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• W2247011864 abstract "Satellite cells are the major myogenic stem cells residing inside skeletal muscle and are indispensable for muscle regeneration. Satellite cells remain largely quiescent but are rapidly activated in response to muscle injury, and the derived myogenic cells then fuse to repair damaged muscle fibers or form new muscle fibers. However, mechanisms eliciting metabolic activation, an inseparable step for satellite cell activation following muscle injury, have not been defined. We found that a noncanonical Sonic Hedgehog (Shh) pathway is rapidly activated in response to muscle injury, which activates AMPK and induces a Warburg-like glycolysis in satellite cells. AMPKα1 is the dominant AMPKα isoform expressed in satellite cells, and AMPKα1 deficiency in satellite cells impairs their activation and myogenic differentiation during muscle regeneration. Drugs activating noncanonical Shh promote proliferation of satellite cells, which is abolished because of satellite cell-specific AMPKα1 knock-out. Taken together, AMPKα1 is a critical mediator linking noncanonical Shh pathway to Warburg-like glycolysis in satellite cells, which is required for satellite activation and muscle regeneration. Satellite cells are the major myogenic stem cells residing inside skeletal muscle and are indispensable for muscle regeneration. Satellite cells remain largely quiescent but are rapidly activated in response to muscle injury, and the derived myogenic cells then fuse to repair damaged muscle fibers or form new muscle fibers. However, mechanisms eliciting metabolic activation, an inseparable step for satellite cell activation following muscle injury, have not been defined. We found that a noncanonical Sonic Hedgehog (Shh) pathway is rapidly activated in response to muscle injury, which activates AMPK and induces a Warburg-like glycolysis in satellite cells. AMPKα1 is the dominant AMPKα isoform expressed in satellite cells, and AMPKα1 deficiency in satellite cells impairs their activation and myogenic differentiation during muscle regeneration. Drugs activating noncanonical Shh promote proliferation of satellite cells, which is abolished because of satellite cell-specific AMPKα1 knock-out. Taken together, AMPKα1 is a critical mediator linking noncanonical Shh pathway to Warburg-like glycolysis in satellite cells, which is required for satellite activation and muscle regeneration. Skeletal muscle is the main component in animal locomotion system. It is also the major tissue sustaining respiration and the primary peripheral tissue utilizing glucose and fatty acids, important in preventing obesity and type 2 diabetes (1Schenk S. Horowitz J.F. Acute exercise increases triglyceride synthesis in skeletal muscle and prevents fatty acid-induced insulin resistance.J. Clin. Investig. 2007; 117: 1690-1698Crossref PubMed Scopus (295) Google Scholar, 2Youn J.Y. Cai H. Fueling up skeletal muscle to reduce obesity: a TrkB story.Chem. Biol. 2015; 22: 311-312Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar, 3Kennedy J.W. Hirshman M.F. Gervino E.V. Ocel J.V. Forse R.A. Hoenig S.J. Aronson D. Goodyear L.J. Horton E.S. Acute exercise induces GLUT4 translocation in skeletal muscle of normal human subjects and subjects with type 2 diabetes.Diabetes. 1999; 48: 1192-1197Crossref PubMed Scopus (289) Google Scholar). Skeletal muscle fibers are frequently damaged during exercise and because of physical trauma or diseases such as Duchenne muscular dystrophy (4Irintchev A. Wernig A. Muscle damage and repair in voluntarily running mice: strain and muscle differences.Cell Tissue Res. 1987; 249: 509-521Crossref PubMed Scopus (110) Google Scholar, 5Webster C. Silberstein L. Hays A.P. Blau H.M. Fast muscle fibers are preferentially affected in Duchenne muscular dystrophy.Cell. 1988; 52: 503-513Abstract Full Text PDF PubMed Scopus (450) Google Scholar). Efficient regeneration following muscle injury is critical for maintaining the normal physiological function of skeletal muscle. On the other hand, insufficient muscle regeneration replaces muscle fibers with fibrotic tissue and weakens the contractile function of muscle, which is a key etiological factor leading to progressive muscle weakness associated with aging and muscle dystrophic diseases (6Uezumi A. Fukada S. Yamamoto N. Takeda S. Tsuchida K. Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle.Nat. Cell Biol. 2010; 12: 143-152Crossref PubMed Scopus (809) Google Scholar, 7Bernasconi P. Torchiana E. Confalonieri P. Brugnoni R. Barresi R. Mora M. Cornelio F. Morandi L. Mantegazza R. Expression of transforming growth factor-β1 in dystrophic patient muscles correlates with fibrosis: pathogenetic role of a fibrogenic cytokine.J. Clin. Invest. 1995; 96: 1137-1144Crossref PubMed Scopus (251) Google Scholar, 8Li H. Malhotra S. Kumar A. Nuclear factor-κB signaling in skeletal muscle atrophy.J. Mol. Med. 2008; 86: 1113-1126Crossref PubMed Scopus (301) Google Scholar). Despite the presence of multiple types of myogenic cells in skeletal muscle, satellite cells are the major postnatal myogenic cells indispensable for muscle regeneration (9Sambasivan R. Yao R. Kissenpfennig A. Van Wittenberghe L. Paldi A. Gayraud-Morel B. Guenou H. Malissen B. Tajbakhsh S. Galy A. Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration.Development. 2011; 138: 3647-3656Crossref PubMed Scopus (595) Google Scholar). Satellite cells maintain in a quiescent stage and become activated when muscle regeneration process is triggered (10Montarras D. L'Honoré A. Buckingham M. Lying low but ready for action: the quiescent muscle satellite cell.FEBS J. 2013; 280: 4036-4050Crossref PubMed Scopus (140) Google Scholar, 11Kuang S. Kuroda K. Le Grand F. Rudnicki M.A. Asymmetric self-renewal and commitment of satellite stem cells in muscle.Cell. 2007; 129: 999-1010Abstract Full Text Full Text PDF PubMed Scopus (958) Google Scholar). Activated satellite cells proliferate to expand their population and undergo further myogenic differentiation orchestrated by sequential expression of myogenic regulatory factors, Myf5, MyoD, myogenin, and MRF4 (12Sabourin L.A. Rudnicki M.A. The molecular regulation of myogenesis.Clin. Genet. 2000; 57: 16-25Crossref PubMed Scopus (557) Google Scholar). Recent studies show that stem cells including satellite cells rely on glycolysis to provide energy (13Kondoh H. Lleonart M.E. Nakashima Y. Yokode M. Tanaka M. Bernard D. Gil J. Beach D. A high glycolytic flux supports the proliferative potential of murine embryonic stem cells.Antioxid. Redox Signal. 2007; 9: 293-299Crossref PubMed Scopus (259) Google Scholar, 14Folmes C.D. Dzeja P.P. Nelson T.J. Terzic A. Metabolic plasticity in stem cell homeostasis and differentiation.Cell Stem Cell. 2012; 11: 596-606Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar), likely because of their limited access to oxygen because of deep location in the tissue and the need to prevent the damage from reactive oxygen species (15Ochocki J.D. Simon M.C. Nutrient-sensing pathways and metabolic regulation in stem cells.J. Cell Biol. 2013; 203: 23-33Crossref PubMed Scopus (108) Google Scholar, 16Eliasson P. Jönsson J.I. The hematopoietic stem cell niche: low in oxygen but a nice place to be.J. Cell Physiol. 2010; 222: 17-22Crossref PubMed Scopus (343) Google Scholar, 17Clarke L. van der Kooy D. Low oxygen enhances primitive and definitive neural stem cell colony formation by inhibiting distinct cell death pathways.Stem Cells. 2009; 27: 1879-1886Crossref PubMed Scopus (65) Google Scholar). Satellite cells have relatively small cytoplasm and few mitochondria (10Montarras D. L'Honoré A. Buckingham M. Lying low but ready for action: the quiescent muscle satellite cell.FEBS J. 2013; 280: 4036-4050Crossref PubMed Scopus (140) Google Scholar, 18Bracha A.L. Ramanathan A. Huang S. Ingber D.E. Schreiber S.L. Carbon metabolism-mediated myogenic differentiation.Nat. Chem. Biol. 2010; 6: 202-204Crossref PubMed Scopus (70) Google Scholar), resulting in low metabolic rates. However, metabolism of stem cells rapidly elevates during wakening from their quiescent state, providing energy needed for stem cell proliferation and further differentiation (10Montarras D. L'Honoré A. Buckingham M. Lying low but ready for action: the quiescent muscle satellite cell.FEBS J. 2013; 280: 4036-4050Crossref PubMed Scopus (140) Google Scholar). Warburg glycolysis is a primarily source of energy for certain cancer cells, which allows their fast proliferation (19Warburg O. Posener K. Negelein E. Metabolism of carcinoma cells.Biochemische Zeitschrift. 1924; 152: 319-344Google Scholar, 20Warburg O. On the origin of cancer cells.Science. 1956; 123: 309-314Crossref PubMed Scopus (9554) Google Scholar). Cancer cells and stem cells share metabolic similarity, and recently, Warburg-like glycolysis was identified in induced stem cells (21Vazquez-Martin A. Corominas-Faja B. Cufi S. Vellon L. Oliveras-Ferraros C. Menendez O.J. Joven J. Lupu R. Menendez J.A. The mitochondrial H+-ATP synthase and the lipogenic switch: new core components of metabolic reprogramming in induced pluripotent stem (iPS) cells.Cell Cycle. 2013; 12: 207-218Crossref PubMed Scopus (69) Google Scholar). Moreover, a Warburg-like glycolysis was discovered in differentiated C2C12 myotubes and brown fat (22Teperino R. Amann S. Bayer M. McGee S.L. Loipetzberger A. Connor T. Jaeger C. Kammerer B. Winter L. Wiche G. Dalgaard K. Selvaraj M. Gaster M. Lee-Young R.S. Febbraio M.A. Knauf C. Cani P.D. Aberger F. Penninger J.M. Pospisilik J.A. Esterbauer H. Hedgehog partial agonism drives Warburg-like metabolism in muscle and brown fat.Cell. 2012; 151: 414-426Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). AMP-activated kinase (AMPK) 2The abbreviations used are: AMPKAMP-activated protein kinaseOXPHOSoxidative phosphorylationPFKFB36-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3SAGsmoothened agonistTAtibialis anteriorCTXcardiotoxinIHCimmunohistochemicalAICAR5-aminoimidazole-4-carboxamide ribonucleotide. is a master regulator of metabolism that has an α catalytic subunit with two isoforms, α1 and α2 (23Zhang B.B. Zhou G. Li C. AMPK: an emerging drug target for diabetes and the metabolic syndrome.Cell Metab. 2009; 9: 407-416Abstract Full Text Full Text PDF PubMed Scopus (842) Google Scholar). Here, we report that AMPKα1 is the dominant isoform in satellite cells, and AMPK α1 mediates Warburg-like glycolysis needed for satellite cell activation following muscle injury. Consequently, satellite cell-specific AMPKα1 KO impairs muscle regeneration, characterized by reduced satellite cell activation and muscle structure restoration. AMP-activated protein kinase oxidative phosphorylation 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 smoothened agonist tibialis anterior cardiotoxin immunohistochemical 5-aminoimidazole-4-carboxamide ribonucleotide. All animals were handled in accordance with protocols approved by the Animal Use and Care Committees of Washington State University. Wild-type C57BL/6 mice, B6.129S-Pax7tm1(cre/ERT2)Gaka/J mice (catalog no. 017763) in which tamoxifen-inducible Cre recombinase is driven by the endogenous mouse Pax7 promoter, B6(Cg)-Prkaa1tm1.1Sjm/J mice (catalog no. 014141) in which AMPKα1 exon 3 was flanked by two loxP sites, and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J mice (catalog no. 007576) in which a membrane-targeted tdTomato is floxed and EGFP is expressed when cross-bred to Cre recombinase-expressing mice, were obtained from Jackson Laboratory (Bar Harbor, ME). B6.129S-Pax7tm1(cre/ERT2)Gaka/J mice were cross-bred with Prkaa1tm1.1Sjm/J mice to generate tamoxifen-inducible satellite cell-specific AMPKα1 KO mouse strain (Pax7cre/AMPKα1fl/fl). Pax7cre/AMPKα1fl/fl mice were cross-bred with Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J mice to generate tamoxifen-inducible satellite cell-specific AMPKα1 KO mouse strain with tamoxifen-inducible satellite cell-specific EGFP expression (Pax7Cre/AMPKα1fl/fl/tdtomato,EGFP). Antibodies against AMPKα (antibody 2532), and phospho-AMPKα at Thr-172 (antibody 2535), rabbit anti-Ki67 Alexa Fluor 488 (antibody 11882), rabbit anti-PFKFB3 (antibody 13123), rabbit anti-phospho-AMPK substrate motif (antibody 5759), goat anti-mouse Alexa Fluor 555 (antibody 4409), and goat anti-rat Alexa Fluor 488 (antibody 4416) were purchased from Cell Signaling (Danvers, MA). Anti-embryonic myosin heavy chain (F1.652), anti-Pax7 (PAX7), anti-MHC (MF20), and anti-β-tubulin (E7) mouse monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA). Rabbit anti-AMPKα1 antibody (ABIN737886) and rabbit anti-AMPKα2 (ABIN680458) antibody were obtained from Antibodies-Online Inc. (Atlanta, GA). Mouse anti-MyoD antibody (sc32758), smoothened agonist (SAG), and cyclopamine were purchased from Santa Cruz (Dallas, TX). Rat anti-laminin antibody (4H8-2) was purchased from Enzo (Faringdale, NY). Goat anti-mouse IgG1 Alexa Fluor 555 (A-21127) was purchased from Life Technologies Inc. IRDye 800CW goat anti-rabbit secondary antibody and IRDye 680 goat anti-mouse secondary antibody were purchased from LI-COR Biosciences (Lincoln, NE). Cardiotoxin, tamoxifen and Oil-Red O were purchased from Sigma. Basic FGF2 (233-FB-025) and anti-mouse integrin α7 APC was purchased from R&D Systems (Minneapolis, MN). Anti-mouse CD45 PE-Cy7, anti-mouse CD16/CD32, and flow cytometry buffer were purchased from eBioscience (San Diego, CA). Anti-mouse TER-119 PE-Cy7, anti-mouse Sca-1 APC-Cy7, and anti-mouse CD31 PE-Cy7 antibodies were purchased from BioLegend (San Diego, CA). Gill's hematoxylin (catalog no. 26030-10) and Eosin Y-Phloxine B (catalog no. 26051-21) were purchased from Electron Microscopy Sciences (Hatfield, PA). AICAR was purchased from Toronto Research Chemicals (Toronto, Canada). Tibialis anterior (TA) muscle was digested in DMEM with collagenase D and dispase II as previously described (24Fu X. Zhao J.X. Zhu M.J. Foretz M. Viollet B. Dodson M.V. Du M. AMP-activated protein kinase alpha1 but not alpha2 catalytic subunit potentiates myogenin expression and myogenesis.Mol. Cell Biol. 2013; 33: 4517-4525Crossref PubMed Scopus (47) Google Scholar). Cells were blocked in anti-mouse CD16/CD32 antibody and then stained with anti-mouse CD45 PE-Cy7, anti-mouse TER119 PE-Cy7, anti-mouse CD31 PE-Cy7, anti-mouse Sca-1 APC-Cy7, and anti-mouse integrin α7 APC antibodies. Stained cells were sorted on FACSaria (BD Biosciences, San Jose, CA) and analyzed by FlowJo (Treestar, Inc., San Carlos, CA). Gates were made based on fluorescence minus one control. Satellite cells were resuspended in F-10 medium with 20% FBS, 1% antibiotic mixture and 5 ng/ml FGF2, and seeded on collagen-coated plates. Myogenic differentiation of satellite cells was induced by switching medium to DMEM supplemented with 2% horse serum and 1% antibiotic mixture. Nonmyogenic cells were cultured in DMEM with 10% FBS and 1% antibiotic mixture. Satellite cells were isolated as previously described (25Rando T.A. Blau H.M. Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy.J. Cell Biol. 1994; 125: 1275-1287Crossref PubMed Scopus (799) Google Scholar) with modifications. Muscle was removed from the hind limbs of 3–4-month-old mice. Muscle was cut into small pieces and digested in digestion buffer containing collagenase D and dispase II for about 30 min. Muscle slurry was passed through a 100-μm cell strainer. Filtrate was centrifuged for 5 min at 350 × g. Cell pellet was resuspended and cultured in F-10 medium with 20% FBS, 5 ng/ml FGF2, and 1% antibiotic mixture on collagen-coated plates. Satellite cells were enriched by preplating. Fast attaching nonmyogenic cells were also collected. Five thousand satellite cells were seeded in each well of 12 well plates. Cells were then trypsinized and counted at 1, 2, and 3 days after to determine the cell proliferation rate. Single muscle fibers were isolated as previously described (26Pasut A. Jones A.E. Rudnicki M.A. Isolation and culture of individual myofibers and their satellite cells from adult skeletal muscle.J. Vis. Exp. 2013; 73e50074Google Scholar) with modification. The extensor digitorum longus muscle was removed from 1-month-old Pax7Cre/AMPKα1fl/fl/tdtomato,EGFP and Pax7Cre/tdtomato,EGFP mice that had been treated with tamoxifen. Extensor digitorum longus muscle was digested in digestion buffer containing collagenase D. Extensor digitorum longus muscle was then carefully flushed to release single muscle fibers. Intact single muscle fibers were then transferred to 24-well plates with one muscle fiber in each well and cultured in high glucose DMEM with 20% FBS, 5 ng/ml FGF2, 110 mg/ml sodium pyruvate, and 1% antibiotic mixture. Glucose uptake test was performed using glucose uptake cell base assay kit from Cayman (Ann Arbor, MI) following the manufacturer's protocol. The cells were seeded onto 96-well plates at a density of 1 × 104 cells/well. Cells were cultured with fluorescently labeled deoxyglucose analog, and fluorescence was detected using Synergy H1 hybrid reader (BioTek, Winooski, VT). Total RNA was extracted using TRIzol (Sigma) followed by DNase (New England BioLabs Inc., Ipswich, MA) treatment, and cDNA was synthesized using a reverse transcription kit (Bio-Rad). Real time PCR was carried out using CFX real time PCR detection system (Bio-Rad) with a SYBR Green real time PCR kit from Bio-Rad. After amplification, a melting curve (0.01 °C/s) was used to confirm product purity, and agarose gel electrophoresis was performed to confirm that only a single product of the right size was amplified. Relative mRNA content was normalized to 18S rRNA content (24Fu X. Zhao J.X. Zhu M.J. Foretz M. Viollet B. Dodson M.V. Du M. AMP-activated protein kinase alpha1 but not alpha2 catalytic subunit potentiates myogenin expression and myogenesis.Mol. Cell Biol. 2013; 33: 4517-4525Crossref PubMed Scopus (47) Google Scholar). Primer sequences and their respective PCR fragment lengths are listed below. 18S rRNA (110 bp), forward 5′-TGCTGTCCCTGTATGCCTCT-3′ and reverse 5′-TGTAGCCACGCTCGGTCA-3′; Pax7 (115 bp), forward 5′-TTGGGGAACACTCCGCTGTGC-3′ and reverse 5′-CAGGGCTTGGGAAGGGTTGGC-3′; MyoD (100 bp), forward 5′-TCTGGAGCCCTCCTGGCACC-3′ and reverse 5′-CGGGAAGGGGGAGAGTGGGG-3′; Myf5 (125 bp), forward 5′-AAACTCCGGGAGCTCCGCCT-3′ and reverse 5′-GGCAGCCGTCCGTCATGTCC-3′; Myogenin (97 bp), forward 5′-GAGATCCTGCGCAGCGCCAT-3′ and reverse 5′-CCCCGCCTCTGTAGCGGAGA-3′; Smo (121 bp) forward 5′-GGCCTGACTTTCTGCGTTGCACACC-3′ and reverse 5′-GGGTTGTCTGTTCGCACCAAGG-3′; Shh (182 bp) forward 5′-CAGCGGCAGATATGAAGGGAAGA-3′ and reverse 5′-CAGGCCACTGGTTCATCACAGA-3′; Gli1 (188 bp) forward 5′-AGGTCTGCGTGGTAGAGGGAA-3′ and reverse 5′-GTTGGCTTGGTGGCAAAAGGG-3′; Ptch1 (121 bp) forward 5′-GCAAGTTTTTGGTTGTGGGTCTCC-3′ and reverse 5′-TCTCGACTCACTCGTCCACCAA-3′; AMPKα1 (246 bp) forward 5′-TGTCTCTGGAGGAGAGCTATTTGA-3′ and reverse 5′-GGTGAGCCACAGCTTGTTCTT-3′; and AMPKα2 (150 bp) forward 5′-CAGAAGATTCGCAGTTTAGATGTTGT-3′ and reverse 5′-ACCTCCAGACACATATTCCATTACC-3′. Immunoblotting analysis was performed as previously described using an Odyssey Infrared Imaging System (LI-COR Biosciences) (27Zhao J.X. Yue W.F. Zhu M.J. Du M. AMP-activated protein kinase regulates β-catenin transcription via histone deacetylase 5.J. Biol. Chem. 2011; 286: 16426-16434Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Band density was normalized to β-tubulin content. Cells grown on multiple well plates were fixed in cold methanol for 10 min, permeabilized with 0.1% Triton X-100 for 5 min, blocked with 1% BSA, and incubated with primary antibodies at 4 °C overnight. Cells were then stained with corresponding secondary antibodies (1:1,000) for 1 h. Images were taken using a EVOS microscope. TA muscle was fixed in cold 4% paraformaldehyde and frozen in isopentane cooled in liquid nitrogen. Frozen tissue was sectioned (5–10 μm thick). Sections were heated in citrate buffer for 20 min, blocked in 5% goat serum in TBS containing 0.3% Triton X-100, and stained with primary antibodies and corresponding fluorescent secondary antibodies. Sections were then mounted in a mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). Pax7+ cells with nuclei identified by DAPI staining were classified as satellite cells. For each TA muscle sample, the number of satellite cells and EMH+ muscle fibers on four randomly picked microscopic fields of each of three sections at different depths of the muscle were counted (four fields/section, three sections/muscle). Average numbers obtained from the three examined sections of each muscle sample were used as a biological replicate for comparative analysis. TA muscle frozen sections were rinsed in PBS, stained with Gill's hemotoxylin, and counterstained with eosin Y following the manufacturer's protocol. Ten thousand cells were seeded in each well of 96-well plates. 24 h after seeding, cell culture medium was collected and tested for lactate content using an l-lactate assay kit from Eton Bioscience, Inc. (San Diego, CA) following the manufacturer's instruction. 200,000 cells were seeded in each well of 6-well plates. One day after seeding, cell culture medium was changed with fresh medium. Oxygen content in medium was measured after 30 min of incubation with Orion 3-Star Pus Dissolved Oxygen Meter (Thermo Scientific, Waltham, MA). Oxygen consumption was calculated from the difference between the oxygen content in medium after 30 min of incubation and the oxygen content in fresh medium. For all studies, at least three independent experiments were conducted. All data are expressed as means ± S.D. The data were analyzed using the general linear model of SAS (SAS Inst. Inc., Cary, NC), and t test or Tukey range test was used to determine significance of differences among means. p < 0.05 was considered significant. To test the role of AMPKα1 in muscle regeneration, we first measured the expression of AMPKα1 during the proliferation and differentiation of isolated satellite cells. AMPKα1 expression profoundly increased (∼6-fold) during the activation and proliferation of satellite cells. After induction of myogenesis, the expression of AMPKα1 dropped first, followed by a slight increase (Fig. 1A), which is consistent with a previous report showing AMPKα1 activity in muscle increases during muscle regeneration, whereas the activity of AMPKα2 remain unchanged (24Fu X. Zhao J.X. Zhu M.J. Foretz M. Viollet B. Dodson M.V. Du M. AMP-activated protein kinase alpha1 but not alpha2 catalytic subunit potentiates myogenin expression and myogenesis.Mol. Cell Biol. 2013; 33: 4517-4525Crossref PubMed Scopus (47) Google Scholar, 28Mounier R. Théret M. Arnold L. Cuvellier S. Bultot L. Göransson O. Sanz N. Ferry A. Sakamoto K. Foretz M. Viollet B. Chazaud B. AMPKα1 regulates macrophage skewing at the time of resolution of inflammation during skeletal muscle regeneration.Cell Metab. 2013; 18: 251-264Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). Moreover, AMPKα1 expression was consistently and significantly higher than AMPKα2 in quiescent, activated, and differentiating satellite cells (Fig. 1B). These data support our hypothesis that AMPKα1 is important in activating satellite cell proliferation. To test whether AMPKα1 is critical for muscle regeneration following injury, we analyzed the expression of AMPKα1 and AMPKα2 in TA muscle injured by cardiotoxin (CTX) injection using immunohistochemical (IHC) staining after injury. AMPKα2 was found to be expressed primarily in the cytoplasm of well differentiated myogenic cells and regenerating muscle fibers (Fig. 1C). In contrast, AMPKα1 was found mainly in mononucleated cells including satellite cells (Fig. 1D). Moreover, we found that AMPKα1 was also expressed in satellite cells in uninjured muscle (Fig. 1D). However, p-AMPKα was only detected in satellite cells after injury, indicating AMPKα1 activation in satellite cells during muscle regeneration, which might be involved in satellite cell activation and proliferation (Fig. 1E). These data further suggest that AMPKα1 has a regulatory role in satellite cell activation, whereas AMPKα2 plays a major role in regulating metabolism in differentiated muscle fibers. Therefore, we focused further studies on the role of AMPKα1 in satellite activation and proliferation. To better understand the influence of AMPKα1 KO on satellite cell activation and proliferation, we then tested the proliferation of satellite cells isolated from tamoxifen-treated Pax7cre/AMPKfl/fl mice in which AMPKα1 gene is specifically deleted in satellite cells. Satellite cell-specific AMPKα1 KO was verified by different assays (Fig. 2, A–E). Indeed, AMPKα1 KO satellite cells proliferated slower than WT myoblasts (Fig. 3A). Because isolated satellite cells lack the niche environment, we further prepared single muscle fibers from tamoxifen-treated Pax7cre/tdomato,EGFP mice and Pax7cre/AMPKα1fl/fl/tdomato,EGFP mice, in which satellite cells express membrane-located EGFP upon tamoxifen treatment accompanied with AMPKα1 KO specifically in satellite cells. 48 h after muscle fiber isolation, satellite cells on muscle fibers were compared. Although 38 of 45 observed WT satellite cells had activated and started to proliferate, only 9 of 41 observed AMPKα1 KO satellite cells showed sign of proliferation, which suggested less efficient satellite cell activation and proliferation because of AMPKα1 KO (Fig. 3B).FIGURE 3AMPKα1 KO satellite cells proliferated slower than WT cells in vitro. A, proliferation of satellite cells isolated from tamoxifen-treated AMPKα1fl/fl (WT) mice and tamoxifen-treated Pax7cre/AMPKα1fl/fl mice (AMPKα1 KO) mice. B, proliferation of satellite cell on single muscle fibers isolated from tamoxifen-treated Pax7Cre/tdtomato,EGFP (WT) and tamoxifen-treated Pax7Cre/AMPKα1fl/fl/tdtomato,EGFP (AMPKα1 KO) mice at 48 h after isolation. *, p < 0.05 versus control; means ± S.D.; n ≥ 3. Scale bars, 200 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It has been recently reported that noncanonical Sonic Hedgehog (Shh) signaling promotes a Warburg-like glycolysis in differentiated C2C12 myotubes (22Teperino R. Amann S. Bayer M. McGee S.L. Loipetzberger A. Connor T. Jaeger C. Kammerer B. Winter L. Wiche G. Dalgaard K. Selvaraj M. Gaster M. Lee-Young R.S. Febbraio M.A. Knauf C. Cani P.D. Aberger F. Penninger J.M. Pospisilik J.A. Esterbauer H. Hedgehog partial agonism drives Warburg-like metabolism in muscle and brown fat.Cell. 2012; 151: 414-426Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Therefore, we questioned whether this pathway is also present in undifferentiated myoblasts and satellite cells and whether AMPKα1 has a mediatory role in eliciting Warburg-like glycolysis. We first tested the effect of Smoothened (Smo) agonist (SAG), an activator of Shh pathway, on AMPK activity in C2C12 myoblasts and WT satellite cells. C2C12 myoblasts and WT satellite cells were treated with 200 nm SAG for only 10 min to avoid the activation of canonical Shh signaling (22Teperino R. Amann S. Bayer M. McGee S.L. Loipetzberger A. Connor T. Jaeger C. Kammerer B. Winter L. Wiche G. Dalgaard K. Selvaraj M. Gaster M. Lee-Young R.S. Febbraio M.A. Knauf C. Cani P.D. Aberger F. Penninger J.M. Pospisilik J.A. Esterbauer H. Hedgehog partial agonism drives Warburg-like metabolism in muscle and brown fat.Cell. 2012; 151: 414-426Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 29Chen J.K. Taipale J. Young K.E. Maiti T. Beachy P.A. Small molecule modulation of Smoothened activity.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 14071-14076Crossref PubMed Scopus (830) Google Scholar). In both C2C12 cells and WT satellite cells, 10 min of SAG treatment activated AMPK (Fig. 4, A and B). l-Lactate assay revealed increased glycolysis rates in C2C12 cells and WT satellite cells in response to SAG treatment, which was absent in AMPKα1 KO satellite cells (Fig. 4C). In addition, SAG promoted the proliferation of both C2C12 cells and purified WT satellite cells but failed to promote proliferation of purified AMPKα1 KO satellite cells (Fig. 4D), suggesting that the proliferative effects of SAG treatment on satellite cells require AMPKα1. Skeletal muscle contains multiple cell types that interact with satellite cells and participate in muscle regeneration (30Joe A.W. Yi L. Natarajan A. Le Grand F. So L. Wang J. Rudnicki M.A. Rossi F.M. Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.Nat. Cell Biol. 2010; 12: 153-163Crossref PubMed Scopus (1013) Google Scholar). To better understand the potential effect of SAG treatment on satellite cell activation and proliferation in the presence of other cell types, muscle tissue slurry from tamoxifen-treated Pax7cre/tdomato,EGFP mice expressing EGFP in Pax7+ cells was obtained by enzymatic digestion of muscle tissue and plated without sorting. 48 h later, all cells were harvested, and EGFP-positive satellite cells were quantified. We found that SAG treatment increased the number of EGFP+ satellite cells, further supporting the promotive effect of Shh signaling on satellite activation and proliferation (Fig. 4E). To further test whether the observed effects of SAG on satellite cells was through noncanonical Shh signaling, WT primary myoblasts were treated with cyclopamine, a noncanonical Shh specific activator, which is also known to inhibit canonical Shh (22Teperino R. Amann S. Bayer M. McGee S.L. Loipetzberger A. Connor T. Jaeger C. Kammerer B. Winter L. Wic" @default.
• W2247011864 created "2016-06-24" @default.
• W2247011864 creator A5000428250 @default.
• W2247011864 creator A5049104586 @default.
• W2247011864 creator A5049277889 @default.
• W2247011864 creator A5074270918 @default.
• W2247011864 date "2015-10-01" @default.
• W2247011864 modified "2023-10-02" @default.
• W2247011864 title "AMP-activated Protein Kinase Stimulates Warburg-like Glycolysis and Activation of Satellite Cells during Muscle Regeneration" @default.
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