**1. Introduction**

Lysine (Lys) is the first limiting essential amino acid for mammals consuming a predominantly cereal-based diet [1,2]. The important role of Lys in promoting skeletal muscle growth has already been demonstrated in animal husbandry, and this effect was attributed to increased protein synthesis [3,4]. Moreover, the functions of Lys in preventing human illnesses, such as osteoporosis and maldevelopment, have been intensively studied to protect human health [5,6]. In contrast, a low Lys diet has been used to treat glutaric aciduria type I and pyridoxine-dependent epilepsy [7,8], despite the fact that Lys deficiency causes severe body growth restriction and a reduction in body weight [9]. Furthermore, to study the mechanism of Lys in governing skeletal muscle growth, it has been reported that the mammalian target of rapamycin complex 1 (mTORC1) pathway is activated by Lys in the skeletal muscle of rats [10]. Additionally, Lys suppresses protein degradation in C2C12 myotubes via greater

mTORC1 pathway phosphorylation [11]. However, protein synthesis is controlled by DNA in the nucleus, such that a higher number of cell nuclei in myofibers means greater protein synthesis efficiency [12].

As muscle stem cells are involved in skeletal muscle growth, satellite cells (SCs) are distributed in the basal lamina and sarcolemma of skeletal muscle fibers [13,14]. It has already been established that through proliferation [14], migration [15] and fusion into myotubes to form new nuclei, SCs contribute considerably to muscle fiber hypertrophy [16]. Moreover, in the study of SCs, the mTORC1 pathway is an invaluable index [17,18]. First, mTORC1 is critical for SC participation in skeletal muscle regeneration [19]. Another study showed that mTORC1 is also necessary for RNA-induced mitochondrial restoration in SC activation [17]. Furthermore, the addition of leucine (Leu) could promote proliferation in rat SCs via increasing mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase 1 (S6K1) phosphorylation [20]. Alway et al. found that a metabolite of Leu, β-hydroxy-β-methylbutyrate (HMB), promotes SC proliferation but does not activate the mTORC1 pathway [21]. Thus, investigating whether Lys could function as a signal regulatory factor that regulates SC proliferation through the mTORC1 pathway to promote skeletal muscle growth is an important endeavor.

In the current work, we aimed to expand our understanding of the role of Lys in governing skeletal muscle growth. Our research was designed to determine the specific skeletal muscle growth of piglets with dietary Lys supplementation in greater detail than a previous study [22]. Importantly, isobaric tag for relative and absolute quantitation (iTRAQ) analysis of the longissimus dorsi muscle displayed differentially expressed proteins related to SCs and the mTORC1 pathway, indicating the potential communication between Lys, the mTORC1 pathway and SCs in skeletal muscle growth. Then, we investigated the changes in proliferation and protein synthesis by accurately controlling Lys supplementation in medium to demonstrate that SC proliferation relies on mTORC1 pathway activation. Moreover, rapamycin was used to confirm the indispensable role of the mTORC1 pathway in the proliferation of SCs with Lys re-supplementation.

#### **2. Materials and Methods**

#### *2.1. Ethics Statement*

All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of South China Agricultural University (Guangzhou, China), and the experiments were approved by the Animal Ethics Committee (SCAU#0158Ethic Committee Approval Number) of South China Agricultural University (Guangzhou, China).
