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Role of the α2 subunit of AMP-activated protein kinase and its nuclear localization in mitochondria and energy metabolism-related gene expressions in C2C12 cells.
Metabolism. 2019 01; 90:52-68.M

Abstract

BACKGROUND

AMP-activated protein kinase (AMPK), a heterotrimer with α1 or α2 catalytic subunits, acts as an energy sensor and regulates cellular homeostasis. Whereas AMPKα1 is necessary for myogenesis in skeletal muscle, the role of AMPKα2 in myogenic differentiation and energy metabolism-related gene expressions has remained unclear. We here examined the specific roles of AMPKα1 and AMPKα2 in the myogenic differentiation and mitochondria and energy metabolism-related gene expressions in C2C12 cells.

MATERIALS AND METHODS

Stable C2C12 cell lines expressing a scramble short hairpin RNA (shRNA) or shRNAs specific for AMPKα1 (shAMPKα1), AMPKα2 (shAMPKα2), or both AMPKα1 and AMPKα2 (shPanAMPK) were generated by lentivirus infection. Lentiviruses encoding wild-type AMPKα2 (WT-AMPKα2) or AMPKα2 with a mutated nuclear localization signal (ΔNLS-AMPKα2) were also constructed for introduction into myoblasts. Myogenesis was induced by culture of C2C12 myoblasts for 6 days in differentiation medium.

RESULTS

The amount of AMPKα2 increased progressively, whereas that of AMPKα1 remained constant, during the differentiation of myoblasts into myotubes. Expression of shPanAMPK or shAMPKα1, but not that of shAMPKα2, attenuated the proliferation of myoblasts as well as the phosphorylation of both acetyl-CoA carboxylase and the autophagy-initiating kinase ULK1 in myotubes. Up-regulation of myogenin mRNA, a marker for the middle stage of myogenesis, was attenuated in differentiating myotubes expressing shPanAMPK or shAMPKα1. In contrast, up-regulation of gene expression for muscle creatine kinase (MCK), a late-stage differentiation marker, as well as for genes related to mitochondrial biogenesis including the transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1α1 and α4 (PGC-1α1 and PGC-1α4) and mitochondria-specific genes such as cytochrome c were attenuated in myotubes expressing shAMPKα2 or shPanAMPK. The diameter of myotubes expressing shPanAMPK or shAMPKα2 was reduced, whereas that of those expressing shAMPKα1 was increased, compared with myotubes expressing scramble shRNA. A portion of AMPKα2 became localized to the nucleus during myogenic differentiation. The AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) and 2-deoxyglucose (2DG) each induced the nuclear translocation of WT-AMPKα2, but not that of ΔNLS-AMPKα2. Finally, expression of WT-AMPKα2 increased the mRNA abundance of PGC-1α1 and MCK mRNAs as well as cell diameter and tended to increase that of PGC-1α4, whereas that of ΔNLS-AMPKα2 increased only the abundance of MCK mRNA, in myotubes depleted of endogenous AMPKα2.

CONCLUSION

TAMPKα1 and AMPKα2 have distinct roles in myogenic differentiation of C2C12 cells, with AMPKα1 contributing to the middle stage of myogenesis and AMPKα2 to the late stage. AMPKα2 regulates gene expressions including MCK, PGC-1α1 and PGC-1α4 and mitochondria-specific genes such as cytochrome c during the late stage of differentiation. Furthermore, the nuclear translocation of AMPKα2 is necessary for maintenance of PGC-1α1 mRNA during myogenesis.

Authors+Show Affiliations

Division of Endocrinology and Metabolism, Department of Homeostatic Regulation, National Institute for Physiological Sciences, 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan; Department of Physiological Sciences, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan; Second Department of Internal Medicine (Endocrinology, Diabetes and Metabolism, Hematology, Rheumatology), Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.Division of Endocrinology and Metabolism, Department of Homeostatic Regulation, National Institute for Physiological Sciences, 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan; Department of Physiological Sciences, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan.Division of Endocrinology and Metabolism, Department of Homeostatic Regulation, National Institute for Physiological Sciences, 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan.Division of Endocrinology and Metabolism, Department of Homeostatic Regulation, National Institute for Physiological Sciences, 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan.Division of Endocrinology and Metabolism, Department of Homeostatic Regulation, National Institute for Physiological Sciences, 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan; Department of Physiological Sciences, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan. Electronic address: minokosh@nips.ac.jp.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

30359677

Citation

Okamoto, Shiki, et al. "Role of the Α2 Subunit of AMP-activated Protein Kinase and Its Nuclear Localization in Mitochondria and Energy Metabolism-related Gene Expressions in C2C12 Cells." Metabolism: Clinical and Experimental, vol. 90, 2019, pp. 52-68.
Okamoto S, Asgar NF, Yokota S, et al. Role of the α2 subunit of AMP-activated protein kinase and its nuclear localization in mitochondria and energy metabolism-related gene expressions in C2C12 cells. Metabolism. 2019;90:52-68.
Okamoto, S., Asgar, N. F., Yokota, S., Saito, K., & Minokoshi, Y. (2019). Role of the α2 subunit of AMP-activated protein kinase and its nuclear localization in mitochondria and energy metabolism-related gene expressions in C2C12 cells. Metabolism: Clinical and Experimental, 90, 52-68. https://doi.org/10.1016/j.metabol.2018.10.003
Okamoto S, et al. Role of the Α2 Subunit of AMP-activated Protein Kinase and Its Nuclear Localization in Mitochondria and Energy Metabolism-related Gene Expressions in C2C12 Cells. Metabolism. 2019;90:52-68. PubMed PMID: 30359677.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Role of the α2 subunit of AMP-activated protein kinase and its nuclear localization in mitochondria and energy metabolism-related gene expressions in C2C12 cells. AU - Okamoto,Shiki, AU - Asgar,Nur Farehan, AU - Yokota,Shigefumi, AU - Saito,Kumiko, AU - Minokoshi,Yasuhiko, Y1 - 2018/10/23/ PY - 2018/07/27/received PY - 2018/10/07/revised PY - 2018/10/16/accepted PY - 2018/10/26/pubmed PY - 2019/5/24/medline PY - 2018/10/26/entrez KW - AMP-activated protein kinase (AMPK) KW - C2C12 cells KW - Muscle creatine kinase KW - Muscle differentiation KW - Nuclear localization signal KW - PGC-1α SP - 52 EP - 68 JF - Metabolism: clinical and experimental JO - Metabolism VL - 90 N2 - BACKGROUND: AMP-activated protein kinase (AMPK), a heterotrimer with α1 or α2 catalytic subunits, acts as an energy sensor and regulates cellular homeostasis. Whereas AMPKα1 is necessary for myogenesis in skeletal muscle, the role of AMPKα2 in myogenic differentiation and energy metabolism-related gene expressions has remained unclear. We here examined the specific roles of AMPKα1 and AMPKα2 in the myogenic differentiation and mitochondria and energy metabolism-related gene expressions in C2C12 cells. MATERIALS AND METHODS: Stable C2C12 cell lines expressing a scramble short hairpin RNA (shRNA) or shRNAs specific for AMPKα1 (shAMPKα1), AMPKα2 (shAMPKα2), or both AMPKα1 and AMPKα2 (shPanAMPK) were generated by lentivirus infection. Lentiviruses encoding wild-type AMPKα2 (WT-AMPKα2) or AMPKα2 with a mutated nuclear localization signal (ΔNLS-AMPKα2) were also constructed for introduction into myoblasts. Myogenesis was induced by culture of C2C12 myoblasts for 6 days in differentiation medium. RESULTS: The amount of AMPKα2 increased progressively, whereas that of AMPKα1 remained constant, during the differentiation of myoblasts into myotubes. Expression of shPanAMPK or shAMPKα1, but not that of shAMPKα2, attenuated the proliferation of myoblasts as well as the phosphorylation of both acetyl-CoA carboxylase and the autophagy-initiating kinase ULK1 in myotubes. Up-regulation of myogenin mRNA, a marker for the middle stage of myogenesis, was attenuated in differentiating myotubes expressing shPanAMPK or shAMPKα1. In contrast, up-regulation of gene expression for muscle creatine kinase (MCK), a late-stage differentiation marker, as well as for genes related to mitochondrial biogenesis including the transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1α1 and α4 (PGC-1α1 and PGC-1α4) and mitochondria-specific genes such as cytochrome c were attenuated in myotubes expressing shAMPKα2 or shPanAMPK. The diameter of myotubes expressing shPanAMPK or shAMPKα2 was reduced, whereas that of those expressing shAMPKα1 was increased, compared with myotubes expressing scramble shRNA. A portion of AMPKα2 became localized to the nucleus during myogenic differentiation. The AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) and 2-deoxyglucose (2DG) each induced the nuclear translocation of WT-AMPKα2, but not that of ΔNLS-AMPKα2. Finally, expression of WT-AMPKα2 increased the mRNA abundance of PGC-1α1 and MCK mRNAs as well as cell diameter and tended to increase that of PGC-1α4, whereas that of ΔNLS-AMPKα2 increased only the abundance of MCK mRNA, in myotubes depleted of endogenous AMPKα2. CONCLUSION: TAMPKα1 and AMPKα2 have distinct roles in myogenic differentiation of C2C12 cells, with AMPKα1 contributing to the middle stage of myogenesis and AMPKα2 to the late stage. AMPKα2 regulates gene expressions including MCK, PGC-1α1 and PGC-1α4 and mitochondria-specific genes such as cytochrome c during the late stage of differentiation. Furthermore, the nuclear translocation of AMPKα2 is necessary for maintenance of PGC-1α1 mRNA during myogenesis. SN - 1532-8600 UR - https://www.unboundmedicine.com/medline/citation/30359677/Role_of_the_α2_subunit_of_AMP_activated_protein_kinase_and_its_nuclear_localization_in_mitochondria_and_energy_metabolism_related_gene_expressions_in_C2C12_cells_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0026-0495(18)30213-0 DB - PRIME DP - Unbound Medicine ER -