Guanidinoacetic Acid Regulates Myogenic Differentiation and Muscle Growth Through miR-133a-3p and miR-1a-3p Co-mediated Akt/mTOR/S6K Signaling Pathway

Abstract

Guanidinoacetic acid (GAA), an amino acid derivative that is endogenous to animal tissues including muscle and nerve, has been reported to enhance muscular performance. MicroRNA (miRNA) is a post-transcriptional regulator that plays a key role in nutrient-mediated myogenesis. However, the effects of GAA on myogenic differentiation and skeletal muscle growth, and the potential regulatory mechanisms of miRNA in these processes have not been elucidated. In this study, we investigated the effects of GAA on proliferation, differentiation, and growth in C2C12 cells and mice. The results showed that GAA markedly inhibited the proliferation of myoblasts, along with the down-regulation of cyclin D1 (CCND1) and cyclin dependent kinase 4 (CDK4) mRNA expression, and the upregulation of cyclin dependent kinase inhibitor 1A (P21) mRNA expression. We also demonstrated that GAA treatment stimulated myogenic differentiation 1 (MyoD) and myogenin (MyoG) mRNA expression, resulting in an increase in the myotube fusion rate. Meanwhile, GAA supplementation promoted myotube growth through increase in total myosin heavy chain (MyHC) protein level, myotubes thickness and gastrocnemius muscle cross-sectional area. Furthermore, small RNA sequencing revealed that a total of eight miRNAs, including miR-133a-3p and miR-1a-3p cluster, showed differential expression after GAA supplementation. To further study the function of miR-133a-3p and miR-1a-3p in GAA-induced skeletal muscle growth, we transfected miR-133a-3p and miR-1a-3p mimics into myotube, which also induced muscle growth. Through bioinformatics and a dual-luciferase reporter system, the target genes of miR-133a-3p and miR-1a-3p were determined. These two miRNAs were shown to modulate the Akt/mTOR/S6K signaling pathway by restraining target gene expression. Taken together, these findings suggest that GAA supplementation can promote myoblast differentiation and skeletal muscle growth through miR-133a-3p- and miR-1a-3p-induced activation of the AKT/mTOR/S6K signaling pathway.

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“…MicroRNAs (MiRNAs) are a class of non-coding RNAs which modulate the expression of target genes via inhibiting translation at the post-transcriptional level or mediating mRNA degradation [6]. It has been revealed that miR-133a-3p as a tumor inhibitor in several malignant neoplasias and overexpression of miR-133a-3p can inhibit the growth of colorectal cancer (CRC) cells [7].…”

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“…It has been shown that miR‐26a‐5p facilitates chicken ovarian theca cell proliferation by targeting the TNRC6A gene (Kang et al, 2017), and gga‐miR‐200a‐3p, gg‐amiR‐101‐3p, gga‐miR‐458b‐5p and gga‐miR‐32‐5p may play vital roles in ovarian development (W. Wang et al, 2018). Previous reports have revealed that apla‐mir‐1‐13 (also named miR‐1a‐3p in other species) alleviated the symptoms of isoproterenol‐induced heart failure in mice by enhancing mitochondrial ND1 and COX1 (He et al, 2019); in addition, apla‐mir‐1‐13 regulated skeletal muscle growth (X. Wang et al, 2018; Xie et al, 2018) and played important roles in breast muscle development in chickens (Fu et al, 2018). However, there are no reports of apla‐mir‐1‐13 regulating ovarian development.…”

The Potential of Guanidino Acetic Acid to Reduce the Occurrence and Severity of Broiler Muscle Myopathies

“…Guanidinoacetic acid has been related to muscle function mainly by its role in the formation of creatine. Nevertheless, an in vitro study conducted by Wang et al (2018) using C2C12 cells, which are an immortalized mouse myoblast cell line, indicated that GAA stimulated myogenic differentiation 1 (MyoD) and myogenin (MyoG) mRNA expression increasing the myotube fusion rate. Myoblast fusion is a mechanism of increasing muscle mass without increasing muscle myofiber number.…”