Free Amino Acid Profile and Expression of Genes Implicated in Protein

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Free amino acid profile and expression of genes related to protein metabolism in skeletal muscle of growing pigs fed lowprotein diets supplemented with branched-chain amino acids Yehui Duan, Qiuping Guo, Chaoyue Wen, Wenlong Wang, Yinghui Li, Bie Tan, Fengna Li, and Yulong Yin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03966 • Publication Date (Web): 22 Nov 2016 Downloaded from http://pubs.acs.org on November 28, 2016

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Running title: Branched chain amino acid and protein metabolism

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Free amino acid profile and expression of genes implicated in protein metabolism

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in skeletal muscle of growing pigs fed with low-protein diets supplemented with

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branched-chain amino acids

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Yehui Duan ,‡, Qiuping Guo ,‡, Chaoyue Wen§, Wenlong Wang§, Yinghui Li ,‡, Bie

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Tan , Fengna Li ,$* and Yulong Yin ,§*

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†

†

†

†

†

†

†

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

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Subtropical Agriculture, Chinese Academy of Sciences; Hunan Provincial

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Engineering Research Center for Healthy Livestock and Poultry Production;

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Scientific Observing and Experimental Station of Animal Nutrition and Feed Science

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in South-Central, Ministry of Agriculture, Changsha 410125, China;

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‡

University of Chinese Academy of Sciences, Beijing 100039, China;

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§

Laboratory of Animal Nutrition and Human Health, School of Biology, Hunan

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Normal University, Changsha Hunan 410018, China;

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$

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Collaborative Innovation Center for Utilization of Botanical Functional Ingredients,

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Changsha, Hunan, China

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* Corresponding author: Fengna Li and Yulong Yin; E-mail: [email protected],

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[email protected]; Tel: (+86-731) 8461-9703; Fax: (+86-731) 8461-2685.

Hunan Co-Innovation Center of Animal Production Safety, CICAPS; Hunan

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ABSTRACT: Revealing the expression patterns of genes involved in protein

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metabolism as affected by diets would be useful for further clarifying the importance

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of the balance among the branched-chain amino acids (BCAAs), which include

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leucine (Leu), isoleucine (Ile), and valine (Val). Therefore, we used growing pigs to

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explore the effects of different dietary BCAA ratios on muscle protein metabolism.

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The Leu: Ile: Val ratio was 1:0.51:0.63 (20% crude protein, CP), 1:1:1 (17% CP),

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1:0.75:0.75 (17% CP), 1:0.51:0.63 (17% CP), and 1:0.25:0.25 (17% CP), respectively.

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Results showed that compared with the control group, low-protein diets with the

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BCAA ratio ranging from 1:0.75:0.75 to 1:0.25:0.25 elevated muscle free amino acid

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(AA) concentrations and AA transporters expression, and significantly activated the

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mammalian target of rapamycin complex 1 pathway, and decreased serum urea

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nitrogen content and the mRNA expression of genes related to muscle protein

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degradation (P < 0.05). In conclusion, these results indicated that maintaining the

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dietary Leu: Ile: Val ratio within 1:0.25:0.25-1:0.75:0.75 in low-protein diets (17% CP)

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would facilitate the absorption and utilization of free AA, and resulted in improved

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protein metabolism and muscle growth.

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KEYWORDS：branched-chain amino acid ratio, free amino acid, amino acid

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transceptor, mTORC1 pathway, low-protein diets, growing pig

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INTRODUCTION

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Post-weaning pigs often experience intestinal dysfunction, leading to diarrhea and

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mortality.

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levels, which can improve gastrointestinal health and function after weaning and thus

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contribute to overall health and growth of pigs.

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reduced-protein diets are often at the expense of impaired muscle protein synthesis

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and growth, partly by suppressing the mammalian target of rapamycin (mTOR)

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signaling.

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metabolically adapt to chronic protein insufficiency.

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acids (AAs) exert a key role in growth response by modulating muscle protein

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turnover.

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the crude protein (CP) level in pig diets, which maintains sufficient essential AA

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supply and muscle growth. 9, 15

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Branched-chain amino acids (BCAAs), which consist of leucine (Leu), isoleucine

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(Ile), and valine (Val), have anabolic properties apart from the function as components

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of proteins in animal metabolism. 16 It is well-established that supplementation of Leu

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alone to a low-protein diet is sufficient to promote protein synthesis via stimulating

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mTOR signaling pathway in (weaned and finishing) pigs and adults, suggesting that

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the effect of Leu to improve anabolism under dietary protein restriction is not lost

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with age. 17-20 Notably, Val and Ile fail to induce the activation of the mTOR pathway

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and to promote protein synthesis.

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alone to a low-protein diet did not improve growth performance in either young

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An effective strategy to solve this problem is to reduce dietary protein

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5-8

However, the positive effects of

Because organisms need reduce the protein synthesis rate to 12

Intriguingly, functional amino

The use of functional AAs in animal nutrition allows the reduction of

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In support of this view, supplementation with Ile


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piglets or growing pigs.

However, apart from the first four limiting AAs L-lysine

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(Lys), L-methionine (Met), L-threonine (Thr), and L-tryptophan (Trp), Val and/or Ile

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may become limiting when the dietary protein content is reduced by more than 4%

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units, and dietary supplementation with these AA enables pigs fed protein-restricted

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diets to maintain growth performance.

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need to be supplied in reduced-protein diets with supplemental Leu.

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A diet deficient in Val with excessive supply of Leu leads to a rapid decline in feed

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intake in pigs, which may further impair muscle protein synthesis and growth. 24-27 In

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these studies, dietary BCAA imbalance occurs, because pigs refused to injest a BCAA

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unbalanced diet within 2 d of exposure to the diet.

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enzymes in their catabolic pathways, the supply of one BCAA may influence the

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requirement of the other BCAA. 26 As revealed in rats, supplementation of 5% Leu to

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a reduced-protein diet decreased the plasma Val concentration and increased Val

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oxidation within 1 h after ingestion.

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protein-restricted diets is of tremendous nutritional importance.

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Although recent years have witnessed growing interest in the use of a mixture of

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crystalline BCAAs in pigs fed low-protein diets. 10, 29 Research to estimate the optimal

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ratio of all the three BCAA for growing pigs is sparse, particularly in low protein diets.

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Furthermore, most experiments on dietary BCAA supplementation in pigs have

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focused on performance parameters. 23, 26, 29, 30 So data on free AA concentrations, AA

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transporters, and expression of muscle protein metabolism-related genes in skeletal

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muscle are sparse or absent. This study is part of a series of experiments to investigate

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9, 11, 15, 23

In this context, all the three BCAA

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The BCAAs share the same

Therefore, balancing the three BCAA ratio in



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the optimal ratio of BCAAs in growing pigs (10 to 30 kg body weight). We previously

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reported that low-protein diets supplemented with optimal Leu: Ile: Val ratio (from

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1:0.75:0.75 to 1:0.25:0.25) contribute to improving the growth performance of the

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growing pigs.

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with the reduced CP diets (Leu: Ile: Val = 1:0.75:0.75, 1:0.51:0.63, 1:0.25:0.25) may

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at least partly result from an augment in protein synthesis and a decline in protein

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degradation of skeletal muscle. Therefore, the aim of the current study was to extend

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our previous studies and to study: (1) the effect of different Leu: Ile: Val ratios in

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protein-restricted diets on the composition of free AAs and selected AA transporters in

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selected muscles, and (2) whether supplementing balanced BCAAs to a

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protein-restricted diet for growing pigs could be effective in maintaining adequate

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protein synthesis and inhibiting protein degradation, and to examine the underlying

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mechanism. This research will offer an intriguing new approach to understand the

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application of low-protein diets as a nutrition strategy for swine or other young

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mammals where normal meal feeding is not possible or protein intake is restricted.

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We hypothesized that the improved growth performance of pigs fed

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MATERIALS AND METHODS

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Animals and Experimental Diets. The experiment was performed according to the

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Chinese guidelines for animal welfare and experimental protocols, and approved by

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the committee on animal care of the Institute of Subtropical Agriculture, the Chinese

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Academy of Sciences. Forty Large White × Landrace pigs (9.85 ± 0.35 kg) were


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randomly allotted into 5 dietary treatments. Each dietary treatment contained eight

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replicates (n=8). Pigs were raised individually in cages. Diets were isoenergetic and

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met the nutritional needs for growing pigs according to the National Research Council

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(NRC, 2012 32) (Table S1). The Leu: Ile: Val ratio of the five dietary treatments were

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as following: diet A = 1:0.51:0.63 (20% CP, the positive control group), diet B = 1:1:1

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(17% CP), diet C = 1:0.75:0.75 (17% CP), diet D = 1:0.51:0.63 (17% CP), and diet E

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= 1:0.25:0.25 (17% CP). The dietary BCAA ratio and CP level of the control group fit

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well with the recommendation of the 2012 NRC 32. The total BCAA amount of diets

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was of the same value in all treatments. Pigs had ad libitum access to diets and clean

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drinking-water. The experiment lasted for 45 d.

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Sample Collection. Feed intake and final body-weight gain were recorded on a daily

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and weekly basis, respectively, to calculate the feed: gain ratio as previously described.

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slaughtered by electrically stunning (250V, 0.5 A, for 5~6s) and exsanguinating.

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is noteworthy that before slaughter, blood samples were collected from all pigs for the

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determination of serum metabolites and hormone levels. Serum was separated and

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stored as previously described.

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psoas major muscle (PM), biceps femoris muscle (BM), and longissimus dorsi muscle

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(LM) were immediately and rapidly excised from the left side of the carcass. The

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samples were then either stored at -20°C or placed in liquid N2 and then stored at

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-80°C, respectively, until further analysis.

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Measurement of Serum Metabolites and Hormone Levels. The concentration of

When the feeding test ended, all the pigs were fasted overnight and then

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It

After slaughter, skeletal muscle samples including



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blood urea nitrogen (BUN), albumin (ALB), total protein (TP), and creatinine (CREA)

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were determined using the Biochemical Analytical Instrument (Beckman CX4) and

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commercial kits (Sino-German Beijing Leadman Biotech Ltd., Beijing, China). The

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concentrations of interleukin-15 (IL-15) and insulin-like growth factor 1 (IGF-1) were

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analyzed with the corresponding commercial ELISA kits (CUSABIO, Wuhan, China)

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following the recommended procedures.

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Free AA Profile. Free AA profile was determined in the LM, BM, and PM of growing

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pigs as described previously. 33

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Quantitative RT-PCR Analysis. The reverse transcription and real-time quantitative

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PCR were conducted as previously described.

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from PM, BM, and LM of growing pigs using the TRIzol reagent (Invitrogen-Life

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Technologies, CA, USA). The primer sequences for the target genes are shown in

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Table 1. The amplification of the housekeeping gene β-actin in each sample was used

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to normalize the mRNA expression levels of target genes. The relative quantification

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of gene amplification by RT-PCR was performed using the value of the threshold

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cycle (Ct). Relative expression of target genes were determined by the 2-△△Ct method. 31

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Western Blotting Analysis. Relative protein levels for mTOR, regulatory associated

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protein of mTOR (Raptor), and p70S6 kinase (S6K1), obtained from LM, BM, and

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PM, were determined by the western blotting technique as we described previously. 34

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The primary antibodies used in the present study were as follows: anti-phosphor

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(p)-mTOR and total (t)-mTOR, anti-p-S6K1 and t-S6K1, anti-p-Raptor and t-Raptor,

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Briefly, total RNA was isolated


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and anti-β-actin. P-/t-mTOR and p-/t-S6K1 were purchased from Cell Signaling

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Technology (Danvers, MA), while p-/t-Raptor and β-actin were from Santa Cruz

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Biotechnology. The bands of the protein were visualized using a chemiluminescent

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reagent (Pierce, Rockford, USA) with a ChemiDoc XRS system (Bio-Rad,

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Philadelphia, PA, USA). The resultant signals were quantified using Alpha Imager

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2200 software (Alpha Innotech Corporation, CA, USA) and the data were normalized

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with the inner control.

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Statistical Analysis. Data obtained from the present study were analyzed by one-way

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ANOVA using the SAS 8.2 software package, followed by a Duncan’s multiple range

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test to determine treatment effects. The results were regarded to achieve statistical

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significance at P < 0.05.

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RESULTS

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Serum Parameters. As presented in Table 2, no differences were detected in the

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concentrations of ALB, TP, CREA, and IGF-1 among the treatments (P > 0.05). The

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BUN concentration in the diet B group was the same as that in the control group,

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while BUN concentrations in other 17% CP groups tended to be lower, with the

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greatest decrease observed in the diet C and D groups (P = 0.09). Moreover, relative

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to the positive control group, 17% CP diets displayed increased IL-15 concentrations,

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with the greatest increase observed in the diet E group (P < 0.05).

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Muscle Free AA Profile. The results regarding the free AA profile of the LM of



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growing pigs fed diets supplemented with different BCAA ratios are revealed in Table

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3. The concentrations of most AA in the LM were strongly influenced by the dietary

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BCAA ratio. Specifically, relative to the positive control, the diets B to D groups

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decreased the concentrations of L-histidine (His), L-arginine (Arg), L-glutamic acid

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(Glu), and L-tyrosine (Tyr) (P < 0.05), which were restored in the diet E group to the

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level as the control. Meanwhile, the concentrations of Thr, Val, and L-serine (Ser) in

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the dies B, C, and D groups were similar to those in the control, which significantly

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increased in the diet E group (P < 0.05). Except for the above-mentioned AAs, other

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AAs, essential AA (EAA), and nonessential AA (NEAA) remained unaffected by the

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diet treatments (P > 0.05).

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As depicted in Table 4, in BM, compared with the positive control group, most AA

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levels (especially for Leu, Lys, Met, Thr, Val, Glu, Ser, and Tyr) increased with the

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decreasing of the dietary Leu: Ile: Val ratio in 17% CP groups, with the greatest

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increase noted in the diet E group (P < 0.05). Similarly, the concentrations of EAA (P

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= 0.07) and NEAA (P = 0.10) tended to augment from diet A to E. Diet treatments

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had no effect on other AAs.

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As shown in Table 5, diet treatments influenced most of EAA in PM. Specifically, the

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concentrations of most EAA levels (Leu, Lys, Thr, Val) increased from diet A to E, as

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was the case to the total EAA levels (P < 0.05). However, no change in response to

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diet treatments was detected concerning the concentrations of all the NEAA.

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Muscle AA Transceptors Gene Expression. The mRNA levels of L-type amino acid


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transporter 1 (LAT1), the sodium-coupled neutral amino acid transporter 2 (SNAT2),

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and solute carrier family 1 member 5 (SLC1A5) were measured in LM, BM, and PM

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(Fig.1). In LM, the mRNA abundance of all AA transceptors genes studied here in

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diets C, D, and E groups was the same as or higher than (P < 0.05) that of the control

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group, with the greatest increase observed in diet C and E groups. Of note, diet A

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group significantly decreased the SNAT2 mRNA level compared to the control (P
0.05). In PM, the mRNA

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expression of IL-15 in the diet E group (1:0.25:0.25, 17% CP) was markedly

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up-regulated relative to the positive control group (P < 0.05), and the difference was

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not statistically significant between the positive control group and other 17% CP

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groups. The mRNA expression of myostatin in PM was similar to that in BM.

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Moreover, the mRNA expression of MyoD and MyoG in diets B and C groups were

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significantly down-regulated relative to the positive control group (P < 0.05), whereas

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the difference was not statistically significant between the positive control group and

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other 17% CP groups (P > 0.05).

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Muscle Proteolysis-Related Genes Expression. The mRNA abundance of the

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muscle atrophy F-box (MAFbx), muscle ring finger 1 (MuRF1), and forkhead

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transcription factor 1 (FOXO1) were measured in LM, BM, and PM (Fig.3). In LM,

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the diets C and D groups exhibited similar mRNA expression of MuRF1 and MAFbx

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relative to the control group, while the diets B and E groups significantly

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down-regulated the mRNA abundance of MuRF1 and MAFbx (P < 0.05). No

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significant difference in the FOXO1 mRNA expression was detected between the


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positive control group and the 17% CP groups except for the diet C group

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(1:0.75:0.75) (P > 0.05). In BM, compared with the positive control group, the

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mRNA expression of MuRF1 in the 17% CP groups was significantly up-regulated (P

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< 0.05), the mRNA expression of MAFbx in diet B group was significantly

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up-regulated (P < 0.05), but no differences were detected between other 17% CP diets

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and the control. Diets treatment did not affect the mRNA expression of FOXO1 in

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BM (P > 0.05). In PM, compared with the control group, the mRNA expression of

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MuRF1 and FOXO1 in diet C group (1:0.51:0.63, 17% CP) was greatly

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down-regulated (P < 0.05).

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Abundance of mTOR Pathway Proteins. As presented in Fig.4, the protein

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abundances of p-mTOR, p-Raptor, and p-S6K1 in LM decreased in diet B, C, and D

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groups (1:1:1, 1:0.75:0.75, 1:0.51:0.63, 17% CP) relative to the positive control (P

Free Amino Acid Profile and Expression of Genes Implicated in Protein

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