Marked Phenotypic Differences Of Endurance Performance And

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Am J Physiol Endocrinol Metab 305: E213–E229, 2013. First published May 21, 2013; doi:10.1152/ajpendo.00114.2013. Marked phenotypic differences of endurance performance and exercise-induced oxygen consumption between AMPK and LKB1 deficiency in mouse skeletal muscle: changes occurring in the diaphragm Shinji Miura,1,2 Yuko Kai,2 Miki Tadaishi,2 Yuka Tokutake,3 Kimitoshi Sakamoto,4 Clinton R. Bruce,5 Mark A. Febbraio,5 Kiyoshi Kita,4 Shigeru Chohnan,3 and Osamu Ezaki2,6 1 Submitted 28 February 2013; accepted in final form 15 May 2013 Miura S, Kai Y, Tadaishi M, Tokutake Y, Sakamoto K, Bruce CR, Febbraio MA, Kita K, Chohnan S, Ezaki O. Marked phenotypic differences of endurance performance and exercise-induced oxygen consumption between AMPK and LKB1 deficiency in mouse skeletal muscle changes occurring in the diaphragm. Am J Physiol Endocrinol Metab 305: E213–E229, 2013. First published May 21, 2013; doi:10.1152/ajpendo.00114.2013.—LKB1 phosphorylates members of the AMP-activated protein kinase (AMPK) family. LKB1 and AMPK in the skeletal muscle are believed to regulate not only fuel oxidation during exercise but also exercise capacity. LKB1 was also required to prevent diaphragm fatigue, which was shown to affect exercise performance. Using mice expressing dominant negative (DN) mutants of LKB1 and AMPK, specifically in the skeletal muscle but not in the heart, we investigated the roles of LKB1 and AMPK activity in exercise performance and the effects of these kinases on the characteristics of respiratory and locomotive muscles. In the diaphragm and gastrocnemius, both AMPK-DN and LKB1-DN mice showed complete loss of AMPK␣2 activity, and LKB1-DN mice showed a reduction in LKB1 activity. Exercise capacity was significantly reduced in LKB1-DN mice, with a marked reduction in oxygen consumption and carbon dioxide production during exercise. The diaphragm from LKB1-DN mice showed an increase in myosin heavy chain IIB and glycolytic enzyme expression. Normal respiratory chain function and CPT I activity were shown in the isolated mitochondria from LKB1-DN locomotive muscle, and the expression of genes related to fiber type, mitochondria function, glucose and lipid metabolism, and capillarization in locomotive muscle was not different between LKB1-DN and AMPK-DN mice. We concluded that LKB1 in the skeletal muscle contributes significantly to exercise capacity and oxygen uptake during exercise. LKB1 mediated the change of fiber-type distribution in the diaphragm independently of AMPK and might be responsible for the phenotypes we observed. liver kinase B1; AMP-activated protein kinase; exercise; diaphragm; oxygen uptake prevents obesity and diabetes (15, 22), but the molecular mechanisms mediating these favorable effects of exercise remain elusive. AMP-activated protein kinase (AMPK) is a master sensor and IT IS WELL RECOGNIZED THAT INCREASING PHYSICAL ACTIVITY Address for reprint requests and other correspondence: S. Miura, Laboratory of Nutritional Biochemistry, Graduate School of Nutritional and Environmental Sciences, Univ. of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan (e-mail: [email protected]). http://www.ajpendo.org regulator of energy balance at the cellular level (17, 18). In addition, skeletal muscle AMPK activity is required for maintaining exercise capacity. Several studies have demonstrated that exercise capacity is reduced dramatically in a skeletal musclespecific AMPK-deficient mice (14, 33, 51). However, more experiments have now demonstrated that skeletal muscle AMPK is dispensable in modulating the effects of contraction or pharmacological activation on fuel metabolism, raising the possibility that AMPK-independent pathways may regulate glucose and lipid metabolism (2, 11, 13, 26, 28, 33, 37). Although fuel metabolism is an important factor for endurance performance, it is unclear whether these pathways are involved in the decreased exercise capacity observed in genetically modified AMPK model mice. There are 12 protein kinases related to AMPK␣1 and -␣2 (31), and these AMPK-related kinases might play a permissive role in contraction (exercise) or in the pharmacological effects of fuel metabolism in the skeletal muscle. Liver kinase B1 (LKB1) is an upstream kinase regulating the activity of 13 of the 14 AMPKrelated kinases (29). Therefore, it is likely that alteration of LKB1 activity in the skeletal muscle may affect fuel oxidation during exercise. To elucidate the physiological roles of LKB1, skeletal muscle and heart LKB1-knockout (mhLKB1-KO) mice were produced (27, 45, 55, 57). In previous studies, tissue-specific ablation of LKB1 has revealed that LKB1 plays a major role in the regulation of cellular metabolism. Two groups have shown that disruption of LKB1 in the skeletal muscle results in impaired contraction-stimulated glucose transport, suggesting that LKB1 is an important mediator of contraction-stimulated glucose transport in the skeletal muscle and that one or more AMPK-related protein kinases are important in the regulation of contraction-stimulated glucose transport (28, 45). Although markers of mitochondrial content in the muscle are decreased in mhLKB1-KO mice (57), it is not known what effect this has on fat metabolism. Koh et al. (27) reported that intramuscular triglyceride content is elevated in mhLKB1-KO muscles. This finding could result from increased fatty acid uptake or from decreased fatty acid oxidation by the muscle. However, Thomson et al. (55) demonstrated that baseline fatty acid oxidation was not different between wild-type (WT) and mhLKB1-KO muscles. 5-Aminoimidazole-4-carboxamide-3ribonucleoside (AICAR)-mediated increase in fatty acid oxidation was inhibited in mhLKB1-KO extensor digitorum longus (EDL) muscles, but fatty acid oxidation was not altered by AICAR treatment in soleus muscles from either genotype. To the best of 0193-1849/13 Copyright © 2013 the American Physiological Society E213 Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on September 11, 2017 Laboratory of Nutritional Biochemistry, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka, Japan; 2Department of Nutritional Science, National Institute of Health and Nutrition, Tokyo, Japan; 3Department of Bioresource Science, Ibaraki University College of Agriculture, Ibaraki, Japan; 4Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, Tokyo, Japan; 5Cellular and Molecular Metabolism Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Australia; and 6Department of Human Health and Design, Showa Women’s University, Tokyo, Japan E214 ROLE OF MUSCLE LKB1 AND AMPK IN ENDURANCE PERFORMANCE MATERIALS AND METHODS Transgenic mice. Transgenic mice that specifically express an NH2-terminal FLAG epitope-tagged DN-mutant (D194A) mouseLKB1 transgene (46) in the skeletal muscle under control of the human ␣-skeletal actin promoter were constructed (5). Coexpression of this mutant with STE20-related adaptor (STRAD) and mouse protein 25 (MO25) in human embryonic kidney-293T cells yields a catalytically inactive complex (21). Two integration-positive mouse lines, B and C, were studied. Male chimeras harboring the LKB1 (D194A) transgene were mated with C57BL/6J females to obtain F1 offspring. Heterozygous F3 LKB1-DN mice and their WT littermates were compared. Transgenic mice were produced, expressing a DN mutant of AMPK␣1 in the skeletal muscle (AMPK-DN mice), as described previously (34, 54), and heterozygous F7 AMPK-DN were used. Mice were maintained on a 12:12-h light-dark cycle at 22°C and were fed a normal chow diet ad libitum (CE2; CLEA Japan, Tokyo, Japan). All animal procedures were reviewed and approved by the National Institute of Health and Nutrition Ethics Committee on Animal Research. Mice were euthanized by cervical dislocation before the removal of muscles for analysis. Measurement of LKB1, AMPK, MAP/microtuble affinity-regulating kinase 4, and salt-inducible kinase family kinase 3 activity. Muscle lysate protein was incubated at 4°C for 1 h, with the corresponding antibody conjugated to protein G-Sepharose (GE Healthcare BioScience, Uppsala, Sweden). Phosphotransferase activity of the immunoprecipitates was measured as described previously (44). The antibodies against LKB1, MAP/microtuble affinity-regulating kinase 4 (MARK4), and salt-inducible kinase (SIK) family kinase 3 (QSK) were provided by Dr. K. Sakamoto of the University of Dundee, Dundee, UK. The antibodies against the AMPK␣1 or -␣2 catalytic subunits were purchased from Upstate Biotechnology (Lake Placid, NY; cat. nos. 07-350 and 07-363). Western blots. The protein and phosphorylated protein levels in the gastrocnemius muscles were measured by Western blotting with the following antibodies: anti-LKB1 (cat. no. sc-28788; Santa Cruz Biotechnology, Santa Cruz, CA), anti-LYK5 (STRAD) (cat. no. ab64799; Abcam, Tokyo, Japan), anti-MO25 (cat. no. M7070; Sigma, St. Louis, MO), anti-phospho-AMPK (Thr172) (cat. no. 2531; Cell Signaling Technology, Beverly, MA), anti-AMPK␣1 and -␣2 (cat. no. 07-350 and 07-363; Upstate Biotechnology), anti-phospho-ACC (cat. no. 3661; Cell Signaling Technology), and anti-ACC (cat. no. 3662; Cell Signaling Technology). Exercise protocol. Three different modes of treadmill exercise were examined; mice began running on a treadmill at 6 m/min for 15 min, and the speed was then increased by 2 m/min every 3 min until exhaustion. For measuring blood lactate concentrations, mice began running on a treadmill at 10 m/min for 15 min, and the speed was then increased by 5 m/min every 15 min for ⱕ25 m/min. Mice also ran until exhaustion, which is defined as the animal remaining on the shocker plate for more than 10 –15 s. For measuring LKB1, AMPK, MARK4, QSK, and carnitine palmitoyltransferase (CPT I) activities, malonyl-CoA and adenosine phosphate concentrations in muscle, and liver and muscle glycogen concentrations after exercise, mice ran at 10 m/min for 30 min. Measurement of oxygen consumption and carbon dioxide production. Open-circuit indirect calorimetry was performed with an O2/CO2 metabolism-measuring system for small animals (MK-5000RQ; Mu˙ O2 and V ˙ CO2 romachi Kikai, Tokyo, Japan). The system monitored V at 3-min intervals and calculated the respiratory quotient (RQ) ratio ˙ CO2/V ˙ O2). Measurements were performed during the dark period (V (from 1900 to 0700) or light period (from 0700 to 1630). For ˙ O2 and V ˙ CO2 during exercise, mice were allowed to measurement of V acclimatize to the airtight treadmill chamber (Muromachi Kikai) for ˙ O2 and V ˙ CO2 were stable. Measurements were 30 min, at which point V continued for another 30 min while mice were maintained in a sedentary state. Mice were then exercised as described above. The substrate utilization rate and energy production rate were calculated with the formula used by Ferrannini (12), where the rate of glucose ˙ CO2 (l/min) ⫺ 3.21 ⫻ V ˙ O2 (l/min) ⫺ 2.87 oxidation (g/min) ⫽ 4.55 ⫻ V ˙ O2 ⫺ ⫻ N (mg/min) and the rate of lipid oxidation (g/min) ⫽ 1.67 ⫻ (V ˙ CO2) ⫺ 1.92 ⫻ N and where N is the rate of urinary nitrogen V excretion used to estimate protein oxidation. However, considering that only a small portion of resting and exercise energy expenditure arises from protein oxidation, the contributions of protein oxidation were ignored (64). Quantitative real-time RT-PCR. Methods of RNA preparation, quantitative real-time RT-PCR, and primers were as described previously (34, 35). The other mouse-specific primer pairs used are shown in Table 1. Histological evaluation. The tissue blocks were rapidly frozen in isopentane cooled by liquid nitrogen. Transverse cross sections of 10 ␮m were made with a cryostat (CM1510; Leica) at ⫺20°C. The sections were stained for detection of succinate dehydrogenase (SDH) activity, following the staining protocol described by Nishizaka et al. (38). The SDH activity was determined by incubation in the following medium: 0.9 mM 1-methoxyphenazine methylsulfate, 1.5 mM nitroblue tetrazolium, 5.6 mM ethylenediaminetetraacetic acid disodium AJP-Endocrinol Metab • doi:10.1152/ajpendo.00114.2013 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on September 11, 2017 our knowledge, there are no reports of whether LKB1 is required for exercise- or contraction-stimulated fatty acid oxidation. Interestingly, mhLKB1-KO mice exhibit diminished AMPK activity, reduced phosphorylation of acetyl-CoA carboxylase (ACC), decreased voluntary wheel running, and lower levels of mitochondrial proteins, suggesting that a reduction in oxidative capacity of the muscle might be the reason for the reduced running volume. However, heart LKB1 was also reduced in mhLKB1-KO mice, and a considerable decrease in citrate synthase activity in mhLKB1-KO mice was observed; these changes in the heart might be another reason for the reduced running volume (57). Recently, the diaphragm of mhLKB1-KO mice, which fatigues more quickly and has an impaired ability to recover compared with WT, was shown to have a lower percentage of type IIx fibers and an elevated percentage of type IIb fibers (6). Respiratory muscle fatigue might be involved in limiting exercise tolerance, because the time to exhaustion was decreased when the respiratory muscles were prefatigued, and the time was increased when exercise-induced diaphragm fatigue was prevented (43). Therefore, rapid diaphragm fatigue could be an additional reason for the decrease in endurance performance observed in mhLKB1-KO mice. Although LKB1 is an important factor for endurance performance, it is unclear whether the decrease in the performance of mhLKB1-KO mice was due to 1) a defect of skeletal muscle or heart LKB1 activity, 2) fatigue of the diaphragm rather than the LKB1-mediated changes in the locomotive muscle, or 3) a decrease in skeletal muscle (diaphragm and locomotive muscle) AMPK activity. The generation of transgenic mice overexpressing a dominant negative (DN) form of LKB1 in the skeletal muscle, but not in the heart, may help to elucidate the role of skeletal muscle LKB1 in exercise performance. In this study, using mice expressing a skeletal muscle-specific DN mutant of LKB1 or AMPK, the role of these kinases on exercise performance was examined. Furthermore, the effects of these kinases on diaphragm or locomotive muscle characterization were compared using these animal models. As a result, we found that a deficiency of LKB1, specifically that in the skeletal muscle, greatly affected exercise capacity and changed the characteristics of the diaphragm. E215 ROLE OF MUSCLE LKB1 AND AMPK IN ENDURANCE PERFORMANCE Table 1. Mouse-specific primer pairs used for quantitative RT-PCR Reverse 5=-GGCCCTGCACTCTCGCTTTC-3= 5=-CCAAGGGCCTGAATGAGGAG-3= 5=-AAGCGAAGAGTAAGGCTGTC-3= 5=-AGGCCAGGGTCCGTGAA-3= 5=-ACAAGCTGCGGGTGAAGAGC-3= 5=-GGAATGTGGAGCGTGCTAAAA-3= 5=-CCGACTAAATCAAGCAACAGTAACA-3= 5=-CTATGTGTATGGCCCCATCC-3= 5=-GGGTGACCCCACTATTTGTC-3= 5=-CGGAAATCATATCCAACCAG-3= 5=-CCTGAACATCGAGTGTCGAATAT-3= 5=-CTCTTCATCGCGGCCATCATTCT-3= 5=-GCCGGTGTGCATTCCAA-3= 5=-TTTCTCCCATCATGACAGAGCA-3= 5=-TGGGAAACTGTCCTCCATGAA-3= 5=-CCCCCAACTCCGGAACTG-3= 5=-AGCGTTCGTCCCACTGATTC-3= 5=-GCATCAAAAGGGAGCCCAAT-3= 5=-ATGGCTGTCGCTGGTTTCTC-3= 5=-GGAACCCAGCTGTTTGACCA-3= 5=-GCCATCGCCGTGTTGAC-3= 5=-TTGACTCTGCCCCCATCAC-3= 5=-AGCTGGTGAAGAGCTGGTATATCC-3= 5=-CCATGGATGGACGGTAACG-3= 5=-CGGAACTGTGGGCTCATTG-3= 5=-TGACAGTGCCACCAACAAGAA-3= 5=-CGAGCAGGGCATCAATGTCT-3= 5=-ACTCGGTGTGGGCTTTGC-3= 5=-GTGCAAGCAGCCCGTCTAG-3= 5=-TGGATCTGTGCAGCGGATT-3= 5=-CGAGATAGAGTACATCTTCAAGCC-3= 5=-AAAAAAAAAGGAGAGTGCTGTGAAG-3= 5=-GGTCAGCGCCGAGGG-3= 5=-AAGGAGCAAGGAGGCCATAT-3= 5=-AAGGAAGTAGCCAATGCAGTGAA-3= 5=-TGCCAGGACGCGCTTGT-3= 5=-GCAAAGGCTCCAGGTCTGAG-3= 5=-GTGATTGCTTGCAAAGGAAC-3= 5=-CCACGTTGCGCTTCTGTTC-3= 5=-CAGGACAGTGACAAAGAACG-3= 5=-TGCTGGAAAAACACTTCGGAATA-3= 5=-AAATTTCAGAGCATTGGCCATAG-3= 5=-AGCGGGCTCTCACTTCTTC-3= 5=-CGGAAGAGATCTCGAGTTGG-3= 5=-TGAGGACCGCTAGCAAGTTTG-3= 5=-GGTCTTCTTCTGAATCTTGCAGCT-3= 5=-TCTGCCATCTTCTGCAGCAGCTT-3= 5=-CACTCCGGAACCCCAACAG-3= 5=-AATGCACGTCCCCATCTCC-3= 5=-TCAAAGCGTGTTCTTTCCTTCTC-3= 5=-AATACACCTTACAACCCTTGCTGTT-3= 5=-GCTCCAACAGCACCAATCTCA-3= 5=-GCCTTGCCTCCAATCATGA-3= 5=-ACCCATGCCGACAATGAAGT-3= 5=-CAGGGGAACGAGAAGGTGAAA-3= 5=-GCCCTGACGGCAGCATT-3= 5=-GCAGGCCCAATGGTACAAAT-3= 5=-TCTGGTCTTCTGGGCTCTTCTC-3= 5=-TACAGGGCGGCCACAAGT-3= 5=-GCATGAGAATGCCTCCAAACA-3= 5=-GCGCTATCGCCCTTTCG-3= 5=-CCGTACAGGCCCATGTTCTT-3= 5=-ATGATGGTAGTAGGCTTGGTCATG-3= 5=-TTGCGGCGATACATGATCAT-3= 5=-GGGTCACCATAGAGCTGAAGACA-3= 5=-TCATCGTTACAGCAGCCTGC-3= 5=-TCCCAGCCCGGAACAGA-3= 5=-GCGGATGGAGTGGTCGC-3= 5=-ATATGTTCTGAGGGTGACCCC-3= 5=-TCACACGCTTCGCCATCA-3= MHC, myosin heavy chain; mtTFA, mitochondrial transcription factor A; COX, cytochrome c oxidase; PGC, peroxisome proliferator-activated receptor-␥ coactivator; PPAR, peroxisome proliferator-activated receptor; Foxo, forkhead transcriptional factor; Sox, SRY box-containing gene; GYS, glycogen synthase; PHKA, phosphorylase kinase-␣; PYGM, muscle glycogen phosphorylase; GLUT, glucose transporter; HK, hexokinase; PFKM, muscle phosphofructokinase; PKM, muscle pyruvate kinase; PDK, pyruvate dehydrogenase kinase; LPL, lipoprotein lipase; FATP, fatty acid transport protein; FABPpm, plasma membrane fatty acid-binding protein; FABP, fatty acid-binding protein; CPT Ib, carnitine palmitoyltransferase Ib; MCAD, medium chain acyl-CoA dehydrogenase; Mb, myoglobin; VEGF, vascular endothelial growth factor; PDGF-B, platelet-derived growth factor-B; nNOS, neuronal nitric oxide synthase; eNOS, endothelial nitric oxide synthase. salt, and 48 mM succinate disodium salt, pH 7.6, in 100 mM phosphate buffer. The reaction time was 10 min, after which the reaction was arrested using distilled water, and then sections were dehydrated in a graded series of ethanol and passed through xylene. The cross-sectional areas were measured by tracing fiber outlines of ⬃150 fibers of the muscle section. The images were analyzed using Image J software (National Institutes of Health, Bethesda, MD), and the thresholded pixels in grayscale were set at 170 to distingish SDH negative. Measurement of malonyl-CoA. Malonyl CoA in the gastrocnemius was measured as described previously (9, 10, 58). Malonyl-CoA in the skeletal muscle was extracted by homogenization in 400 ␮l of 0.6 M sulfuric acid per 100 mg of tissue, and then the homogenized muscles were kept at 4°C overnight. After centrifugation at 9,000 g at 4°C for 10 min, 1 M Tris (0.05 vol of the supernatant) was added to the supernatant, and the resulting solution was carefully adjusted to an approximate pH of 6.5 with NaOH on ice. The neutralized solution was kept at ⫺80°C overnight, and the clear extract was recovered by centrifugation after thawing. First, acetyl-CoA in the extracts was eliminated with citrate synthase (EC 4.1.3.7), and then the remaining malonyl-CoA was measured using the acyl-CoA cycling method with malonate decarboxylase (10, 58). The reaction mixture for the citrate synthase treatment contained 50 mM Tris·HCl (pH 7.2), 10 mM MgSO4, 2 mM oxaloacetate, 1 U of citrate synthase from porcine heart (Roche Diagnostics, Mannheim, Germany), and the neutralized extract in 1 ml. The reaction was carried out at 25°C for 20 min and terminated by placing the reaction tube on ice slush. An aliquot of the reaction mixture was transferred to the mixture of the acyl-CoA cycling method. The reaction mixture of the cycling method contained 50 mM Tris·HCl (pH 7.2), 1 mM 2-mercaptoethanol, 10 mM MgSO4, 50 mM malonate, 10 mM ATP, 1 U of malonate decarboxylase, and the extract that was treated with citrate synthase in 400 ␮l. Malonate decarboxylase was prepared from Pseudomonas putida JCM 20089 (9). The cycling reaction was initiated by adding malonate decarboxylase, and the mixture was incubated at 30°C for 20 min, followed by the addition of 1 U of acetate kinase from Escherichia coli (EC 2.7.2.1; Roche Diagnostics). After 20 min of incubation, 0.2 ml of 2.5 M of neutralized hydroxylamine was added, and the incubation was continued for an additional 20 min at 30°C. The reaction was terminated by adding 0.6 ml of 10 mM ferric chloride that was dissolved in 25 mM trichloroacetic acid and 1 M HCl. The absorbance at 540 nm of the acetohydroxamate was measured. Every assay was performed in duplicate. Measurement of adenine nucleotides. The ATP, ADP, and AMP concentrations in quadriceps were determined using high-performance liquid chromatograms (36). The skeletal muscle was homog- AJP-Endocrinol Metab • doi:10.1152/ajpendo.00114.2013 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on September 11, 2017 36B4 MHC1 MHC2A MHC2X MHC2B mtTFA COX2 COX4 Nur77 PGC-1␣ PPAR␣ PPAR␦ MyoD Foxo1 Sox6 GYS1 PHKA1 PYGM GLUT4 HK2 PFKM PKM2 PDK4 LPL CD36 FATP1 FABPpm FABP3 CPT Ib MCAD VEGF-A VEGF-B PDGF-B nNOS eNOS Forward E216 ROLE OF MUSCLE LKB1 AND AMPK IN ENDURANCE PERFORMANCE A Mouse LKB1 (D194A) BGHpA cDNA (1308 bp) Human skeletal actin Exon IVS promoter (2 kb) 1 1 D C-line B-line WT LKB1-DN WT LKB1-DN LKB1 STRAD B 0.4 Gastro. Heart AMPK 1 0.3 0.2 AMPK 2 0.1 Phospho-AMPK *** *** 0 WT WT ACC LKB1-DN LKB1-DN Phospho-ACC Activities (pmol min-1 mg protein-1) C 7 AMPK 1 AMPK 2 6 5 4 3 2 1 *** *** 0 WT WT LKB1-DN Activities (pmol min-1 mg protein-1) LKB1-DN 0.6 MARK4 QSK 0.5 0.4 0.3 0.2 *** *** *** 0.1 *** 0 WT WT LKB1-DN LKB1-DN Fig. 1. Skeletal muscle-specific overexpression of liver kinase B1-dominant negative (LKB1-DN) reduces the activity of LKB1 and its target enzyme and the amounts of phosphorylated (phospho) AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). A: map of the DN mutant of LKB1 (D194A) transgene construct used for the microinjection of fertilized eggs. B: the activity of LKB1 in the skeletal muscles (gastrocnemius) and heart from lines B and C transgenic mice (LKB1-DN) and wild-type (WT) littermates. The LKB1 activity was measured in immunoprecipitates. Each value is the mean ⫾ SE of 4 mice from each of the B and C lines (8 wk old). C: activities of the AMPK␣1 and -␣2 subunits, MAP/microtuble affinity-regulating kinase 4 (MARK4), and salt-inducible kinase family kinase (QSK) in the skeletal muscle (gastrocnemius) from lines B and C transgenic mice and WT littermates. These activities were measured in immunoprecipitates. Each value is the mean ⫾ SE of 4 mice from each of the B and C lines (8 wk old). D: typical data from Western blot analyses of LKB1, STE20-related adaptor (STRAD), mouse protein 25 (MO25), AMPK␣1, AMPK ␣2, phospho-AMPK, ACC, and phospho-ACC in the skeletal muscle (gastrocnemius) from LKB1-DN (lines B and C) mice and WT littermates. ***P ⬍ 0.001 vs. WT littermates. AJP-Endocrinol Metab • doi:10.1152/ajpendo.00114.2013 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on September 11, 2017 LKB1 activities (pmol min-1 mg protein-1) MO25 E217 ROLE OF MUSCLE LKB1 AND AMPK IN ENDURANCE PERFORMANCE Activity (pmol min-1 mg protein-1) LKB1 activity 0.14 WT 0.12 AMPK-DN 0.10 LKB1-DN 0.08 ** 0.06 0.04 *** 0.02 0 Heart Diaphragm Gastro. 8 6 * * 4 2 * Activity (pmol min-1 mg protein-1) Activity (pmol min-1 mg protein-1) 10 3.5 3.0 2.5 2.0 1.5 1.0 0.5 *** *** *** 0 0 Heart Diaphragm Heart Gastro. Diaphragm *** Gastro. Fig. 2. Effect of skeletal muscle-specific overexpression of AMPK-DN or LKB1-DN on activities of LKB1 and AMPK in the heart, diaphragm, and gastrocnemius. Activities of LKB1, AMPK␣1, and AMPK␣2 in the heart, diaphragm, and gastrocnemius (Gastro.) from WT, AMPK-DN, and LKB1-DN mice. The activities were measured in immunoprecipitates. Each value is the mean ⫾ SE of 4 – 6 mice from each group (24 wk old). *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 vs. WT mice. enized with 1 N perchloric acid and centrifuged at 10,000 g for 15 min. After neutralization of the supernatant with calcium carbonate, the ATP, ADP, and AMP concentrations were determined by highperformance liquid chromatogram. A Luna 5 ␮ NH2 column (100 A, 250 mm ⫻ 4.60 mm ID; Phenomenex, Torrance, CA) was used for separation. The flow rate was 1.0 ml/min, and the detection wavelength was 260 nm. Mobile phases were 90% of 20 mM sodium phosphate buffer (pH 7.0, mobile phase A) and 10% of 500 mM sodium phosphate buffer (pH 6.0, mobile phase B). Their compositions changed to 0:100 (mobile phase A/mobile phase B) gradually from 0 to 6 min. Each standard reagent (Sigma Aldrich, St. Louis, MO) was used for peak identification and quantitative determination of adenine nucleotides. All samples were stored at ⫺80°C until analysis. Preparation of the mitochondrial fraction from skeletal muscle. To obtain the mitochondrial fraction, differential centrifugation was used as described previously (7). Enzymatic activity of the respiratory complex. NADH-cytochrome c reductase, succinate-cytochrome c reductase, and cytochrome c oxidase (COX) activities in the mitochondrial fraction from the tibialis anterior muscle were measured as described previously (4, 60). Citrate synthase activity in skeletal muscle homogenate (gastrocnemius) or the mitochondrial fraction (tibialis anterior) was measured as described previously (49). Measurement of mitochondrial respiration. Mitochondrial oxygen consumption was measured polarographically using Oxygraph (Hansatech Instruments, Kings Lynn, UK) at 30°C in a medium containing 225 mM mannitol, 75 mM sucrose, 10 mM KCl, 10 mM Tris·HCl, and 5 mM KH2PO4 at pH 7.2, as described previously (60). Measurement of CPT I activity. The determination of CPT I activity was measured as described by McGarry et al. (32), with minor modifications (3, 7). Other assays. Lactate levels were measured by Lactate Pro (Arkray, Kyoto, Japan). Blood samples were obtained by cutting the tail tip. Glycogen content in the skeletal muscle and liver was measured as glycosyl units after acid hydrolysis (30). Statistical analysis. Data were analyzed by one-way or two-way ANOVA. Where differences were significant, each group was compared with the other by Student’s t-test (JMP 5.1.2; SAS, Cary, NC). In the exercise tolerance test, a Kaplan-Meier survival curve was obtained, and a comparison of groups was performed using the log-rank test (StatView 5.0). Statistical significance was defined as P ⬍ 0.05. Values are shown as means ⫾ SE. RESULTS Production of skeletal muscle-specific LKB1-DN mice. LKB1DN mice were made with a DNA construct containing the 5=-flanking skeletal muscle-specific regulatory region and promoter of the human ␣-skeletal actin gene and a cDNA encoding a DN mutant of LKB1 (Fig. 1A). Northern blots probed by LKB1 cDNA showed that line B and C transgenic mice expressed one transcript (1.9 kb) from the LKB1-DN transgene in the skeletal muscles (gastrocnemius) but not in the white adipose tissue, liver, spleen, kidney, heart, lung, brown adipose tissue, or brain (data not shown). LKB1 activity in the skeletal muscles was not detected in LKB-DN mice, whereas AJP-Endocrinol Metab • doi:10.1152/ajpendo.00114.2013 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on September 11, 2017 2-AMPK activity 1-AMPK activity 12 E218 ROLE OF MUSCLE LKB1 AND AMPK IN ENDURANCE PERFORMANCE Table 2. Body and tissue weights of LKB1-DN mice and WT littermates (3 mo old) Male Female Weight, g WT LKB1-DN WT LKB1-DN Body weight Gastrocnemius Quadriceps TA and EDL Soleus Diaphragm Gonadal white adipose tissue Liver Heart 25.7 ⫾ 0.4 0.277 ⫾ 0.005 0.348 ⫾ 0.008 0.119 ⫾ 0.003 0.018 ⫾ 0.001 0.093 ⫾ 0.003 0.359 ⫾ 0.027 1.231 ⫾ 0.046 0.112 ⫾ 0.003 24.9 ⫾ 0.7 0.260 ⫾ 0.006 0.310 ⫾ 0.012* 0.124 ⫾ 0.003 0.019 ⫾ 0.001 0.105 ⫾ 0.003* 0.328 ⫾ 0.022 1.229 ⫾ 0.041 0.111 ⫾ 0.005 20.2 ⫾ 0.3 0.211 ⫾ 0.005 0.265 ⫾ 0.008 0.095 ⫾ 0.001 0.016 ⫾ 0.001 0.085 ⫾ 0.002 0.254 ⫾ 0.050 0.869 ⫾ 0.030 0.088 ⫾ 0.004 20.9 ⫾ 0.2 0.207 ⫾ 0.003 0.239 ⫾ 0.009 0.097 ⫾ 0.002 0.016 ⫾ 0.001 0.088 ⫾ 0.005 0.296 ⫾ 0.028 0.976 ⫾ 0.052 0.086 ⫾ 0.003 Values are means ⫾ SE; n ⫽ 6. LKB1-DN, liver kinase B1-dominant negative; WT, wild type; TA, tibialis anterior; EDL, extensor digitorum longus. *P ⬍ 0.05. (Fig. 1D), suggesting that overexpression of LKB1-DN impairs LKB1 activity and subsequent phosphorylation of AMPK and ACC in the skeletal muscle. Because the data from lines B and C showed similar changes, the data were combined and compared with the WT littermates in the following analysis. LKB1 and AMPK activities were measured in the heart, diaphragm, and gastrocnemius (Fig. 2). LKB1 activity in the diaphragm and gastrocnemius was decreased significantly in LKB-DN mice compared with WT littermates, whereas in the heart it was not altered. LKB1 activity in AMPK-DN mice was not altered in the heart, diaphragm, or gastrocunemius. AMPK␣1 activity in the diaphragm was impaired in LKB1-DN and AMPK-DN mice; however, the activity in the gastrocnemius was only slightly impaired in AMPK-DN mice. AMPK␣2 activity in the diaphragm was reduced by 96 and 98% in LKB1-DN and Table 3. Oxygen consumption, carbon dioxide production, RQ ratio, spontaneous motor activity while sedentary, and body weight change after 24 h of fasting Male Fed (dark) ˙ O2, ml 䡠 min⫺1 䡠 kg⫺1 V ˙ CO2, ml 䡠 min⫺1 䡠 kg⫺1 V RQ ratio Activity, counts/min Fed (light) ˙ O2, ml 䡠 min⫺1 䡠 kg⫺1 V ˙ CO2, ml 䡠 min⫺1 䡠 kg⫺1 V RQ ratio Activity, counts/min Fast (dark) ˙ O2, ml 䡠 min⫺1 䡠 kg⫺1 V ˙ CO2, ml 䡠 min⫺1 䡠 kg⫺1 V RQ ratio Activity, counts/min Fast (light) ˙ O2, ml 䡠 min⫺1 䡠 kg⫺1 V ˙ CO2, ml 䡠 min⫺1 䡠 kg⫺1 V RQ ratio Activity, counts/min Body weight, g Before fasting After fasting Difference during fasting Female WT (n ⫽ 5) LKB1-DN (n ⫽ 5) WT (n ⫽ 7) LKB1-DN (n ⫽ 7) 49.3 ⫾ 1.6 40.5 ⫾ 1.6 0.82 ⫾ 0.01 206 ⫾ 37 50.3 ⫾ 1.2 41.8 ⫾ 1.1 0.83 ⫾ 0.01 171 ⫾ 21 66.1 ⫾ 1.7 53.5 ⫾ 3.0 0.81 ⫾ 0.03 258 ⫾ 28 66.1 ⫾ 2.0 53.4 ⫾ 2.5 0.80 ⫾ 0.02 245 ⫾ 28 42.1 ⫾ 1.0 33.3 ⫾ 0.9 0.79 ⫾ 0.01 62 ⫾ 12 42.3 ⫾ 1.5 34.1 ⫾ 1.3 0.80 ⫾ 0.01 45 ⫾ 9 55.8 ⫾ 1.3 45.5 ⫾ 1.2 0.81 ⫾ 0.01 78 ⫾ 8 54.3 ⫾ 1.9 43.6 ⫾ 1.9 0.80 ⫾ 0.01 58 ⫾ 6 42.7 ⫾ 1.4 30.1 ⫾ 0.9 0.70 ⫾ 0.00 274 ⫾ 59 42.7 ⫾ 1.6 30.4 ⫾ 1.0 0.71 ⫾ 0.01 195 ⫾ 19 52.0 ⫾ 2.2 36.3 ⫾ 1.5 0.70 ⫾ 0.01 260 ⫾ 33 51.0 ⫾ 2.0 35.8 ⫾ 1.5 0.70 ⫾ 0.01 217 ⫾ 31 31.5 ⫾ 0.9 21.6 ⫾ 0.6 0.69 ⫾ 0.00 39 ⫾ 14 32.5 ⫾ 0.9 22.4 ⫾ 0.5 0.69 ⫾ 0.01 36 ⫾ 10 34.5 ⫾ 1.1 23.8 ⫾ 0.7 0.69 ⫾ 0.01 64 ⫾ 14 32.8 ⫾ 1.6 22.6 ⫾ 1.2 0.69 ⫾ 0.01 42 ⫾ 9 32.3 ⫾ 0.7 28.7 ⫾ 0.7 ⫺3.6 ⫾ 0.3 31.5 ⫾ 1.8 27.7 ⫾ 1.9 ⫺3.7 ⫾ 0.2 19.7 ⫾ 0.7 17.1 ⫾ 0.6 ⫺2.6 ⫾ 0.2 19.8 ⫾ 0.3 17.3 ⫾ 0.3 ⫺2.6 ⫾ 0.1 Values are means ⫾ SE of LKB1-DN and WT mice (12 wk old). RQ, respiratory quotient. Oxygen consumption, carbon dioxide production, RQ ratio, and spontaneous motor activity were measured using open-circuit indirect calorimetry with an O2/CO2 metabolism-measuring system for small animals that was equipped with an infrared sensor. Measurements were performed during the dark (from 1900 to 0700) or light period (from 0700 to 1630) under ad libitum feeding (fed) or fasting (fast) conditions. In fasting experiments, the remaining food was removed at 1700. No significant differences were observed between LKB1-DN and WT littermates. AJP-Endocrinol Metab • doi:10.1152/ajpendo.00114.2013 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on September 11, 2017 in the heart it was not altered (Fig. 1B). Activities of LKB1target enzymes, namely AMPK, MARK4, and QSK, were measured in the skeletal muscle (Fig. 1C). AMPK␣2 activity was reduced by 94 and 95% in lines B and C, respectively. AMPK␣1 activity was not impaired, which was due possibly to its localization in nonmyocytes of the skeletal muscle (13, 37). MARK4 and QSK activity was also reduced markedly in both lines of LKB1-DN mice. Immunoblot analysis with LKB1 antibody showed that exogenous LKB1-DN protein (55 kDa) in the gastrocnemius was 10to 14-fold greater than endogenous LKB1 protein (Fig. 1D). Levels of STRAD and MO25 were not altered in LKB1-DN mice. The levels of AMPK␣1 and -␣2 proteins did not differ in LKB1-DN mice. The levels of phosphorylated AMPK and ACC protein in LKB1-DN mice were lower than in WT littermates ROLE OF MUSCLE LKB1 AND AMPK IN ENDURANCE PERFORMANCE Glucose (mmo/L) A Male Female 15.0 12.5 10.0 7.5 5.0 WT LKB1-DN 2.5 0 0 30 60 90 120 0 30 60 90 120 B 125 Glucose (% of 0 min) Time after glucose administration (min) 100 Male Female 75 50 WT LKB1-DN 25 0 0 30 60 90 120 0 30 60 90 120 Time after insulin injection (min) Fig. 3. Effect of skeletal muscle-specific overexpression of LKB1-DN on systemic glucose homeostasis. A: oral glucose tolerance tests. B: insulin tolerance tests. Each data point is the mean ⫾ SE. LKB1-DN mice (8 male and 9 female) and WT littermates (7 male and 6 female) were used at 15 wk of age. For some data points, the error bars are smaller than the symbols. No significant differences were observed between LKB1-DN and WT mice. 2 m/min every 3 min until exhaustion (Fig. 4A, left). The exercise capacity of LKB1-DN mice was significantly lower than that of WT mice (P ⬍ 0.001), whereas the capacity of AMPK-DN mice was only slightly lower than that of WT mice (P ⫽ 0.0567). LKB1-DN mice ran for a duration of 34 min at a maximum speed of 20 m/min (total distance was 0.4 km), whereas WT and AMPK-DN mice ran for a duration of 55 and 50 min at a maximum speed of 34 and 30 m/min (total distances were 1.0 or 0.8 km), respectively (Fig. 4A, right). These data suggest that LKB1 activity in the skeletal muscle is required for endurance exercise. To investigate effects on fuel utilization and oxygen con˙ O2 and V ˙ CO2 were monitored simultaneously dursumption, V ˙ O2 and V ˙ CO2 ing exercise until mice reached exhaustion. V increased gradually as speed increased until exhaustion in WT and AMPK-DN mice (Fig. 4B). In WT mice, the RQ ratio increased as the treadmill speed increased, suggesting that glucose oxidation became predominant with increasing exercise intensity, as shown in the calculated glucose and lipid ˙ O2, V ˙ CO2, and the calculated lipid oxidation rates (Fig. 4B). V oxidation rate were lower in LKB1-DN mice than in AMPK-DN and WT mice. We also observed an earlier increase in the RQ ratio in LKB-DN mice with increasing treadmill speed compared with the AMPK-DN and WT mice. The peak oxygen consumption in AMPK-DN and WT mice was 120 ⫾ 17.2 and 115 ⫾ 0.7 ml·min⫺1·kg⫺1, respectively. Oxygen consumption of LKB1-DN mice was 65 ⫾ 2.0 ml·min⫺1·kg⫺1 at exhaustion, and it was not the highest consumption during the running. When the data were expressed as ˙ O 2, V ˙ CO2, and the calcuthe percentage of maximal speed, V lated glucose and lipid oxidation rates were significantly lower in LKB1-DN mice compared with WT mice (Fig. 4C). However, no difference was observed in the increase in RQ ratio of LKB1-DN mice compared with AMPK-DN and WT mice. No significant differences were observed in these parameters between AMPK-DN and WT. These data indicated that LKB1-DN mice could not increase their oxygen consumption and carbon dioxide production to the same extent as WT and AMPK-DN mice. Significant phenotypic differences between AMPK-DN and WT mice were not observed in this exercise protocol. To investigate whether aerobic exercise capacity is decreased in LKB1-DN mice, blood lactate concentration was determined while the mice were started off running on a treadmill at 10 m/min for 15 min, then increasing by 5 m/min every 15 min for ⱕ25 m/min (Fig. 5). Although WT mice did not reach exhaustion in this protocol, LKB1-DN mice were exhausted at 20 m/min. The exhausted speed of LKB1-DN mice was almost the same as the result obtained in the experiment shown in Fig. 4A. An increase in blood lactate concentration was not observed in WT mice. In LKB1-DN mice, the concentration started to increase at 30 min and reached 7.1 ⫾ 0.8 nmol/l at exhaustion. The AMPK-DN mice did not reach exhaustion, but the concentration increased at 60 min (4.4 ⫾ 0.8 nmol/l). This suggested that aerobic exercise capacity was markedly reduced in LKB1-DN mice, and these mice may switch to anaerobic energy pathways at a lower exercise intensity than WT and AMPK-DN mice. The mRNA expression of genes related to muscle fiber type and metabolism in the resipiratory and locomotive muscles. The mRNA expression of genes related to muscle fiber type and metabolism in the diaphragm was determined by quanti- AJP-Endocrinol Metab • doi:10.1152/ajpendo.00114.2013 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on September 11, 2017 AMPK-DN mice, respectively. AMPK␣2 activity in the gastrocnemius was not detected in LKB1-DN and AMPK-DN mice. In the heart, AMPK␣1 and -␣2 activities were not impaired in LKB1-DN and AMPK-DN mice. From previous studies (13, 33), AMPK␣1-DN mice completely lacked AMPK␣2 activity, whereas AMPK␣1 activity decreased only slightly, because it seemed that the ␣1-inactive form predominantly displaced endogenous ␣2 rather than ␣1; additionally, very little AMPK␣1 may have been expressed in skeletal muscle. There were no meaningful differences in tissue weight (Table 2), oxygen consumption, carbon dioxide production, RQ ratio, spontaneous motor activity while sedentary, body weight change after 24 h of fasting (Table 3), or glucose and insulin tolerance (Fig. 3) between LKB1-DN mice and WT littermates. Although agerelated skeletal muscle dysfunction was observed in mhLKB1-KO mice (56), myopathic phenotype was not observed in LKB1-DN mice (data not shown). Exercise capacity of LKB1-DN, AMPK-DN, and WT mice. Previous studies have reported that voluntary running was decreased in both mhLKB1-KO and AMPK-DN mice (14, 33, 51, 57). To investigate the effect of skeletal muscle-specific reduction of LKB1 or AMPK activity on exercise capacity, we compared the exercise capacities of LKB1-DN and AMPK-DN mice. The mice were started off running on a treadmill at 6 m/min for 15 min, and then the speed was increased by E219 E220 ROLE OF MUSCLE LKB1 AND AMPK IN ENDURANCE PERFORMANCE tative real-time RT-PCR (Fig. 6A). Compared with WT, expression of myosin heavy chain (MHC) 2B (fast-twitch glycolytic fibers) was increased 3.1-fold in the LKB1-DN diaphragm. This increase was not observed in the AMPK-DN Protocol Exercise tolerance 42 38 34 30 26 22 18 14 10 6 0 15 30 60 WT AMPK-DN LKB1-DN 40 20 0 60 (min) 45 Log rank test P=0.0567, WT vs AMPK-DN P<0.001, WT vs LKB1-DN 80 0 15 30 60 (min) 45 B 160 140 VO2 (ml min-1 kg-1) 140 120 120 100 100 0.95 VCO2 (ml min-1 kg-1) RQ ratio 0.90 80 0.85 60 0.80 80 60 40 40 WT AMPK-DN LKB1-DN 20 0 -30 140 -15 0 15 30 45 60 (min) Glucose oxidation (mg min-1 kg-1) 120 0.75 20 0 -30 40 35 -15 0 15 30 45 0.70 -30 60 (min) -15 0 15 30 45 Lipid oxidation (mg min-1 kg-1) 30 100 25 80 20 60 15 40 10 20 5 0 -30 -15 0 15 30 45 60 (min) 0 -30 -15 0 15 30 45 60 (min) C 160 VO2 (ml min-1 kg-1) 140 80 60 0 0 140 AUC t-test NS, WT vs AMPK-DN P<0.001, WT vs LKB1-DN 25 50 75 % of EXmax 100 Glucose oxidation (mg min-1 kg-1) 80 RQ ratio 0.90 80 0.85 60 0.80 40 20 0 0 40 AUC t-test NS, WT vs AMPK-DN P<0.001, WT vs LKB1-DN 25 50 75 % of EXmax AUC t-test NS, WT vs AMPK-DN NS, WT vs LKB1-DN 0.75 100 0.70 0 25 50 75 % of EXmax Lipid oxidation (mg min-1 kg-1) 35 120 100 0.95 100 100 20 VCO2 (ml min-1 kg-1) 120 120 40 140 AUC t-test NS, WT vs AMPK-DN P<0.05, WT vs LKB1-DN 30 25 20 60 15 40 10 20 5 0 0 25 50 75 % of EXmax 100 0 0 AUC t-test NS, WT vs AMPK-DN P<0.001, WT vs LKB1-DN 25 50 75 % of EXmax 100 AJP-Endocrinol Metab • doi:10.1152/ajpendo.00114.2013 • www.ajpendo.org 100 60 (min) Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on September 11, 2017 0 100 Percentage still running Speed (m/min) A diaphragm. Expression of MHC 1 (slow-twitch oxidative fibers, rich in mitochondria), MHC 2A (fast-twitch oxidative fibers), and MHC 2X (mouse fast-twitch oxidative fibers) was not significantly different in the diaphragm among the three ROLE OF MUSCLE LKB1 AND AMPK IN ENDURANCE PERFORMANCE E221 WT AMPK-DN LKB1-DN 100 Blood lactate (nmol/L) Proportion still running (%) 10 80 60 WT AMPK-DN LKB1-DN 40 20 10 m/min 0 15 m/min 20 m/min 25 m/min 8 6 4 2 10 m/min 0 0 15 30 60 0 15 20 m/min 30 25 m/min 45 60 Time (min) groups of mice. These data suggested that expression of glycolytic fibers was increased in the diaphragms of LKB1-DN mice, as observed in mhLKB1-KO mice (6). The expression of genes involved in mitochondria biosynthesis and function, such as mitochondrial transcription factor A, COX-2, and COX-4, was not significantly different among the groups. In LKB1-DN mice, the expression of genes involved in glycogenolysis, such as phosphorylase kinase-␣1 and muscle glycogen phosphorylase, was significantly increased 1.6 –1.8 times higher than in WT mice. The key enzyme for glycolysis, muscle phosphofructokinase, was significantly increased to 1.6 times that of WT mice (Fig. 6A). Pyruvate dehydrogenase kinase-4 expression was decreased to 64% of WT in LKB1-DN mice. No significant differences were observed in the AMPK-DN diaphragm compared with WT. These data suggested that LKB1-DN mice had a muscle fiber type shift toward fast-twitch glycolytic fibers and that the decreased LKB1 activity in the diaphragm was associated with increased production of pyruvate and conversion to acetyl-CoA in the mitochondria. On the other hand, the expression of genes encoding proteins involved in fatty acid transport and fatty acid oxidation, such as lipoprotein lipase (LPL) and CD36, was lower in both the AMPK-DN and LKB1-DN mice. One of the NR4A orphan nuclear receptor proteins, Nur77, which regulates expression of genes linked to glucose metabolism (8), was significantly increased in both of AMPK-DN and LKB1-DN mice. PPAR␣, which regulates expression of genes linked to lipid oxidation, was decreased significantly in both AMPK-DN and LKB1-DN mice. The genes related to fast-twitch muscle fiber differentiation, such as MyoD and Sox6, were increased significantly only in the LKB1-DN diaphragm. Furthermore, histochemical analysis of the diaphragm was performed to investigate whether defects in LKB1 activity increased the cross-sectional area of glycolytic fibers in vivo using SDH activity as a marker for oxidative fibers (Fig. 6B). The SDH-positive muscle fiber crosssectional area was the same in all three genotypes. On the other hand, the area of the SDH-negative fibers was increased significantly in LKB1-DN mice. These data suggested an increase in the cross-sectional area of glycolytic fibers in the diaphragm of LKB1-DN mice. The expression of mRNA in the gastrocnemius was determined by quantitative real-time RT-PCR (Fig. 7A). No significant differences were observed in the expression of genes related to fiber type, fast-twitch muscle fiber differentiation, or glucose metabolism among the three groups of mice. Expression of COX-2, COX-4, fatty acid-binding protein 3 (FABP3), medium-chain acyl-CoA dehydrogenase (MCAD), vascular endothelial growth factor (VEGF)-A, and neuronal nitric oxide synthase (nNOS) were decreased significantly in the gastrocnemius from both LKB1-DN and AMPK-DN mice compared with WT mice. Expression of PPAR␥ coactivator-1␣ (PGC1␣) and LPL showed a tendency to decrease in both LKB1-DN and AMPK-DN mice. Among the analyzed genes, changes suggesting an alteration in mitochondria function, lipid metabolism, capillarization, and NOS production were observed in both LKB1-DN and AMPK-DN gastrocnemius. Any specific differences observed in LKB1-DN diaphragm were not observed in the gastrocnemius. In the histochemical analysis, although SDH activity tended to increase in SDH-positive fibers, no significant difference was observed in SDH-negative muscle fiber cross-sectional area of the tibialis anterior among the three genotypes (Fig. 7B). LKB1, AMPK, MARK4, and QSK activities, malonyl-CoA and adenosine phosphate concentrations in locomotive muscle, and liver and locomotive muscle glycogen concentration after exercise. The activities of LKB1, AMPK, MARK4, and QSK, the phosphorylation of AMPK and ACC, and the concentration of malonyl-CoA were measured in the locomotive muscle after low-intensity exercise (10 m/min for 30 min). No significant Fig. 4. Exercise tolerance, oxygen consumption, carbon dioxide production, respiratory quotient (RQ) ratio, and calculated glucose and lipid oxidation rates during exercise. A: AMPK-DN, LKB1-DN, and WT mice (n ⫽ 5–10; each 12 wk old) were exercised by forced running on a treadmill at 6 m/min for 15 min. The speed increased by 2 m/min every 3 min until exhaustion. Mice ran until exhaustion (exercise tolerance test). Exercise tolerance is shown as a Kaplan-Meier survival curve. A significant difference (P ⬍ 0.001, log-rank test) was observed between the exercise tolerances of LKB1-DN mice vs. WT. B: oxygen consumption and carbon dioxide production were monitored using an O2/CO2 metabolism-measuring system for small animals, which was equipped with an airtight treadmill chamber. The RQ ratio and calculated glucose and lipid oxidation rates are also shown. Each value is the mean ⫾ SE. C: oxygen consumption, carbon dioxide production, RQ ratio, and calculated glucose and lipid oxidation rates are plotted against relative exercise intensity, as estimated by the percentage of mean speed at exhaustion for each genotype. Statistical significance was calculated as the area under the curve (AUC), using the mean value at rest for each genotype as a base line. P values are shown in the figure. NS, not significant. AJP-Endocrinol Metab • doi:10.1152/ajpendo.00114.2013 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on September 11, 2017 Time (min) 45 15 m/min Fig. 5. Blood lactate level during exercise. LKB1-DN and AMPK-DN mice and WT littermates (n ⫽ 7– 8; each 23 wk old) were exercised by forced running on a treadmill for 15 min at 10 m/min. The speed increased 5 m/min for ⱕ25 m/min every 15 min. Both WT and AMPK-DN mice completed the running protocol without retiring, which is why the lines in the graph are merged. Blood lactate levels were measured before and every 15 min of exercise. The lactate levels were also measured at the point of exhaustion in the running protocol described above for LKB1-DN mice. E222 A ROLE OF MUSCLE LKB1 AND AMPK IN ENDURANCE PERFORMANCE (%) 350 300 Fiber type *** WT AMPK-DN LKB1-DN (%) 140 (%) 300 Mitochondria 120 250 100 200 80 Transcription factor ** 250 * 200 ** ** 150 150 60 100 40 50 20 MHC1 MHC2A MHC2X MHC2B COX2 COX4 Nur77 PGC-1 Glucose metabolism ** 160 (%) 140 ** PPAR PPAR MyoD Foxo1 Sox6 Lipid metabolism 120 140 100 120 ** 80 100 80 ** 60 * ** 60 40 40 20 20 0 0 GYS1 PHKA1 PYGM GLUT4 HK2 PFKM PKM2 PDK4 LPL CD36 FATP1 FABP- FABP3 CPT1b MCAD pm B AMPK-DN WT 1600 LKB1-DN * Fiber area (µm2) 1400 1200 1000 800 600 400 200 0 100 µm 100 µm 100 µm SDH (+) SDH (-) Fig. 6. Changes in gene expression and histological analysis in the diaphragm. A: results of quantitative RT-PCR analysis of transcripts encoding proteins involved in fiber type, mitochondria function, transcription factor, glucose metabolism, and lipid metabolism in the diaphragm from WT, AMPK-DN, and LKB1-DN mice at 10 wk of age. Values are means ⫾ SE (n ⫽ 4). *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 vs. WT. MHC, myosin heavy chain; mtTFA, mitochondrial transcription factor; COX, cytochrome c oxidase; PGC, peroxisome proliferator-activated receptor-␥ coactivator; PPAR, peroxisome proliferatoractivated receptor; Foxo, forkhead transcriptional factor; Sox, SRY box-containing gene; GYS, glycogen synthase; PHKA, phosphorylase kinase-␣, PYGM, muscle glycogen phosphorylase; GLUT, glucose transporter; HK, hexokinase; PFKM, muscle phosphofructokinase; PFKFB, 6-phosphofructo-2-kinase/fructose2,6-biphosphatase; PKM, muscle pyruvate kinase; PDK, pyruvate dehydrogenase kinase; LPL, lipoprotein lipase; FATP, fatty acid transport protein; FABP-pm, plasma membrane fatty acid-binding protein; FABP, fatty acid-binding protein; CPT, carnitine palmitoyltransferase; MCAD, medium-chain acyl-CoA dehydrogenase. B: histological analysis of the diaphragm from WT, AMPK-DN, and LKB1-DN mice at 10 wk of age. The diaphragm was stained for succinate dehydrogenase (SDH) activity, and typical pictures are shown. The muscle fiber cross-sectional area of SDH-positive (⫹) and -negative (⫺) fibers was measured. Values are means ⫾ SE (n ⫽ 3). *P ⬍ 0.05 vs. WT. changes in any of these parameters were observed in either LKB1-DN or WT mice in response to exercise (Fig. 8, A–E). Although the phosphorylation of ACC was decreased markedly in sedentary LKB1-DN mice, malonyl-CoA concentration in LKB1-DN mice was only slightly lower compared with WT mice. ATP concentration was significantly lower both before and after exercise in LKB1-DN gastrocnemius compared with WT (Fig. 8F). ADP concentration was not different between the groups. AMP concentration before the exercise was similar between the groups. Whereas no change in AMP concentration was observed after exercise in WT mice, a significant increase was observed in the gastrocnemius of LKB1-DN mice after exercise. Glycogen concentrations in the liver and gastrocnemius were measured (Fig. 8G). At rest, glycogen content in the gastrocnemius of LKB1-DN mice was 24% lower than in WT mice, whereas there was no significant difference in liver glycogen content. Skeletal muscle and liver glycogen content were significantly decreased after low-intensity exercise in both LKB1-DN and WT mice. The functions of mitochondria in the locomotive muscle. Markers of mitochondrial content and respiratory chain activity were measured in the locomotive muscles from LKB1-DN mice and WT littermates. Citrate synthase activity per wet tissue weight in the LKB1-DN gastrocnemius was reduced by ⬃20% in male and AJP-Endocrinol Metab • doi:10.1152/ajpendo.00114.2013 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on September 11, 2017 180 * 0 mtTFA *** ** 50 0 0 (%) 200 100 E223 ROLE OF MUSCLE LKB1 AND AMPK IN ENDURANCE PERFORMANCE A (%) 180 (%) 120 Fiber type WT AMPK-DN LKB1-DN 160 140 (%) 200 Mitochondria 180 100 ** ** ** 80 120 160 140 120 100 60 100 80 ** 80 40 60 40 60 40 20 20 20 MHC1 MHC2A MHC2X MHC2B 0 0 mtTFA COX2 COX4 Nur77 PGC-1 PPAR (%) 120 Glucose metabolism PPAR MyoD Foxo1 Sox6 Lipid metabolism 100 200 * 80 150 * * *** ** 60 100 40 50 20 0 0 GYS1 (%) 180 PHKA1 PYGM GLUT4 HK2 PFKM PKM2 LPL PDK4 CD36 FATP1 FABP- FABP3 CPT1b MCAD pm Capillarization and NOS 160 140 120 100 80 * * 60 ****** 40 20 0 VEGF-A VEGF-B PDGF-B nNOS B eNOS AMPK-DN WT 200 µm LKB1-DN 200 µm 200 µm Fig. 7. Changes in gene expression in the gastrocnemius. A: results of quantitative RT-PCR analysis of transcripts encoding proteins involved in fiber type, mitochondria function, transcription factor, glucose metabolism, lipid metabolism, capillarization, and nitric oxide synthase (NOS) in the gastrocnemius from WT, AMPK-DN, and LKB1-DN mice at 10 wk of age. Values are means ⫾ SE (n ⫽ 4). *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 vs. WT. VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; nNOS, neuronal nitric oxide synthase; eNOS, endothelial nitric oxide synthase. B: histological analysis of the tibialis anterior from WT, AMPK-DN, and LKB1-DN mice at 10 wk of age. The tibialis anterior was stained for SDH activity, and typical pictures are shown. AJP-Endocrinol Metab • doi:10.1152/ajpendo.00114.2013 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on September 11, 2017 0 (%) 250 Transcription factor E224 ROLE OF MUSCLE LKB1 AND AMPK IN ENDURANCE PERFORMANCE LKB1 C 0.20 0.15 Phospho AMPK 0.10 AMPK Phospho AMPK/AMPK (% of sedentary WT) Activities (pmol min-1 mg protein-1) 12 LKB1-DN After After Sedentary exercise Sedentary exercise Sedentary After exercise (10 m/min for 30 min) 0.05 0 ** ** LKB1-DN WT 2-AMPK 1-AMPK 14 WT Sedentary After exercise (10 m/min for 30 min) 10 8 Sedentary After exercise (10 m/min for 30 min) 150 125 100 *** 75 ** 50 25 0 WT LKB1-DN 6 4 ** 0 WT D 2 ** *** WT LKB1-DN WT LKB1-DN After After Sedentary exercise Sedentary exercise LKB1-DN Phospho ACC QSK ACC Phospho ACC/ACC (% of sedentary WT) 0.20 0.15 * * 0.10 * * 0.05 0 WT LKB1-DN WT LKB1-DN E Malonyl CoA (nmol/g tissue) Activities (pmol min-1 mg protein-1) MARK4 0.25 175 Sedentary After exercise (10 m/min for 30 min) 150 125 100 75 50 ** 25 ** 0 WT LKB1-DN Sedentary After exercise (10 m/min for 30 min) 0.5 0.4 * 0.3 * 0.2 0.1 0 WT AJP-Endocrinol Metab • doi:10.1152/ajpendo.00114.2013 • www.ajpendo.org LKB1-DN Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on September 11, 2017 B Activities (pmol min-1 mg protein-1) A E225 ROLE OF MUSCLE LKB1 AND AMPK IN ENDURANCE PERFORMANCE Sedentary F After exercise (10 m/min for 30 min) 8 ATP 2.0 ADP 0.3 AMP ### *** ** 0.2 4 1.0 2 0.5 0.1 WT G 30 # LKB1-DN Sedentary After exercise (10 m/min for 30 min) # 25 20 * 15 * 10 5 0 WT 0 WT WT LKB1-DN 300 LKB1-DN ### ## 250 200 150 100 LKB1-DN 50 0 WT LKB1-DN Fig. 8—Continued. female mice (WT, 36.6 ⫾ 1.2 ␮mol·min⫺1·g⫺1 tissue; LKB1-DN, 30.0 ⫾ 0.6 ␮mol·min⫺1·g⫺1 tissue; n ⫽ 6 in each group). However, citrate synthase activity in the mitochondrial fractions taken from LKB1-DN skeletal muscle was not different from WT skeletal muscle (Table 4). The enzyme activities of CPT I, complex I ⫹ III ⫹ IV, and complex II ⫹ III were also similar in LKB1-DN mice and WT mice when expressed relative to citrate synthase activity (Table 4). The respiration rate measured by oxygen consumption in the mitochondrial fraction was similar between LKB1-DN mice and WT mice in the presence and absence of oligomycin (Table 4), suggesting that activity of respiratory enzymes and membrane potential formation were comparable in each mitochondrion in LKB1-DN and WT mice. These data suggested that, although the number of mitochondria was decreased in LKB1-DN locomotive muscle to the same extent as in AMPK-DN mice (41), mitochondrial oxidative phosphorylation per unit mitochondria was similar in the skeletal muscles between the two groups. DISCUSSION In this study, we found that LKB1-DN mice, which were deficient in LKB1 activity specifically in the skeletal muscle, but not in the heart, had significantly diminished exercise capacity with decreased oxygen consumption and carbon dioxide production during exercise. However, the exercise capacity, oxygen consumption, and carbon dioxide production were not diminished in AMPK-DN mice, suggesting that these effects are caused by LKB1 deficiency and may be independent of the AMPK pathway. Fig. 8. LKB1, AMPK, MARK4, and QSK activities, malonyl-CoA and adenosine phosphate concentrations in the locomotive muscle, and liver and locomotive muscle glycogen concentration after exercise. LKB1-DN mice and WT littermates (16 wk old) were assigned to 2 groups. One group was kept sedentary, whereas the other group was subjected to running on the treadmill at a speed of 10 m/min for 30 min. Dissected muscles were immediately used for analysis. A and B: activities of LKB1, AMPK␣1, AMPK␣2, MARK4, and QSK in the skeletal muscle (gastrocnemius) from LKB1-DN and WT littermates were measured in immunoprecipitates. Each value is the mean ⫾ SE (n ⫽ 3). C and D: phospho-AMPK/total AMPK and phospho-ACC/total ACC in the sedentary and exercised gastrocnemius of WT and LKB1-DN mice were measured by Western blotting. Each value, expressed as a percentage of the value for sedentary WT littermates, was the mean ⫾ SE (n ⫽ 4). Typical blots are also shown. Phospho-AMPK is indicated by the arrow in C. E: malonyl-CoA concentrations in sedentary and exercised quadriceps of WT and LKB1-DN mice were measured. Each value is the mean ⫾ SE (n ⫽ 4). F: ATP, ADP, and AMP contents of the quadriceps from WT and LKB1-DN mice were measured. Data represent means ⫾ SE (n ⫽ 4). G: glycogen levels in the gastrocnemius and liver from WT and LKB1-DN mice were measured. Each value is the mean ⫾ SE (n ⫽ 4). *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 vs. WT littermates; #P ⬍ 0.05, ##P ⬍ 0.01, and ###P ⬍ 0.001 vs. sedentary. AJP-Endocrinol Metab • doi:10.1152/ajpendo.00114.2013 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on September 11, 2017 0 0 Gastro. glycogen (µmol/g) 1.5 Liver glycogen (µmol/g) Adenine nucleotide (µmol/g tissue) * 6 E226 ROLE OF MUSCLE LKB1 AND AMPK IN ENDURANCE PERFORMANCE Table 4. Oxidative phosphorylation of mitochondria in the skeletal muscles LKB1-DN 1.98 ⫾ 0.12 2.17 ⫾ 0.13 21.7 ⫾ 1.1 19.0 ⫾ 0.8 0.118 ⫾ 0.019 0.488 ⫾ 0.030 0.103 ⫾ 0.008 0.463 ⫾ 0.011 68.4 15.2 64.8 14.4 66.5 36.0 62.8 33.7 Values are means ⫾ SE from LKB1-DN and WT mice at 20 wk of age (n ⫽ 4). Citrate synthase activity, CPT I activity, and the enzymatic activity of the respiratory chain complex were measured using the mitochondrial fraction that was prepared from the TA. Citrate synthase and CPT I activities were normalized to the protein concentration of the mitochondrial fraction. The enzymatic activity of the respiratory chain complex was normalized to the citrate synthase activity of the mitochondrial fraction. State III or the uncoupling respiration rate of the mitochondrial fraction that was prepared from the skeletal muscles (gastrocnemius, quadriceps, and tibialis anterior) was measured in triplicate. Each data point is the mean value of the measurements normalized to the citrate synthase activity of the fraction. The skeletal muscles were obtained from both WT and LKB1-DN mice at 20 wk of age. In the skeletal muscles, although expression of genes related to fiber type, mitochondria function, glucose and lipid metabolism, and capillarization in the locomotive muscle was not different between LKB1-DN and AMPK-DN mice, we did observe marked differences in expression of MHC IIB and glycolytic enzymes in the diaphragm of LKB1-DN mice, suggesting that the function of the diaphragm might be one reason for the decrease in exercise capacity, oxygen consumption, and carbon dioxide production during exercise found in LKB1-DN mice. In the locomotive muscle, a decrease in citrate synthase activity and an increase in exercise-mediated AMP production were found in LKB1-DN mice compared with WT mice. However, mitochondrial oxidative phosphorylation per unit of isolated mitochondria from the locomotive muscle was similar between LKB1-DN and WT mice, suggesting that the changes in diaphragm characterization are likely to account for the impairment in exercise capacity and oxygen uptake. The respiratory muscles alter air flow to and from the alveolar surface by generating pressure gradients. Because of this task, which does not allow them to rest throughout their entire lifetime, respiratory muscle fibers must be able to work without becoming fatigued. Small fiber size, abundance of capillaries, and a high aerobic oxidative enzyme activity are typical features of diaphragm fibers, giving them the fatigue resistance required by their continuous activity. Changes in the diaphragm fiber type demonstrated one reason for the increased fatigue (48, 63). LKB1 deficiency in the diaphragm caused a fiber type shift from type IIx to type IIb muscle fibers, increasing the rate of fatigue and decreasing the ability to recover after a 10-min training session in vitro (6). Because basic ambulatory activity was not impaired, an increase in the expression of the gene encoding MHC IIB in the AJP-Endocrinol Metab • doi:10.1152/ajpendo.00114.2013 • www.ajpendo.org Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on September 11, 2017 Citrate synthase activity, ␮mol 䡠 min⫺1 䡠 mg protein⫺1 CPT I activity, nmol 䡠 min⫺1 䡠 mg protein⫺1 Enzymatic activity of respiratory chain complex/Citrate synthase activity, mol/mol Enzyme complex I ⫹ III ⫹ IV Enzyme complex II ⫹ III Respiration/Citrate synthase activity, mmol/mol Substrate: pyruvate ⫹ malate State III State III with oligomycin Substrate: succinate State III State III with oligomycin WT diaphragm of LKB1-DN mice, which may shift the fiber type, could cause a decrease in fatigue resistance when the muscle is loaded because of the exercise. It has been reported that LKB1 acts through SIK2 and SIK3 to promote the nucleocytoplasmic trafficking of class IIa histone deacetylases (HDACs). Both SIK2 and SIK3 phosphorylate the deacetylases at the conserved motifs and stimulate 14-3-3 binding, which inactivates HDACs and relieves them from the suppression of the target gene transcription (62). Class IIa HDACs are known to act as corepressors of the myocyte enhancer factor 2 (MEF2) family (16). Walkinshaw et al. (62) showed that SIK2, but not SIK3, derepressed MEF2 transcriptional activity. It has been shown that the inactivation of MEF2 leads to the loss of slow fiber formation and enhanced fast fiber formation (39) . It is tempting to speculate that LKB1 deficiency inactivates MEF2 by increasing the dephosphorylated form of HDACs in the nucleus, which occurs through the inactivation of SIK2, and results in the loss of slow fiber formation, leading to fast fiber formation. Fast fiber formation was suggested by the expression of MyoD, which was accumulated in the fast type IIb/IIx fibers (23). It was demonstrated that MyoD interacted with the proximal E-box within the mouse MHC IIb promoter and activated its expression (1, 65). Recently, Sox6 was identified as a fast myofiber-enriched repressor of slow muscle gene expression in vivo (40). Mice lacking Sox6 specifically in the skeletal muscle had exhibited downregulation of the fast myofiber gene program, resulting in enhanced muscular endurance. A significant increase in MyoD and Sox6 expression was observed in LKB1-DN diaphragm compared with WT and AMPK-DN, and this increase seemed to activate MHC IIb expression and fast fiber formation. However, the reason for these specific changes in the diaphragm is unclear at present. The work of breathing has been shown to be a major contributor to the development of locomotor muscle fatigue and to limit exercise performance (20, 42). For example, loading or unloading the respiratory muscles using inspiratory resistors or a mechanical ventilator affected the severity of quadriceps fatigue and the perception of limb discomfort (42). Respiratory muscle fatigue might be a key factor, triggering a metaboreflex from the diaphragm and expiratory muscles that in turn increases sympathetic vasoconstriction of the limb, reducing limb blood flow and consequently oxygen transport (19, 47, 50). Since deficiency of LKB1 activity in the diaphragm caused an increased in the rate of fatigue, we would expect metaboreflex effects during exercise in LKB1-DN mice and, therefore, great decrease in exercise capacity in these mice with decreased oxygen uptake. Diaphragmatic fatigue caused a decrease in diaphragmatic contractility (61). Since diaphragmatic contractility was strongly related to lung volume, fatigue of the diaphragm is one possible reason for the decrease in oxygen consumption and carbon dioxide production during exercise in LKB1-DN mice. Since deletion and ablation of LKB1 activity in the heart can lead to impaired cardiac function, LKB1-mediated cardiac function might affect exercise capacity (24, 25, 56). In this study, we created a transgenic mouse whose LKB1 activity was reduced specifically in the skeletal muscle and not in the heart. Therfore, the decrease in exercise capacity of LKB1-DN mice was due only to deficient LKB1 activity in skeletal muscle. In addition to the pumping capacity of the heart, mitochondrial number (or volume) in the skeletal muscle is closely correlated with aerobic capacity (52, 59). We have reported previously that increased mitochondrial biogenesis, capillaries, and fatty acid ROLE OF MUSCLE LKB1 AND AMPK IN ENDURANCE PERFORMANCE ACKNOWLEDGMENTS We thank Dr. Kei Sakamoto (University of Dundee, UK) for providing the antibodies against LKB1, MARK4, and QSK and helpful advice. We thank Drs. Edna C. Hardeman and Kim L. Guven (Children’s Medical Research Institute, Australia) for providing the 2.2-kb fragment of the human ␣-skeletal actin promoter. We are grateful to Dr. Yasutomi Kamei (Tokyo Medical and Dental University, Japan) for helpful discussions in preparation of the manuscript. GRANTS S. Miura was supported by grants-in-aid for scientific research (KAKENHI) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (Tokyo, Japan), The Nakatomi Foundation (Tokyo, Japan), and The Takeda Science Foundation (Osaka, Japan). C. R. Bruce and M. A. Febbraio were supported by research fellowships from the National Health and Medical Research Council of Australia. DISCLOSURES No potential conflicts of interest relevant to this article, financial or otherwise, are reported by the authors. AUTHOR CONTRIBUTIONS S.M. and O.E. contributed to the conception and design of the research; S.M., Y.K., M.T., Y.T., K.S., and S.C. performed the experiments; S.M., Y.T., K.S., C.B., M.A.F., K.K., S.C., and O.E. analyzed the data; S.M., C.B., M.A.F., and O.E. interpreted the results of the experiments; S.M. and O.E. prepared the figures; S.M. and O.E. drafted the manuscript; S.M., Y.T., K.S., C.B., M.A.F., K.K., S.C., and O.E. edited and revised the manuscript; S.M., Y.K., M.T., Y.T., K.S., C.B., M.A.F., K.K., S.C., and O.E. approved the final version of the manuscript. REFERENCES 1. Allen DL, Sartorius CA, Sycuro LK, Leinwand LA. Different pathways regulate expression of the skeletal myosin heavy chain genes. 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