Accepted Manuscript

Effect of branched-chain amino acid ratio on the proliferation, differentiation and the
expression levels of key regulators involved in protein metabolism of myocytes

Yehui Duan, Liming Zeng, Fengna Li, Wenlong Wang, Yinghui Li, Qiuping Guo,
Yujiao Ji, Bie Tan, Yulong Yin

PII: S0899-9007(16)30249-0
DOI: 10.1016/j.nut.2016.10.016
Reference: NUT 9869

To appear in: Nutrition

Received Date: 3 August 2016

Revised Date: 11 October 2016
Accepted Date: 30 October 2016

Please cite this article as: Duan Y, Zeng L, Li F, Wang W, Li Y, Guo Q, Ji Y, Tan Be, Yin Y, Effect of
branched-chain amino acid ratio on the proliferation, differentiation and the expression levels of key
regulators involved in protein metabolism of myocytes, Nutrition (2016), doi: 10.1016/j.nut.2016.10.016.

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 ACCEPTED MANUSCRIPT

1 Effect of branched-chain amino acid ratio on the proliferation, differentiation and the

2 expression levels of key regulators involved in protein metabolism of myocytes

3 Running title: Branched-chain amino acid ratio and myocytes

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5 Yehui Duan1,2#, Liming Zeng3#, Fengna Li1,4*, Wenlong Wang5, Yinghui Li1,2, Qiuping

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6 Guo1,2, Yujiao Ji1, Bie Tan1,4, and Yulong Yin1,5*

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7

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8 Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical

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9 Agriculture, Chinese Academy of Sciences; Hunan Provincial Engineering Research Center for

10 Healthy Livestock and Poultry Production; Scientific Observing and Experimental Station of
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11 Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Changsha 410125,

12 China
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13 University of Chinese Academy of Sciences, Beijing 100039, China
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14 Science College of Jiangxi Agricultural University, Nanchang, Jiangxi 330045, China

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15 Hunan Collaborative Innovation Center for Utilization of Botanical Functional Ingredients; Hunan
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16 Co-Innovation Center of Animal Production Safety, CICAPS, Changsha 410128, China

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17 School of Biology, Hunan Normal University, Changsha 410018, China
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18
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19 *Corresponding anthor:
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20 Fengna Li or Yulong Yin

21 Institute of Subtropical Agriculture, the Chinese Academy of Sciences,

22 Hunan, China

23 Tel: 86-731-84619703,

24 Fax: 86-731-84612685,

25 E-mail: lifengna@isa.ac.cn, yinyulong@isa.ac.cn.

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26 ABSTRACT

27 Objectives: Branched-chain amino acids (BCAAs), including leucine (Leu), isoleucine

28 (Ile), and valine (Val), are key regulators of protein synthesis in muscle. This study was

29 conducted to investigate the effect of different BCAAs ratios (Leu:Ile:Val) on the

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30 proliferation, differentiation and expression levels of the regulators related to protein

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31 metabolism of C2C12 myocytes. Methods and Results: The ratio of 1:0.25:0.25 increased

32 cell viability and promoted cell cycle progression from G0/G1 phase to S phase, which was

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33 an indicator of proliferation enhancement (P < 0.05). Moreover, this optimal ratio

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34 (1:0.25:0.25) promoted the differentiation of myocytes into myotubes by up-regulating
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35 myogenin and IL-15 gene expression, and differently regulated the expression of L-type

36 amino acid transporter 1 and 4 (LAT1 and LAT4) and system ASC amino acid transporters 2
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37 (ASCT2). Furthermore, the ratio stimulated mTOR expression at the mRNA and
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38 phosphorylated protein levels, as well as ribosomal protein S6 kinase and

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39 regulatory-associated protein of mTOR (raptor). In contrast, the optimal ratio decreased the

40 amount of ubiquitin ligase muscle-specific RING finger 1 and muscle atrophy F-box during
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41 proliferation and differentiation (P < 0.05). No change was observed in the expression of key
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42 genes related to energy metabolism except for uncoupling protein 3 (P > 0.05). Conclusions:
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43 The results suggested that appropriate BCAAs ratios could enhance proliferation and

44 differentiation of the C2C12 myocytes, also mediate the key regulators related to protein

45 metabolism including the mTORC1 pathway. A proper utilization of balanced BCAAs ratio

46 in food would be beneficial to human and animal nutrition.

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47 Keywords: Branched-chain amino acid ratio; Protein metabolism; Proliferation and

48 differentiation; mTORC1; Myocyte

49 1. Introduction

50 Free amino acids are the building blocks for protein synthesis and pre-requisite for the

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51 synthesis of nitrogenous substances with enormous physiological importance [1, 2].

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52 Branched-chain amino acids (BCAAs), including leucine (Leu), isoleucine (Ile), and valine

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53 (Val), are the key regulators of muscle protein synthesis [3, 4]. Leu, Ile, and Val possess a

54 similar structure with a branched-chain residue, and share a common membrane transport

55
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system and enzymes [5]. BCAAs, especially Leu, function as a signaling molecule by
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56 modulating protein synthesis via mammalian target of rapamycin complex 1 (mTORC1)
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57 signaling other than simple nutrition [3, 6]. The complex of mTORC1 includes mTOR and

58 regulatory-associated protein of mTOR (raptor) [7-9], which is a scaffold protein that plays a
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59 crucial role in recruiting mTORC1 substrates and integrating various signals for mTOR
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60 regulation [10]. Moreover, the stability of raptor-mTOR interaction is the target of upstream
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61 signals [11]. The amino acid-mTORC1-protein synthesis axis has been reviewed extensively

62 [6, 12]. When intracellular amino acids are sufficient, they activate ras-related guanosine
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63 triphosphatases, and translocate mTORC1 to the surface of late endosomes and lysosomes,
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64 resulting in the activation of mTORC1 by ras homolog enriched in brain, which resides on

65 endosomal/lysosomal surface [13, 14]. The activation of mTORC1 positively mediates

66 protein synthesis and cell growth by phosphorylating and activating ribosomal protein S6

67 kinase (S6K1) [15, 16].

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69 The complex of mTORC1 is highly sensitive to changes in local amino acid levels, and

70 excessive amino acids are therefore catabolized [17]. Moreover, BCAAs have identical steps

71 during their respective catabolic pathways [18]. Excessive Leu would cause BCAAs

72 imbalance, leading to impairment in the availability of Val and Ile through branched-chain

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73 -keto acid dehydrogenase resulting in the down-regulation of mTORC1 activity [19, 20].

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74 Thus, the ratio of the three BCAAs needs to be closely managed, because changes in any

75 BCAAs levels will destroy the balance resulting in reduced rates of protein synthesis and

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76 increased rates of protein degradation in cells [21, 22]. Further research is required on the

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77 role of BCAAs (but not just Leu) in muscle protein synthesis via mTORC1-dependent
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78 mechanisms or related functions [23-30]. Effects of a mixture of BCAAs or Leu have been

79 evaluated on muscle biochemical parameters of rats [31]. Recent reports indicate BCAAs
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80 mixture (Leu: Ile: Val=1.7:0.8:0.7) supplementation chronically improves protein synthesis

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81 in older adults [32]. Additionally, BCAAs combination (45.8% Leu, 22.9% Ile, 27.6% Val)
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82 reduces muscle atrophy and protein levels of ubiquitin ligase [33]. Collectively,

83 supplementation with an appropriate BCAAs mixture shows positive effect on muscle protein
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84 metabolism.
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85
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86 The present study was conducted to investigate the effect of different BCAAs ratio in C2C12

87 myocytes during proliferation and differentiation. Furthermore, we also would like to observe

88 the mRNA and protein abundance of key genes involved in protein metabolism and

89 mTORC1 signal pathway.

90 2. Materials and Methods

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91 2.1. Reagents

92 High glucose Dulbeccos modified Eagles medium (DMEM) was purchased from Gibco

93 (Life Technologies, Grand Island, NY, USA). Fetal bovine serum (FBS) and horse serum

94 (HS) were purchased from HyClone (GE healthcare, UT, USA). Primary and secondary

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95 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA) or Santa

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96 Cruz Biotechnology (Dallas, TX, USA). CCK-8 kit was purchased from Dojindo (Osaka,

97 Japan). TRIzol, DNase I, and SYBR Green detection kit were purchased from Invitrogen

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98 (Life Technologies). Protease inhibitor cocktail was purchased from Roche (Basel,

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99 Switzerland). Phosphatase inhibitors were purchased from Thermo Scientific. PVDF
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100 membranes were purchased from Millipore (Merck, Darmstadt, Germany).

101 Chemiluminescence kit was purchased from Applygen Technologies Inc. (Beijing, China).
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102 Alpha Imager 2200 was purchased from Alpha Innotech Corporation (San Leandro, CA,
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103 USA). Leu, Ile, and Val were purchased from Sigma (St. Louis, MO, USA).
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104 2.2. Cell culture

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105 C2C12 myocytes were cultured in proliferation media of high glucose DMEM supplemented

106 with 10% FBS. The cells were incubated at 37C in humidified 95% air with 5% CO2 and
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107 subcultured every 2 or 3 d. The differentiation of the myocytes was induced by differentiation

108 media of high glucose DMEM supplemented with 2% HS, with the media being refreshed

109 every 2 d, and last for 4 d until myotubes were fully formed. Cell status was observed and

110 photographed using Nikon microscope (Tokyo, Japan).

111

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112 BCAAs free media were prepared according to the formula (High glucose, Life

113 Technologies). Leu, Ile, and Val were freshly diluted in BCAAs free media and added into

114 the media before treatment according to the different ratios. Therefore, the treatments were

115 without (blank) or with indicated ratios of the BCAAs (Leu:Ile:Val). Leu concentration was

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116 kept constant of 5 mM [34], and the concentrations of Ile and Val were calculated according

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117 to the setting ratio. When the C2C12 myocytes reached to 70% confluency, the cells were

118 incubated with BCAAs-free media overnight (12 h) and then treated with the fresh media

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119 containing different ratios of the BCAAs for 2 d (proliferation stage), or for 4 d after

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120 changing to differentiation media (differentiation stage).
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121 2.3. Cell viability assay
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122 Cell viability was assessed using cell counting kit CCK-8. Briefly, myocytes were treated

123 with the media containing different ratios of the BCAAs for 0.5-48 h. Then, the culture media
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124 were replaced with 100 L of fresh media containing 10 L reagent from the kit. After 45
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125 min incubation at 37C, absorbance at 450 nm of each well was measured with an ELISA
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126 plate reader (Bio-Tek, Winooski, VT, USA). The results were expressed as optical density

127 (OD450) value. The optimum ratio of BCAAs in the cell viability detection was used in the
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128 following analysis.

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129 2.4. Flow cytometry of cell cycle

130 After incubation with different BCAAs ratios, the media were removed and the adhering cells

131 were washed twice with PBS. Then, cells were harvested by trypsinization and fixed with 70%

132 ice-cold ethanol for 10 min at 4C. After washing, the cell pellet was re-suspended in

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133 PI-staining buffer (50 L/mL PI, RNAse A; Sigma, MO, USA) and incubated for 15 min at

134 37C. Cell cycle distribution was analyzed by FACS Calibur (BD Biosciences, San Diego,

135 CA) using ModFit software.

136 2.5. Determination of free amino acid profile

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137 After treating for the indicated time in both proliferation and proliferation stages, culture

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138 media and cells were both harvested for analysis. The adhering cells were washed twice with

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139 PBS and were dissolved in water with methanol (v:v = 1:1) at 4C for 10 min. Then, cells

140 were harvested to 1.5 mL microcentrifuge tubes and stored at -80C until analyses. After

141
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centrifugation to separate soluble from insoluble material, 40 l of the supernatant were
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142 labeled with iTRAQ reagents (AA 45/32 kit, Applied Biosystems) as recommended by the
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143 manufacturer and analyzed on an Applied Biosystems 3200 Q TRAP LC/MS/MS system

144 equipped with a RP-C18-column (150 mm length, 4.6 mm diameter, 5 mm particle size).
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145 2.6. RNA extraction and real-time RT-PCR

146 Total RNA was isolated from C2C12 using TRIzol reagent and treated with DNase I
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147 according to the manufacturers instructions. The cDNA was reverse transcribed and
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148 amplified by quantitative real-time PCR, performed with an ABI 7900 PCR system (ABI
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149 Biotechnology, MD, USA). Real-time PCR was performed duplicate for each cDNA sample,

150 using SYBR Green I as PCR core reagents in a final volume of 20 L. PCR conditions were

151 as follows: incubation for 10 min at 95C, followed by 40 cycles of denaturation for 15 s at

152 95C, annealing and extension for 60 s at (56-64C). Target genes mRNA expression levels

153 in arbitrary unit were acquired from the value of the threshold cycle (Ct) of the real-time PCR

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154 as related to that of -actin using the comparative Ct method through the formula 2-Ct

155 (Ct = (Ct gene of interest - Ct -actin)treat - (Ct gene of interest - Ct -actin)untreat). -actin house-keeping

156 gene was used as an internal control to normalize the expression of target genes. Primers for

157 the selected genes (Table 1) were designed, using Oligo 6.0 software program.

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158 2.7. Western blotting analysis

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159 The cells were rinsed twice using PBS, harvested, pelleted by centrifugation and lysed in

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160 RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50

161 mM Tris-HCl at PH 7.4), plus a protease inhibitor cocktail and phosphatase inhibitors.

162
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Soluble proteins were subjected to SDS-PAGE, and transferred to PVDF membranes,
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163 blocked with 5% nonfat milk in TBS-with 0.05% Tween-20 for 1 h and incubated overnight
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164 with primary antibodies followed by horseradish peroxidase-linked secondary antibodies. The

165 protein bands were visualized using a chemiluminescent reagent. The density of the protein
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166 bands was determined using the Alpha Imager 2200 software (Alpha Innotech Corporation)
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167 and normalized the data with inner control.

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168 2.8. Statistical analysis

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169 All results are expressed as mean SEM. Statistical analyses were carried out using one-way
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170 ANOVA, SAS 8.2. The differences among group means were compared using the Duncan

171 multiple comparison test. Probability values < 0.05 were considered as statistically significant.

172 All the experiments were repeated independently for three times.

173 3. Results

174 3.1. Different BCAAs ratios regulated cell viability of the myocytes

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175 The results indicated that BCAAs ratios enhanced the cell viability in a dose- and

176 time-dependent manner, and the ratio of 1:0.25:0.25 at 48 h showed the significant promotion

177 effect (P < 0.05) (Fig. 1A and 1B). Based on these results, we employed the BCAAs ratios of

178 1:0.25:0.25 and 1:1:1 and incubated the myocytes for 48 h (proliferation stage) in the

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179 following experiments.

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180 3.2. Different BCAAs ratios regulated cell cycle and differentiation of the myocytes into

181 myotubes

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182 To evaluate whether different ratios of the three BCAAs modulate myocyte cycle, C2C12

183
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cells were treated without (blank) or with the Leu:Ile:Val ratios of 1:0.25:0.25 and 1:1:1 for
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184 48 h and subjected to flow cytometry analysis (Fig. 2A). The results showed that the ratio of
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185 1:0.25:0.25 markedly decreased the myocytes in G0/G1 phase about 20% and 10% compared

186 with the blank and 1:1:1 ratio group (P < 0.05), respectively. Conversely, the BCAAs ratio of
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187 1:0.25:0.25 significantly increased the myocytes in S phase about 20% and 10% compared to
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188 the blank and 1:1:1 group (P < 0.05), respectively. No significant difference was observed in
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189 the G2/M phase among the three treatments (Fig. 2B). Moreover, the BCAAs ratio of

190 1:0.25:0.25 promoted the differentiation of C2C12 myocytes into myotubes (Fig. 3). It was
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191 consistent with the mRNA abundance of myogenin.

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192 3.3. Effects of BCAAs ratios on the free amino acid profile of medium and myocytes

193 Free amino acids were determined in media and C2C12 cells in proliferation and

194 differentiation stages, as detailed in Table 2, respectively. In both stages, the ratio of

195 1:0.25:0.25 and 1:1:1 significantly increased (P < 0.05) BCAAs concentrations in C2C12

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196 cells and media, compared with the blank group. Of note, the ratio of 1:0.25:0.25 lead to a

197 marked decrease of Val concentrations in both cells and media (P < 0.05), compared with the

198 ratio of 1:1:1.

199 3.4. Different BCAAs ratios regulated the mRNA expression levels of the selected genes

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200 during proliferation and differentiation

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201 As shown in Figs. 4 and 5, the candidate genes related to protein metabolism (A), myogenic

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202 factors (B), amino acid transporters (C), and energy metabolism (D) were detected. During

203 proliferation phase, the BCAAs ratio of 1:0.25:0.25 significantly increased (P < 0.05) the

204
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mRNA expression levels of mTOR (Fig. 4A), interleukin-15 (IL-15), myogenin (Fig. 4B),
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205 and uncoupling protein 3 (UCP3, Fig. 4D) compared with the other two groups, and
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206 significantly decreased (P < 0.05) the mRNA expression level of ubiquitin ligase including

207 muscle-specific RING finger 1 (MuRF1), muscle atrophy F-box (MAFbx, Fig. 4A), amino
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208 acid transporter including L-type amino acid transporter 1 (LAT1) and system ASC amino
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209 acid transporters 2 (ASCT2, Fig. 4C), and silent information regulator transcript 1 (Sirt1, Fig.
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210 4D) compared with blank and/or 1:1:1 ratio group. There was no difference in the mRNA

211 abundance of S6K1, MyoD, L-type amino acid transporter 4 (LAT4), AMP-activated protein
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212 kinase (AMPK), and peroxisome proliferator-activated receptor coactivator 1-

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213 (PGC-1) among the three groups (P > 0.05; Fig. 4). During differentiation phase, the BCAA

214 ratio of 1:0.25:0.25 led to a marked increase (P < 0.05) in mRNA expression levels of mTOR,

215 S6K1, IL-15, myoD, myogenin, LAT4 and UCP3, and a significant decrease in the value of

216 MAFbx, LAT1, and ASCT2 relative to blank and/or 1:1:1 ratio group. There was no

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217 difference in the mRNA abundance of MuRF1, and energy metabolism-related genes

218 (AMPK, Sirt1, and PGC-1) among all the groups (P > 0.05; Fig. 5).

219 3.5. Different BCAAs ratios regulated the expression levels of the protein metabolism

220 related regulators during proliferation and differentiation

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221 As shown in Figs. 6 and 7, the selected proteins were related to protein metabolism and

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222 amino acid transporters. During proliferation (Fig. 6), the 1:0.25:0.25 ratio increased the

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223 protein expression levels of p-mTOR, p-raptor, p-S6K1, and decrease the value of MuRF1,

224 MAFbx, LAT4, and ASCT2 in C2C12 cells relative to blank and/or 1:1:1 ratio group (P <

225
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0.05). Furthermore, no difference was observed in LAT1 expression among all groups (P >
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226 0.05). During differentiation (Fig. 7), a similar up-regulation of p-mTOR, p-raptor, p-S6K1
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227 and down-regulation of MuRF1 and MAFbx protein expression levels were noted after

228 treatment with the BCAAs ratio of 1:0.25:0.25 (P < 0.05), which were almost consistent with
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229 the trend of the mRNA expression levels described above. However, the treatment with the
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230 BCAAs ratio of 1:0.25:0.25 significantly reduced the protein expression level of LAT1, but
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231 increased the value of LAT4 and ASCT2, and treatment with the BCAAs ratio of 1:1:1 also

232 promoted the value of LAT4 compared with the blank (P < 0.05).
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233 4. Discussion

234 In this study, we found that the BCAAs ratio (Leu: Ile: Val) of 1:0.25:0.25 significantly

235 increased cell viability at 48 h and promoted C2C12 myocytes from G0/G1 phase to S phase,

236 comparing to other BCAAs ratio groups. The cell cycle is a highly regulated process

237 consisting of G1/G0, S, and G2/M phases [35]. Cell cycle slows during the G0/G1 phase, and

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238 progression to S phase does not occur until DNA is correctly encoded [36]. Our findings

239 suggested that different BCAAs ratios could regulate cell proliferation through cell viability

240 and cell cycle, and the BCAAs ratio of 1:0.25:0.25 exhibited the significant promotion effect.

241 Moreover, we noted that the BCAAs ratio of 1:0.25:0.25 markedly enhanced the

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242 differentiation of C2C12 myocytes into myotubes, consistent with the mRNA abundance of

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243 the specific gene myogenin.

244 The expression levels of amino acid transporters are rapidly and transiently up-regulated

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245 following an increase in essential amino acids availability in skeletal muscle, and the raised

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246 amino acid transporter expression may contribute to enhanced amino acid sensitivity [37, 38].
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247 System L transporters include LAT1 (also known as SLC7A5) and LAT4 (also known as

248 SLC43A2), which are critical because of their role in muscle growth [39, 40]. Amino acid
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249 transporter ASCT2 (also known as SLC1A5) is a bidirectional glutamine transporter, which
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250 mediates the simultaneous efflux of glutamine out of cells and transports Leu into the cells.
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251 Whereas, LAT1 forms a heterodimeric plasma membrane complex exchanging internal

252 glutamine and asparagine for external BCAAs, LAT4 is essential for the movement of
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253 BCAAs and NEAAs across the cell membrane of skeletal muscle cells [41, 42]. Accordingly,
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254 in our experiment, the protein abundance of the amino acid transporters LAT4 and ASCT2
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255 was increased during differentiation in the BCAAs-treated groups. However, the protein

256 abundance of LAT4 during proliferation and LAT1 during differentiation in the group of

257 1:0.25:0.25 were markedly reduced relative to other two groups. This was in line with one

258 previous study, which indicated that human cultured primary muscle in response to Leu did

259 not exert a change in expression levels of either LAT1 or LAT4 [3]. The results showed no

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260 consistent change in the mRNA abundance of ASCT2. Probably, BCAAs ratios could

261 improve the translation efficiency after transcription of the amino acid transporters.

262 Consistent with these data, we observed a significantly higher concentration of intracellular

263 BCAAs in the BCAAs treated groups, indicating the intracellular presence of available

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264 BCAAs is partialy modulated by a coordinated activity of AA transporters. Further study is

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265 required to know the exact mechanism that BCAAs ratios modulate amino acid transporters

266 using isotopic tracer method.

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267 The complex mTORC1 is a Ser/Thr kinase signaling complex with regulatory associated

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268 protein raptor as the core component. Here, the myocytes treated with the BCAAs ratio of
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269 1:0.25:0.25 during either proliferation or differentiation showed increased mRNA abundance

270 and phosphorylated protein levels of mTOR, raptor and S6K1. Using S6K1 and raptor
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271 phosphorylation as downstream effectors for mTORC1 activity, our results demonstrated that
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272 the BCAAs ratio of 1:0.25:0.25 led to a marked increase in activation of mTORC1 signal
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273 pathway. It is also suggested that different ratios of BCAAs modulate mTORC1 differently.

274 There are multiple data indicating that the myogenic regulatory factors (MRFs), MyoD and
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275 myogenin, are relevant to muscle cell development. MyoD acts early in myogenesis to
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276 determine myogenic fate, while myogenin acts later in the terminal differentiation of
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277 myocytes into myotubes. Its function is specific and cannot be replaced by other factors [43].

278 IL-15, as a novel anabolic myokine for skeletal muscle, could stimulate differentiated

279 myocytes and muscle fibers to accumulate increased amounts of proteins [44, 45]. In the

280 present study, we noted that the BCAAs ratio of 1:0.25:0.25 significantly up-regulated

281 myogenin and IL-15 mRNA expression levels in C2C12 myocytes compared to the other two

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282 BCAAs-ratio groups during both proliferation and differentiation, as well as for MyoD

283 mRNA expression level only during differentiation phase. Collectively, our results indicated

284 that the BCAAs ratio of 1:0.25:0.25 could promote myocyte proliferation and differentiation

285 through enhancement of the mTORC1 signal and myogenic factors involved in protein

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286 synthesis.

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287 Protein synthesis and degradation are tightly regulated and interconnected. They are affected

288 by the availability of nutrients and physical activity. Moreover, the proteolytic system

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289 produces alternative energy substrates for cells to maintain internal homeostasis [46]. In our

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290 experiments, during either proliferation or differentiation, MuRF1 and MAFbx mRNA and
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291 protein expression levels were significantly down-regulated in myocytes treated with the

292 BCAAs ratio of 1:0.25:0.25, comparing to the blank or the BCAAs ratio of 1:1:1 group.
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293 MuRF1 and MAFbx are markers for muscle atrophy and are viewed as master genes for
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294 muscle wasting. They are responsible for the increase in protein degradation through the
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295 ubiquitin-proteasomal pathway [46-48]. Our results demonstrated that the BCAAs ratio of

296 1:0.25:0.25 not only up-regulated the key factors involved in protein synthesis but also
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297 down-regulated the key factors related to protein degradation, resulting in C2C12 myocytes
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298 growth and development. Moreover, the BCAAs ratio of 1:1:1 markedly increased the
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299 mRNA and protein abundance of ubiquitin ligase (MuRF1 and MAFbx), indicating that

300 excessive Ile or Val would upset the balance of the BCAAs combination and increase protein

301 degradation of the myocytes.

302 Protein synthesis is another major energy-consuming process in the cell [6, 49]. AMPK,

303 PGC-1, Sirt1 are cellular energy sensors, and are phosphorylated (activated) in response to

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304 low cellular energy status [50, 51]. UCP3 is primarily expressed in skeletal muscle, plays key

305 regulatory roles in mitochondrial fatty acid oxidation [52], and is related to respiration in cell

306 mitochondria. Data from the study showed that there was no difference in mRNA expression

307 levels of AMPK, Sirt1, and PGC-1 among all groups during proliferation and

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308 differentiation. However, the value of UCP3 in the C2C12 myocytes and myotubes was

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309 significantly up-regulated by the BCAAs ratio of 1:0.25:0.25. It was demonstrated that the

310 BCAAs ratio of 1:0.25:0.25 could increase the energy utilization of the cells, presumably for

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311 protein synthesis of C2C12 myocytes and myotubes.

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312 5. Conclusion
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313 In summary, the optimal BCAAs ratio (Leu:Ile:Val) of 1:0.25:0.25 promoted C2C12
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314 myocytes proliferation and differentiation accompanied by activating the nutrient sensor

315 mTORC1 and up-regulating myogenic factors and UCP3 but down-regulating ubiquitin
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316 ligase (MuRF1 and MAFbx), leading to the growth and development of myocytes. The
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317 relationship between different BCAAs ratios and amino acid transporters needs further
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318 research. These results provide new insight into the modulatory function of BCAAs ratio

319 balance in skeletal muscle protein anabolism and catabolism. The concept was benefit for
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320 BCAAs application to improve human and animal nutrition.

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321 7. Acknowledgments

322 Yehui Duan and Liming Zeng equally contributed to this article. The authors have

323 declared that no competing interests exist. This study was jointly supported by National Basic

324 Research Program of China (2013CB127305, 2012CB124704), Youth Innovation Promotion

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325 Association CAS (2015), Nature Science Foundation of Hunan (S2014J504I), and Key

326 Projects in the National Science & Technology Pillar Program (2013BAD21B04).

327

328 References

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332 [3] Gran P, Cameron-Smith D. The actions of exogenous leucine on mTOR signalling and amino acid transporters
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441 Figure legends

442 Fig 1. Effects of different BCAAs ratios on the proliferation of C2C12 cells. C2C12 cells

443 were treated without (blank) or with different BCAAs ratios (Leu:Ile:Val = 1:1:1,

444 1:0.75:0.75, 1:0.5:0.5, and 1:0.25:0.25; 5 mM Leu) for 0.5, 1, 2, 4, 8, 24, and 48 h,

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445 respectively. The proliferation rate of the cells was expressed as OD450 at the indicated time.

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446 Values are means (n = 6), with their standard errors represented by vertical bars. Different

447 letters (a, b, c) are indicated significant differences (P < 0.05).

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448 Fig 2. Effects of different BCAAs ratios on the C2C12 cell cycle. (A) C2C12 cells were

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449 treated without (blank) or with different BCAAs ratios (Leu:Ile:Val = 1:0.25:0.25 and 1:1:1,
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450 5 mM Leu) for 48 h and analyzed by flow cytometry for the cell cycle. The cells in G1/G0, S,

451 or G2/M phase are presented as percentages. (B) Data were expressed as means SE (n = 6).
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452 Values with different letters (a, b, c) indicated significant differences (P < 0.05).
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453 Fig 3. Cellular morphology of C2C12 myocytes differentiated into myotubes. Cells were
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454 treated without (blank) or with different BCAAs ratio of (Leu:Ile:Val = 1:0.25:0.25 and
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455 1:1:1, 5 mM Leu) for 2 or 4 d. Myotubes were examined under a microscope (200

456 magnification). The BCAAs ratio of 1:0.25:0.25 induced an increased differentiation into
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457 myotubes. Scale bar = 500 m. The red arrows point to myotubes.
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458 Fig 4. Relative expression levels of selected protein metabolism- (A), myogenesis- (B),

459 amino acid transporter-(C), and energy metabolism-(D) related genes during proliferation.

460 C2C12 cells were treated without (blank) or with different BCAAs ratios (Leu:Ile:Val =

461 1:0.25:0.25 and 1:1:1, 5 mM Leu). Real-time PCR method was employed. Values are means

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462 (n = 6), with their standard errors represented by vertical bars. Different letters (a, b, c)

463 indicated significant differences (P < 0.05).

464 Fig 5. Relative expression levels of selected protein metabolism- (A), myogenesis- (B),

465 amino acid transporters- (C), and energy metabolism-(D) related genes during differentiation.

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466 C2C12 cells were treated without (blank) or with different BCAAs ratio (Leu:Ile:Val =

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467 1:0.25:0.25 and 1:1:1, 5 mM Leu). Real-time PCR method was employed. Values are means

468 (n = 6), with their standard errors represented by vertical bars. Different letters (a, b, c)

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469 indicated significant differences (P < 0.05).

470
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Fig 6. Relative expression levels of selected protein metabolism and amino acid transporters
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471 related genes during proliferation. C2C12 cells were treated without (blank) or with different
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472 BCAAs ratio (Leu:Ile:Val = 1:0.25:0.25 and 1:1:1, 5 mM Leu). Western blotting method was

473 employed. Values are means (n = 6), with their standard errors represented by vertical bars.
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474 Different letters (a, b, c) indicated significant differences (P < 0.05).

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475 Fig 7. Relative expression levels of selected protein metabolism- and amino acid transporter-
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476 related genes during differentiation. C2C12 cells were treated without (blank) or with

477 different BCAAs ratio (Leu:Ile:Val = 1:0.25:0.25 and 1:1:1, 5 mM Leu). Western blotting
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478 method was employed. Values are means (n = 6), with their standard errors represented by

479 vertical bars. Different letters (a, b, c) indicated significant differences (P < 0.05).

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480 Table 1. Primers used for real-time PCR

Genes Primers Sequences (5-3) Size (bp)

mTOR Forward GCGGCCTGGAAATGCGGAAGTGG 133
Reverse AAAGCCCCAAGGAGCCCCAACA
S6K1 Forward GGCGGGACGGCTTTTACCT 76
Reverse ACCTTTCCATAGCCCCCTTTACC
Myogenin Forward ACCTTCCTGTCCACCTTCAG 230

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Reverse CAGACTTCCTCTTACACACC
MyoD Forward CGCCTGAGCAAAGTGAA 383
Reverse GCGGTGTCGTAGCCATT

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IL-15 Forward TCCATCTCGTGCTACTTGTG 118
Reverse CAGCCAGATTCTGCTACAT
LAT1 Forward GCGGGAGAAGATGTTGGAG 155

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Reverse GAAGATGCCAGAGCCGATGA
LAT4 Forward CCTCCTTCATTGGCATCCTA 165
Reverse CACTGTGGTCACCTGCTTGT

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ASCT2 Forward CTTGACCGCTTTCGCCTTCC 362
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Reverse CGGGCACAGGGGTCTACAAC
MuRF1 Forward CATTGTGTGACTGGCGATTG 150
Reverse TCTGGCTGGCCTGTAACTCT
MAFbx Forward GCAGAGAGTCGGCAAGTC 374
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Reverse CAGGTCGGTGATCGTGAG
AMPK Forward TGAAGATCGGCCACTACATCCT 311
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Reverse CTTGCCCACCTTCACTTTCC
Sirt1 Forward ACCTCCCAGACCCTCAAG 114
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Reverse TTCCTTCCTTATCTGACAAAGC
PGC-1 Forward AAACCACACCCACAGGATCAG 265
Reverse TCTTCGCTTTATTGCTCCATGA
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UCP3 Forward ACTGCTGCTGACACGAGA 260

Reverse GACAACACTGTGGGCTGA
-actin Forward GACCTCTATGCCAACACA 216
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Reverse TCAGTAACAGTCCGCCTA
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486 Table 2. Effects of Leu:Ile:Val ratio on the free amino acid profile of C2C12 cells and

487 media in proliferation and differentiation stages

Cells Media
Amino acids
( uM/L ) Blank 1:0.25:0.25 1:1:1 Blank 1:0.25:0.25 1:1:1
Proliferation stage
L-leucine 1.340.17b 20.044.45a 17.112.27a 2.8926.50b 33.982.86a 88.623.56a

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c b a b a
L-isoleucine 0.910.12 6.491.56 17.542.15 1.4126.44 36.923.73 92.608.65a
L-valine 1.350.12c 9.491.69b 25.783.01a 1.5645.98c 70.1751.49b 202.103.03a
Differentiation stage

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L-leucine 1.420.29b 29.093.40a 30.853.00a 2.431.30b 79.106.23a 87.332.25a
L-isoleucine 0.900.21c 9.961.38b 32.432.47a 1.580.93b 86.334.48a 93.557.92a
c b a c b
L-valine 1.130.28 12.501.53 42.382.79 3.811.19 163.052.46 188.097.22a

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Highlights

Optimal BCAAs ratio promoted myocytes proliferation and differentiation.

Optimal BCAAs ratio activated the nutrient sensor mTORC1 pathway of myocytes.

The expression level of myogenic factors of myocytes was regulated by different

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BCAAs ratios.

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