*3.2. Lys-induced Skeletal Muscle Growth in Relation to SC Activation Level*

In addition to the great change in longissimus dorsi muscle mass with dietary Lys supplementation (Tables S1 and S4), we also found that the Lys concentration in the longissimus dorsi muscle was significantly reduced by dietary Lys deficiency for 14 d (Table S5), whereas it could be rescued by dietary Lys re-supplementation for an additional 14 d (Table 2). Furthermore, the concentrations of threonine (Thr), serine (Ser), glutamate (Glu) and arginine (Arg) showed the same changes as Lys (Tables S2 and S5).

Considering these findings, we collected muscle samples for iTRAQ analysis to learn more about the role of Lys in governing skeletal muscle growth (Figure 1). After GO enrichment analysis, we found that proteins related to the transition between slow and fast fibers, filamin binding, mitogen-activated protein kinase binding, cytoskeletal protein binding, actin cytoskeleton, microtubule

binding, cytoskeleton organization, tubulin binding and muscle myosin complexes were enriched in the Lys deficiency versus control downregulated proteins (Figure 1A), indicating that the muscle structure was changed.

**Table 2.** Effect of dietary Lys re-supplementation on the concentrations of amino acids in the longissimus dorsi muscle on day 28 (freeze-dried basis, %) 1.


<sup>1</sup> Values without the same small letters within the same line indicate a significant difference (*p* < 0.05).

In addition, in the Lys deficiency versus control upregulated proteins, the enrichment of lipase inhibitor activity, negative regulation of homotypic cell-cell adhesion, negative regulation of cell activation, response to nutrients and negative regulation of wound healing were observed (Figure 1B), and these results suggested that the functions of the muscle cells were disturbed.

Furthermore, proteins related to protein kinase C inhibitor activity, protein Ser/Thr kinase inhibitor activity and cellular response to amino acid starvation were enriched in the Lys rescue versus control downregulated proteins (Figure 1C). Thus, the mTORC1 pathway may play a crucial role in Lys-controlled skeletal muscle growth.

More importantly, the results for the Lys rescue versus control upregulated proteins showed that the proteins related to the negative regulation of fat cell differentiation, regulation of fibroblast proliferation, regulation of lens fiber cell differentiation, positive regulation of cell proliferation, sarcolemma, basal plasma membrane, basolateral plasma membrane and cell-cell adhesion were enriched (Figure 1D). These data suggest that SCs may be activated.

Moreover, the results for the Lys rescue versus Lys deficiency downregulated proteins showed that the positive regulation of fibril organization, regulation of fibril organization and positive regulation of gap junction assembly were enriched (Figure 1E). The results for the Lys rescue versus Lys deficiency upregulated proteins showed that the muscle system process, negative regulation of fat cell differentiation, muscle hypertrophy, positive regulation of myoblast differentiation, regulation of myoblast differentiation, regulation of fibroblast proliferation, regulation of cell differentiation, positive regulation of muscle cell differentiation, regulation of muscle cell differentiation and regulation of developmental process were enriched (Figure 1F). In summary, these results indicate that skeletal muscle growth regulation by dietary Lys supplementation is possibly connected with the mTORC1 pathway and SCs.

**Figure 1.** GO enrichment analysis of differentially expressed proteins in the longissimus dorsi muscle on day 28 according to iTRAQ analysis. (**A**) GO enrichment analysis results (Lys deficiency versus control) for downregulated proteins. (**B**) GO enrichment analysis results (Lys deficiency versus control) for upregulated proteins. (**C**) GO enrichment analysis results (Lys rescue versus control) for downregulated proteins. (**D**) GO enrichment analysis results (Lys rescue versus control) for upregulated proteins. (**E**) GO enrichment analysis results (Lys rescue versus Lys deficiency) for downregulated proteins. (**F**) GO enrichment analysis results (Lys rescue versus Lys deficiency) for upregulated proteins. The *x*-axis represents the different GO terms, and the *y*-axis represents the enrichment ratio (the ratio between the protein number enriched in the GO term and the protein number annotated to the GO term; the greater the ratio was, the greater the enrichment found was), \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

#### *3.3. SCs and mTORC1 Activity Are Enhanced in Lys-induced Skeletal Muscle Growth*

The specific marker Pax7 and the proliferation marker Ki67 were detected in SCs in the longissimus dorsi muscle on days 14 and 28 by immunohistochemical analysis (Figures S1A and S2A). Based on the total cells (stained with DAPI), dietary Lys deficiency for 14 d or 28 d significantly decreased the numbers of Pax7-positive cells and Pax7 + Ki67-positive cells compared with the control cells (Figures S1B and S2B). Interestingly, compared with those in the Lys deficiency group, the numbers of Pax7-positive cells, Ki67-positive cells and Pax7 + Ki67-positive cells were all increased in the Lys rescue group and were even higher than those in the control group (Figure 2B). In addition, compared with the control group and Lys rescue group, the Lys deficiency group had a decreased ratio of Pax7 + Ki67-positive cells to Pax7-positive cells, regardless of deficiency for 14 d or 28 d (Figures 1C and 2C). Taken together, these observations suggest that the status of SCs in terms of proliferation in the longissimus dorsi muscle is regulated by dietary Lys supplementation.

In addition, we observed that the key proteins in the mTORC1 pathway, such as p-mTOR (Ser2448), p-S6K1 (Thr389), p-S6 (Ser235), p-4EBP1 (Thr470) and eIF4E (*p* = 0.083), were all inhibited by dietary Lys deficiency for 14 d (Figure 1D,E), and this reduction was observed for samples from the group fed Lys-deficient diets for 28 d (Figure 2D,E). Fortunately, when dietary Lys deficiency was re-established to the control level at 14 d and sustained for another 14 d, the restricted key protein levels of the mTORC1 pathway were all increased (Figure 2D,E). In general, these data indicate that SCs, along with the mTORC1 pathway, are required for Lys-induced skeletal muscle growth.

#### *3.4. Cell Proliferation Was Rescued with Increased mTORC1 by Lys Re-supplementation.*

To gain further insight into the role of Lys in SCs, Lys supplementation treatment was designed as shown in Figure 3A. In addition, the Lys concentrations in cell culture are shown in Table S3. The MTT assay results showed that cell viability was significantly reduced under Lys deficiency for 48 h (Figures S2A and S3B), whereas cell viability was rescued by Lys re-supplementation for 48 h compared with Lys deficiency for 96 h (Figure 3B). The number of cells detected by an automated cell counter showed that SC proliferation was significantly decreased under Lys restriction conditions for 24 to 120 h (Figures S2B and S3C). In contrast, the number of SCs was increased after Lys was re-supplemented for 24 h after Lys deficiency (Figure 3C). However, cell viability and the number of SCs continued to increase at a slow rate in the deficiency group. This phenomenon might be explained by the existence of Lys in FBS (Figure S3). Because proliferation is determined by mitosis and because cell cycle distribution is a typically evaluated endpoint [25], we further investigated cell cycle distribution using flow cytometry after Lys deficiency for 48 h (Figure 2C–F). Compared with the control conditions, Lys deficiency resulted in an increased percentage of G1 cells at 48 h and 72 h, while the number of cells in the S phase was decreased (Figure 2E,F).

Moreover, because Lys plays an important role in protein synthesis, the SUnSET assay [26,27] was used to measure changes in protein synthesis in SCs. After Lys deficiency for 48 h, SCs evaluated by puromycin analysis showed an obvious decrease in protein synthesis (Figure S4A). However, protein synthesis was obviously increased after Lys was sufficiently supplemented for another 48 h (Figure 3D). Coomassie blue staining was used to verify equal protein loading (Figures 3E and 4B) [27]. Furthermore, a BCA assay was used to confirm the protein synthesis rate [28], and the results showed that total protein lysis in SCs cultured in Lys-deficient medium was extremely restricted at 48 h (Figure S4C) and was then enhanced after 48 h of Lys supplementation (Figure 3F).

Importantly, to investigate whether Lys-stimulated cell proliferation and protein synthesis were mediated by the mTORC1 pathway, the related proteins were analyzed by western blotting (Figures S4D,E and S3G,H). Compared with the levels in the control group, the levels of phosphorylated mTOR (Ser2448) and its downstream targets, phosphorylated S6K1 (Thr389), phosphorylated ribosomal protein S6 (S6, Ser235) and phosphorylated 4EBP1 (Thr70), and the levels of eukaryotic translation initiation factor 4E (eIF4E) were significantly decreased in the Lys deficiency group at 48 h and 96 h (Supplemental Figures 4E and 3H). However, compared with those under Lys deficiency for 96 h, the phosphorylated protein levels of mTOR and its downstream targets, such as 4EBP1 and S6K1, were restored by Lys supplementation for 48 h after Lys deficiency for 48 h (Figure 3H). Moreover, immunofluorescence staining further demonstrated that p-mTOR (Ser2448) expression was also increased after Lys supplementation (Figure S5). These data indicate that Lys-dependent SC proliferation and protein synthesis are related to mTORC1 pathways.

**Figure 2.** Activation of SCs and protein level of the mTORC1 pathway in the longissimus dorsi muscle on day 28. (**A**) Ki67 (red) and Pax7 (green) staining represents activated SCs during the proliferation period. Bar: 200×. (**B**) Percentage of cells positively stained for Ki67 (red), Pax7 (green) and Ki67 (red) + Pax7 (green) of the total cells (blue, DAPI). (**C**) Percentage of SCs positively stained for Ki67 (red) + Pax7 (green) to Pax7 (green). (**D**) Representative images of key proteins in the mTORC1 pathway detected by western blotting. (**E**) The values represent the ratio of the protein levels of p-mTOR (Ser2448), p-S6K1 (Thr389), p-S6 (Ser235) and p-4EBP1 (Thr470) to the total protein levels and the protein level of eIF4E to that of β-actin, n = 3. The results are shown as the means ± S.E.M. of three independent preparations. Statistical significance was assessed by ANOVA with Tukey's test, \* *p* < 0.05.

**Figure 3.** Changes in cell viability, proliferation, protein synthesis and mTORC1 pathway activation after Lys supplementation to sufficient levels. (**A**) Lys supplementation was changed from deficient to sufficient at 48 h, and the cells cultured for another 72 h. (**B**) MTT assays were used to measure cell viability, n = 20. (**C**) Cell proliferation was measured by cell counting assays, n = 10. (**D**) Representative image of the western blotting analyses for puromycin at 96 h, n = 3. (**E**) Coomassie blue staining was used to verify equal protein loading for puromycin measurements at 96 h, n = 3. (**F**) Total protein quantitation using bicinchoninic acid assays at 96 h, n = 3. (**G**) mTORC1 pathway-related proteins were measured by western blotting after Lys supplementation from deficiency for another 48 h (total 96 h). (**H**) The values represent the ratio of the phosphorylated protein levels to the total protein or β-actin level, n = 3. The bars are the means ± S.E.M. from the representative results of three independent experiments. Statistical significance was assessed by ANOVA and Tukey's test, \* *p* < 0.05, \*\* *p* < 0.01.

#### *3.5. Lys Rescue of SC Proliferation and mTORC1 Pathway Activation Were Inhibited by Rapamycin*

To validate that mTORC1 pathway activity was crucial for Lys-regulated SC functions, SCs were treated with rapamycin under Lys rescue conditions for 48 h. As shown in Figure 4A, we found that the rescued cell viability by Lys re-supplementation was inhibited by the simultaneous addition of rapamycin and reduced to the Lys deficiency level. Moreover, Lys re-supplementation with rapamycin suppressed SC proliferation, and 50 nM rapamycin showed greater inhibition than 20 nM (Figure 4B). Furthermore, the SUnSET assay showed that 20 and 50 nM rapamycin restricted protein synthesis, which was rescued by Lys re-supplementation (Figure 4C). Protein amounts were also verified by Coomassie blue staining, and the results showed equal sample loading (Figure 4D). Apart from that, compared with the control group, all four other groups showed reductions in total protein concentrations (Figure 4E). Importantly, compared with Lys deficiency, Lys re-supplementation increased the total protein concentrations, whereas the increased protein concentrations were decreased by 20 (*p* = 0.055) and 50 nM (*p* < 0.05) rapamycin supplementation (Figure 4E). In addition, compared with control and Lys rescue conditions, Lys re-supplemented with rapamycin significantly decreased the protein levels of phosphorylated mTOR (Ser2448), phosphorylated S6K1 (Thr389), phosphorylated S6 (Ser235) and eIF4E (Figure 4F, G). Although there was no significant difference, the protein level of phosphorylated 4EBP1 (Thr70) was also decreased by 33.18% and 35.17% in the 20 and 50 nM rapamycin groups, respectively, compared to the Lys rescue group. Collectively, these results indicate that Lys-regulated SC functions are mediated by the mTORC1 pathway.

**Figure 4.** The increased cell viability, proliferation and protein synthesis induced by Lys rescue were inhibited by rapamycin, along with mTORC1 pathway downregulation. After Lys deficiency for

48 h, Lys was added to the medium alone or in combination with 20 or 50 nM rapamycin for another 48 h. (**A**) Cell viability was measured by MTT assays at 96 h, n = 20. (**B**) Cell counting assays were used to measure cell proliferation at 96 h, n = 10. (**C**) SUnSET measurements of protein synthesis were performed by incubating SCs in medium containing puromycin at 96 h. A representative image from the western blotting analyses for puromycin is shown, n = 3. (**D**) Coomassie blue staining was used to verify equal protein loading for puromycin measurements at 96 h, n = 3. (**E**) Total protein quantitation using bicinchoninic acid assays at 96 h is shown, n = 3. (**F**) Western blotting was used to detect the key proteins in the mTORC1 pathway at 96 h. (**G**) The values represent the ratio of the phosphorylated protein levels to the total protein or β-actin level, n = 3. The bars are the means ± S.E.M. from the representative results of three independent experiments. Statistical significance was assessed by ANOVA and Tukey's test, \* *p* < 0.05, \*\* *p* < 0.01. \*\*\* *p* < 0.001.

#### **4. Discussion**

Lys is well known to be one of the most important essential amino acids for body growth [1,3,10,22,29]. However, the mechanisms through which Lys governs muscle mass are still debated. In addition, traditionally recognized muscle mass maintenance has been expanded from protein turnover to cell turnover [12,16]. Thus, the balance between myonuclear accretion and reduction is also an important factor in determining muscle mass, and the function of SC fusion into myotubes is important to study [16,30].

Early studies suggested that changes in whole-body weight were the main responses to dietary Lys supplementation or restriction [3,22,31]. Few studies have focused specifically on skeletal muscle growth. In our study, we found that the growth of almost all separated skeletal muscle was restricted during dietary Lys deficiency, while compensatory growth was shown after Lys supplementation was changed from deficient to sufficient [3,32]. In addition, some weights of specific skeletal muscles were unchanged, which could be caused by differences in myofiber type composition.

In previous studies, skeletal muscle mass accumulation was attributed to the relative efficiency between muscle protein synthesis and degradation [3,4,33]. Nevertheless, our GO enrichment analysis results obtained from the iTRAQ analysis showed that there were great changes in skeletal muscle structure and muscle cell function. Furthermore, SCs were found to participate in Lys-induced skeletal muscle growth. This is consistent with what was mentioned in a previous study, which did not provide compelling evidence [34].

To confirm the implications of the GO enrichment analysis for the longissimus dorsi muscle, we found that the SC proliferation ratios (indicated by Pax7 and Ki67 [35–37]) were accurately controlled by dietary Lys supplementation. Consistent with the compensatory growth of skeletal muscle, proliferation also showed the same tendency, especially Pax7 + Ki67-positive SCs. Taking these results together, we believe that SC turnover is required for Lys-induced skeletal muscle growth.

In the case of cell turnover, proliferation is the typical process by which cell numbers are increased by mitosis [14,24]. In this study, the suppressed Lys supply led to reduced cell numbers via changed cell cycle distribution such that there was an increased percentage of G1 phase cells and a decreased percentage of S phase cells. Similarly, a reduction in SC proliferation has also been detected in methionine (Met)- and cysteine (Cys)-restricted cell culture medium [38]. Furthermore, protein synthesis was suppressed directly by Lys deficiency and may thus ultimately cause muscle mass loss [31,32].

In addition to what we found under Lys deficiency conditions, the decrease in cell proliferation was suppressed by changing Lys supplementation from deficiency to sufficiency. These effects may be attributed to the function of Lys as an activator of cell mitotic activity [34]. Furthermore, protein synthesis was rescued by supplementing Lys to Lys-deficient cells. Despite the rescue growth effects of Lys supplementation found in our research, glycine seemed to suppress protein degradation weakly in cells in Lys-deficient medium [11]. These results could explain why Lys seems to play some special functions in life maintenance that have not been previously illustrated, that is, Lys is not used for only protein synthesis [29].

To obtain further insight from our findings, the mTORC1 pathway was measured to investigate the regulatory mechanisms of the Lys relationship with SCs and skeletal muscle growth. Notably, studies have shown that the mTORC1 pathway can be activated by energy, growth factors or nutrients, especially amino acids [38,39]. However, the function of Lys is not as defined as it is for Leu or Arg, as diets supplemented with Leu [40] or Arg [41] promote muscle growth and increase mTORC1 pathway activation. In fact, there has been little research on Lys interactions with mTORC1 in promoting muscle growth. In contrast, the key protein levels in the mTORC1 pathway were not altered after oral Lys administration in rats [33]. More importantly, previous studies showed that the mTORC1 pathway was activated by Lys to suppress protein degradation in vivo and in vitro [10,11]. In the present study, we found that the mTORC1 pathway was inhibited by reducing dietary Lys, and SC proliferation was likewise inhibited in Lys-deficient medium. Furthermore, the related proteins in the mTORC1 pathway were reactivated with complete Lys supplementation. In addition, the indispensable role of the mTORC1 pathway in Lys-governed SC function was verified by rapamycin [20]. We found that the inhibited proliferation and protein synthesis in the Lys deficiency group could be rescued by Lys re-supplementation, whereas these increases were suppressed by the simultaneous addition of rapamycin. Similar to our study, previous studies demonstrated that mTORC1 plays a crucial role in SC function [14,17–19]. Therefore, the mTORC1 pathway is necessary for Lys-induced SC activation in vivo and in vitro. However, the molecular mechanisms, such as extracellular Lys sensing in SCs and intracellular mTORC1 activation, need to be further studied.

#### **5. Conclusions**

In conclusion (Figure 5), our findings demonstrate that Lys supplementation exerts compensatory growth effects and that the functions of Lys in muscle mass accumulation are mediated by SCs and the mTORC1 pathway. Thus, Lys is not only a molecular building block for protein synthesis but also a signal that activates SCs to regulate muscle growth via the mTORC1 signaling pathway. These findings can provide us with a new target and therapeutic strategy for skeletal muscle regeneration and disease.

**Figure 5.** mTORC1 mediates Lys-induced SC activation to promote skeletal muscle growth. Lys supplementation activates the mTORC1 pathway to increase SC proliferation to enhance myogenic potential to promote skeletal muscle growth.

**Supplementary Materials:** The supplementary materials are available online at http://www.mdpi.com/2073-4409/ 8/12/1549/s1.

**Author Contributions:** Data curation, C.-l.J.; Formal analysis, C.-l.J. and J.-l.Y.; Funding acquisition, H.-c.Y. and X.-q.W.; Investigation, C.-l.J.; Methodology, C.-l.J., J.Y. and H.-c.L.; Project administration, C.-l.J. and C.-q.G.; Resources, X.-q.W.; Writing—original draft, C.-l.J.; Writing—review & editing, J.-l.Y., J.Y., C.-q.G., H.-c.L. and X.-q.W.

**Funding:** This research was funded by the National Key R&D Program of China (2018YFD0500403) and the Science and Technology Planning Project of Guangzhou, China (201704030005).

**Conflicts of Interest:** The authors declare no conflict of interest.
