exercise increases skeletal muscle energy turnover and one of the important substrates for the working muscle is glucose taken up from the blood. Despite extensive efforts, the signalling mechanisms vital for glucose uptake during exercise are not yet fully understood, although the GTPase Rac1 is a candidate molecule. The present study investigated the role of Rac1 in muscle glucose uptake and substrate utilization during treadmill exercise in mice in vivo. Exercise-induced uptake of radiolabelled 2-deoxyglucose at 65% of maximum running capacity was blocked in soleus muscle and decreased by 80% and 60% in gastrocnemius and tibialis anterior muscles, respectively, in muscle-specific inducible Rac1 knockout (mKO) mice compared to wild-type littermates. By developing an assay to quantify endogenous GLUT4 translocation, we observed that GLUT4 content at the sarcolemma in response to exercise was reduced in Rac1 mKO muscle. Our findings implicate Rac1 as a regulatory element critical for controlling glucose uptake during exercise via regulation of GLUT4 translocation. (Received 15 December 2015; accepted after revision 30 March 2016; first published online 9 April 2016) Corresponding author L. Sylow: Universitetsparken 13, 2100 Copenhagen O, Denmark. Email: [email protected] Abbreviations AMPK, AMP-activated protein kinase; 2-DG, 2-deoxyglucose; mKO, muscle-specific inducible Rac1 knockout; PBS, phosphate-buffered saline; Rac1, Ras-related C3 botulinum toxin substrate 1; TA, tibialis anterior; WT, wild-type.
Introduction Exercise promotes a large increase in skeletal muscle energy turnover, and one of the important substrates C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
for the working muscle is glucose taken up from the blood. Glucose uptake during exercise is regulated in three main steps: (i) increased glucose delivery; (ii) increased transport of glucose molecules across the muscle plasma DOI: 10.1113/JP272039
4998
L. Sylow and others
membrane; and (iii) augmented intracellular metabolism of glucose (Richter & Hargreaves, 2013). The increase in glucose transport across the plasma membrane is mediated by translocation of the glucose transporter, GLUT4, from intracellular storage sites to the sarcolemma and t-tubules, both in response to insulin (Marette et al. 1992; Kristiansen et al. 1996) or to exercise (Douen et al. 1990; Lauritzen et al. 2010). Despite intense research, the signalling mechanisms that mediate exercise-stimulated glucose uptake are still incompletely understood. Our group recently identified the Rho family GTPase, Rac1 (Ras-related C3 botulinum toxin substrate) as a previously unrealized signalling protein activated by muscle contractions (Sylow et al. 2013b). Rac1 regulates various cellular processes, including NADPH oxidase dependent reactive oxygen species production (Morel et al. 1991; Hordijk, 2006) and reorganization of the actin cytoskeleton (Ridley et al. 1992; Hall & Nobes, 2000; Chiu et al. 2011). A connection between Rac1 and glucose transport was originally observed in L6 muscle cells. Here Rac1 was found to be activated rapidly in response to insulin and contribute to insulin-induced GLUT4 translocation and glucose transport. This was brought about through the induction of cortical actin filament remodelling (JeBailey et al. 2007). Rac1 also regulates insulin-stimulated glucose uptake in mature skeletal mouse muscle. Notably, insulin-stimulated phosphorylation of PAK1/2Thr423/402 , a downstream target of Rac1, is impaired in skeletal muscle of insulin resistant human subjects (Sylow et al. 2013a) and in ob/ob mice (Sylow et al. 2014). Although the contribution of Rac1 to the regulation of glucose transport by in vivo exercise has not been examined, we recently showed that ex vivo electrically-induced contraction increases glucose transport to a lesser extent in muscles with decreased Rac1 signalling (Sylow et al. 2013b). However, electrical stimulation ex vivo induces intense supraphysiological isometric muscle contraction and recruits all fibres in the stimulated muscle, and is quite different from the conditions during in vivo exercise at a submaximal intensity. Hence, there is a need to examine the possible contribution of Rac1 to glucose uptake, as well as to GLUT4 in particular, in exercising skeletal muscles. Exercise-stimulated glucose uptake depends in part on the activation of AMP-activated protein kinase (AMPK) (Mu et al. 2001; O’Neill et al. 2011; Jensen et al. 2014), although this connection has been questioned in some studies (Maarbjerg et al. 2009; Fentz et al. 2015). In addition or on top of AMPK signalling, several other proteins and processes, including protein kinase C, LKB1, mechanical stretching, reactive oxygen species and nitric oxide, may participate in the regulation of exercise-stimulated glucose uptake in skeletal muscle (Richter & Hargreaves, 2013). Other as yet unidentified mechanisms probably also contribute to the regulation
J Physiol 594.17
of glucose uptake during muscle contraction (Jessen & Goodyear, 2005; Rose & Richter, 2005). Based on the arguments outlined above, Rac1 is one such candidate. The present study therefore aimed to investigate the involvement of Rac1 in in vivo exercise-induced glucose uptake, as well as the underlying mechanisms. Methods Tetracycline-inducible muscle-specific Rac1 knockout (mKO) mice
Inducible muscle-specific male and female Rac1 mKO mice were generated as described previously (Sylow et al. 2013b). In brief, Rac1 floxed mice (Chrostek et al. 2006) were crossed with mice containing a tetracycline-controlled transactivator coupled to the human skeletal muscle α-actin promoter, which drives the muscle-specific expression of the Cre recombinase (Rao & Monks, 2009). Control wild-type (WT) mice were littermates carrying the Cre recombinase or the floxed Rac1 gene on none, one or both alleles (excluding combined Cre and floxed Rac1). Rac1 mKO mice were homozygous for the floxed Rac1 gene and either homozygous or heterozygous for the Cre recombinase. Rac1 mKO was induced at 10–14 weeks of age by adding doxycycline in the drinking water (1 g l−1 ; Sigma-Aldrich, St Louis, MO, USA) for 3 weeks followed by a washout period of 3 weeks. All animals were maintained under a 12:12 h light/dark cycle and received standard rodent chow diet (Altromin no. 1324; Chr. Pedersen, Ringsted, Denmark) and water ad libitum. All experiments were approved by the Danish Animal Experimental Inspectorate and conform with the principles of UK regulations as described by Grundy (2015). Acclimatization to treadmill and maximal running capacity test
All mice were acclimatized to the treadmill for 3 × 5 min at 10 m min−1 and 2 × 5 min at 16 m min−1 at 0° incline during the week prior to the maximal running capacity test. The test was performed at 10° incline beginning with 5 min of warm up at 10 m min−1 , after which speed was increased by 1.2 m min−1 every 1 min until exhaustion. Testing was performed blinded. Glycogen-depleted maximal running capacity test
To investigate the effects of low muscle glycogen on maximal running capacity, WT and Rac1 mKO mice were exercised for 30 min at 75% of their maximal running capacity. This exercise bout almost depletes muscle glycogen stores to an equal extent in WT and C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
J Physiol 594.17
Rac1 in exercise-stimulated glucose uptake in muscle
Rac1 KO mice (data not shown). Following exercise, mice were left to recover for 2.5 h without access to food and a maximal running capacity test was performed as described. Tissue specific 2-deoxyglucose (DG) uptake measurements during treadmill running
Each mouse was exercised (16 m min−1 on average) at a relative work load corresponding to 65% of its maximum running capacity for 20 min at 10° incline. To determine 2-DG uptake in muscle and brain tissue, 2-[3 H]DG (Perkin Elmer, Boston, MA, USA) was injected I.P. in a bolus of saline containing 0.1 mM 2-DG and 60 μCi ml−1 2-[3 H]DG corresponding to 12 μCi/mouse (800 μl injected saline volume/100 g body weight) into fed WT or Rac1 mKO mice immediately before the onset of exercise. Control mice were placed on a still treadmill for the same amount of time. During exercise or rest, mice were removed from the treadmill (1 min) and blood samples were collected from the tail vein prior to and after 10 and 20 min and analysed for glucose concentration. At 10 and 20 min, blood samples were also analysed for specific 2-[3 H]DG tracer activity. After 20 min, mice were killed by cervical dislocation, and soleus, quadriceps, tibialis anterior and gastrocnemius muscles were excised and quickly frozen in liquid nitrogen and stored at −80°C until further processing. For the high-intensity experiment, mice were acclimatized as described above and exercised for 10 min at 20° incline at 85% of their maximal running capacity. Tissue specific 2-DG-6-phosphate accumulation was measured as described previously (Fueger et al. 2004) by precipitation of phosphorylated 2-DG, using 0.1 M Ba(OH)2 and 0.1 M ZnSO4 . The total tissue 2-[3 H]DG tracer activity found in 2-DG-6-phosphate was divided by the area under the curve of the mean specific activity at 10 min and 20 min and, for glucose, at 0, 10 and 20 min [for the high-intensity experiment, specific activity only at (end) 10 min was used for the calculations]; injected 3 H labelled 2-DG tracer or plasma glucose was identical between genotypes (data not shown). This was related to muscle weight and the time to obtain the tissue-specific 2-DG uptake (μmol g−1 h−1 ). Percentage increase by exercise was calculated by relating the exercise-stimulated values to the basal control values for the specific genotype group. Immunohistochemistry on cryosections for GLUT4 analysis
Staining of GLUT4 and α-sarcoglycan was carried out on 12 μm cryosections obtained from Tissue-Tek (Sakura Finetek USA, Inc., Torrance, CA, USA) embedded tibialis anterior muscle of basal or treadmill exercised WT and Rac1 mKO mice (from a subgroup of the mice C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
4999
that were used for 2-[3 H]DG uptake measurements). Cryosections were fixed for 30 min in ice-cold 4% Zamboni buffer (4% paraformaldehyde, 0.15% picric acid, 0.1 M Sorensen’s phosphate buffer, pH 7.3) and washed 3 × 10 min with phosphate-buffered saline (PBS) and incubated for 2 h with primary antibodies raised against GLUT4 (rabbit polyclonal; #PA523052; Thermo Scientific, Waltham, MA, USA) and α-sarcoglycan (mouse monoclonal, #IVD3(1)A9; Developmental Studies Hybridoma Bank, Iowa City, IA, USA). After primary antibody incubation, sections were washed 3 × 10 min in PBS and incubated with secondary antibody conjugated to Alexa 488 (anti-rabbit) and 568 (anti-mouse) for 1 h followed by another 3 × 10 min wash. Subsequently, the cryosections were mounted in Vectashield (Vector Laboratories, Inc., Burlingame, CA, USA). Negative controls were performed by incubating cryosections in the absence of primary antibodies. Microscope alignment was tested prior to image collection on each day using 5 μm fluorescent beads and images were acquired on the confocal microscope by recording a z-stack from the top to the bottom of a bead, images spaced 0.10 μm apart in the z-plane. Images were collected using a LSM710 microscope (Carl Zeiss, Oberkochen, Germany) through a 63×/1.40 oil DIC Plan-Apochromat objective at 20°C and analysed using Zen software (Carl Zeiss). The amount of GLUT4 protein at the plasma membrane was calculated at eight different locations across the plasma membrane of one muscle fibre by taking the ratio of the quantified mean intensity of GLUT4 to α-sarcoglycan staining in a 5 μm line drawn perpendicular through the plasma membrane. Care was taken not to quantify areas of the membrane containing unfused GLUT4 vesicles (visible as distinct green dots in close vicinity to the plasma membrane at the resolution of the confocal microscope) such that only the fraction of GLUT4 that was fused with the plasma membrane was quantified. The average of eight fibres from each mouse was used for data analysis. All image collections and quantifications were performed blinded. Following analysis, Tissue-Tek was mechanically removed from the tibialis anterior muscles and 2-DG uptake (n = 5 or 6 for each condition) was measured as described above. Immunohistochemistry on cryosections for capillary/fibre ratio analysis
Staining of capillaries was performed on 12 μm cryosections (cross-sections) on Tissue-Tek embedded tibialis anterior muscle of WT and Rac1 mKO mice using Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin 1 (Vector Laboratories) as primary label and Streptavidin/FITC (Dako, Glostrup, Denmark) as secondary label. Images were collected using an Axioplan 2 Universal microscope with Axiophot 2 Photo Module (Carl Zeiss) through a Plan-NEOFLUAR 20×/0.05 lens
5000
L. Sylow and others
J Physiol 594.17
(Carl Zeiss). The mean capillary-to-fibre ratio was calculated using ImageJ, version 1.47 (National Institutes of Health, Bethesda, MD, USA) as described previously (Baum et al. 2013). Image collections and quantifications were performed blinded.
the AMARA peptide (HAMARAASAAAIARRR; 100 μM) as substrate (Hayashi et al. 2000). α2 β2 γ 1 activity was measured by overnight α2 immunoprecipitation. The remaining lysates were then incubated with the α1 antibody to pull down the α1 β2 γ 1 complex.
Muscle analysis
L6 and C2C12 cell culture and Rac1 activity
Muscle tissue was pulverized in liquid nitrogen and homogenized 2 × 30 s at 30 Hz using a Tissuelyser II with stainless steel grinding balls (Qiagen, Valencia, CA, USA) in 50 mM Hepes (pH 7.5), 150 mM NaCl, 20 mM sodium pyrophosphate, 20 mM β-glycerophosphate, 10 mM NaF, 2 mM sodium orthovanadate, 2 mM EDTA, 1% NP-40, 10% glycerol, 2 mM phenylmethanesulfonyl fluoride, 1 mM MgCl2 , 1 mM CaCl2 , 10 μg ml−1 leupeptin, 10 μg ml−1 aprotinin and 3 mM benzamidine. After rotation end-over-end for 30 min, lysate supernatants were collected by centrifugation (13,000 g) for 20 min at 4°C.
L6 muscle cells were maintained and differentiated into multinucleated myotubes as described previously (JeBailey et al. 2004). C2C12 muscle cells were maintained and differentiated as reported previously (Miyatake et al. 2016). For all experiments, myotubes were incubated in serum-free α-minimal essential medium (Dulbecco’s modified Eagle’s medium for C2C12) containing 5 mmol l−1 glucose for 3 h prior to stimulation with insulin (100 nM, 10 min stimulation), isoproterenol (20 μM, 20 min stimulation), caffeine (5 mM, 20 min stimulation) or electrical pulse stimulation (24 ms pulse, 1 Hz repetition for 1 h). Following stimulation, cells were rinsed in PBS and the cells were lysed in lysis buffer (Cytoskeleton Inc., Denver, CO, USA) and centrifuged for 1 min at 10,000 g. Rac1 activities were measured in the supernatant using a commercially available Rac1 activation assay kit (BK 126; Cytoskeleton Inc.). In brief, immediately after centrifugation, the protein concentration in the lysates were determined (
