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Inhibition of leucine aminopeptidase affects myocyte proliferation and differentiation
Author(s) -
Osana Shion,
Murayama Kazutaka,
Suzuki Naoki,
Takada Hiroaki,
Kubota Takahiro,
Nagatomi Ryoichi
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.02075
Subject(s) - leucine , aminopeptidase , myocyte , c2c12 , myosin , skeletal muscle , sarcopenia , gene knockdown , protein degradation , downregulation and upregulation , proteolysis , biochemistry , biology , microbiology and biotechnology , chemistry , amino acid , endocrinology , gene , myogenesis , enzyme
Background Loss of skeletal muscle mass in the elderly, namely sarcopenia, is a rapidly growing global health problem. Understanding the mechanism of muscle mass regulation is essential to confront this problem. The balance of muscle protein synthesis and degradation is thought to be one of the key issues. Previous reports showed that muscle‐specific deletion of a crucial proteasomal gene, Rpt3 (also known as Psmc4 ), resulted in severe skeletal muscle atrophy (Kitajima Y et al ., 2014). Although proteasomal proteolysis plays an essential role in the maintenance of muscle mass, the underlying mechanism remains unknown. Proteasome generates oligopeptides in the cytoplasm, which are further degraded quickly to amino acids depending on several aminopeptidases (Hershko A et al ., 1992, Saric T et al ., 2004). Although aminopeptidases may have a significant role in the maintenance of muscle mass, not much is known. Microarray analysis of transcripts in Rpt3 knockdown myocytes revealed an upregulated leucine aminopeptidase related genes. In this study, therefore, we examined the contribution of leucine aminopeptidase in the proliferation and differentiation of cultured myocytes. Methods We examined the consequence of leucine aminopeptidase inhibition on C2C12 myocytes under different culture conditions. Leucine aminopeptidase was inhibited by bestatin methyl ester (Bes‐ME; 100 μM), an aminopeptidase inhibitor. Proliferation was evaluated by counting the number of Ki67 (proliferation marker) positive cells. Differentiation was evaluated by myosin heavy chain (MyHC) staining and fusion index. Fusion index was calculated as the ratio of the nuclei number in myocytes with two or more nuclei versus the total number of nuclei. Additionally, phosphorylation of mammalian (or mechanistic) target of rapamycin (mTOR) and eukaryotic translation initiation factor 4E‐binding protein (4EBP1), were evaluated by Western Blotting because they are key factors for muscle protein synthesis. Results Leucine aminopeptidase inhibition by Bes‐ME significantly suppressed the activity of leucine aminopeptidase. The proliferation of C2C12 myoblasts was significantly reduced and the differentiation of C2C12 myoblasts was also markedly restricted after Bes‐ME administration. The protein expression levels of MyHC, Myogenin, and MyoD responsible for myodifferentiation were also significantly reduced. Furthermore, we also discovered that the phosphorylation of mTOR was significantly suppressed after leucine aminopeptidase inhibition. Conclusions Leucine aminopeptidase is essential for myoblast proliferation and differentiation; however, further studies are required to clarify the role of aminopeptidase in skeletal muscle growth. Support or Funding Information Acknowledgements This work was supported by JSPS KAKENHI Grant Number 18H04080.