Leucine Promotes the Growth of Fetal Pigs by Increasing Protein

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Bioactive Constituents, Metabolites, and Functions

Leucine promotes the growth of fetal pigs by increasing protein synthesis through the mTOR signaling pathway in longissimus dorsi muscle at late gestation Chaoxian Wang, Fang Chen, Wenfei Zhang, Shihai Zhang, Kui Shi, Hanqing Song, Yijiang Wang, Sun Woo Kim, and Wutai Guan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00330 • Publication Date (Web): 27 Mar 2018 Downloaded from http://pubs.acs.org on March 29, 2018

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Leucine promotes the growth of fetal pigs by increasing protein synthesis through the

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mTOR signaling pathway in longissimus dorsi muscle at late gestation

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Chao-xian Wang,†,# Fang Chen,†,# Wen-fei Zhang,† Shi-hai Zhang,† Kui Shi,† Han-qing Song,†

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Yi-jiang Wang,† Sung Woo Kim,§ and Wu-tai Guan†,*

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†

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§

College of Animal Science, South China Agricultural University, Guangzhou 510642, China Department of Animal Science, North Carolina State University, Raleigh, NC, United States

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Corresponding Authors：

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*

Telephone: 86-020-85284837. Fax: 86-020-85284837. E-mail: [email protected]

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Chao-xian Wang and Fang Chen are joint first authors.

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ORCID：

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Chao-xian Wang: 0000-0003-3802-0544

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ABSTRACT

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Leucine (Leu) plays an important role in protein synthesis and metabolism. The present

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study tested whether Leu supplementation in the diet for sows during late pregnancy could

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improve piglet birth weight, and also investigated the possible underlying mechanism. Two

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hundreds sows at day 70 of pregnancy were selected and assigned to four groups fed with

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following four diets until farrowing respectively: corn and soybean meal-based diet group

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(CON), CON + 0.40% Leu, CON + 0.80% Leu and CON + 1.20% Leu. We found that

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supplementing with 0.80% Leu significantly increased mean piglet birth weight (P < 0.05).

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Supplementation with 0.40%, 0.80% and 1.20% Leu increased the plasma concentration of

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Leu, while decreased the plasma concentrations of valine (Val) and isoleucine (Ile) in both

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farrowing sows and newborn piglets (P < 0.05). The protein expressions of amino acid

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transporters (including LAT1, SNAT1, SNAT2, 4F2hc and rBAT) in duodenum, jejunum,

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ileum, longissimus dorsi muscle of newborn piglets, and placenta of sows showed difference

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among CON group and Leu supplemented groups. Expressions of p-mTOR, p-4E-BP1, and

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p-S6K1 in longissimus dorsi muscle were also enhanced in each of the supplemental Leu

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groups compared to CON (P < 0.05). Collectively, these results indicated that 0.40-0.80%

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Leu supplementation during late gestation enhanced birth weight of fetal pigs by increasing

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protein synthesis through modulation of plasma amino acids profile, amino acid transporters

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expression and mTOR signaling pathway.

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Key words: fetal pigs, growth performance, late gestation, leucine, mTOR

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INTRODUCTION

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Intrauterine growth retardation and the subsequent high post-natal morbidity and

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mortality are important reasons for small numbers of weaned pigs per litter per year, which

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presents a major challenge to the pig industry and increases the cost of animal production. 1-4

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Tissue accretion in fetal pigs is not a uniform process. Typically, 60% of the total body tissue

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of neonatal pigs, of which more than 60% is protein, is deposited within the last 40 days of

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gestation in fetal pigs.5,6 Furthermore, the gain in protein mass is primarily in skeletal muscle

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compared to other tissues in the body.7 High lean meat accretion and growth rates of pigs are

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closely associated with the skeletal muscle content and protein deposition in skeletal muscle.

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Therefore, strategies to promote the deposition of protein in fetal pigs during late gestation by

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optimizing sow nutrition are critically important for improving the birth weight of piglets,

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neonatal health and survival, and piglet growth during the early postnatal period.8

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In recent years, a large number of studies have shown that Leu is an important inducer of

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skeletal muscle protein synthesis as well as an inhibitor of the degradation of skeletal muscle

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proteins9-13 and these processes are largely mediated by mammalian target of rapamycin

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(mTOR) complex 1 (mTORC1), which is a key regulator of cell growth, protein and lipid

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synthesis, and autophagy et al.14-16 Both of p70-S6 kinase 1 (S6K1) and eukaryotic initiation

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factor (eIF) 4E binding protein 1 (4E-BP1) are two downstream targets of mTORC1.17-19

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mTOR phosphorylates S6K1, an activator of S6, and this protein has been reported to

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promote mRNA translation of proteins involved in the regulation of translation.20 In addition,

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Leu positively regulates mTOR signaling by promoting 4E-BP1 phosphorylation, inhibiting

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4EBP1 × eIF4E complex formation, and increasing the formation of the active eIF4E × eIF4G

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complex, which, in association with eIF4A, mediates the binding of the mRNA to the 43S

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ribosomal complex and promotes the translation of all mRNAs.21-23

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Dietary amino acids are transported by the corresponding amino acid carriers in intestine

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and other tissues into the cell before been utilized by the body.24-29 It has been reported that

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branched-chain amino acids (BCAA, Leu, Ile, and Val) share common transport systems for 3


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absorption.30 Specifically, major transporters of BCAA (system B0) are expressed along the

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entire small intestine.31 A number of Leu transporters have been identified on the mammalian

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small intestine that is responsible for the intestinal absorption of Leu. 32 The cell uses a

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combination of amino acid transporters to selectively increase intracellular Leu levels. The

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system L (LAT1) and system A (SNAT2) type transporters are amino acid exchangers that

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function primarily to import the BCAA in exchange for other intracellular amino acids. While

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due to low capacity to metabolize Leu in the liver, Leu concentrations in the blood reflect its

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availability in the feed, thus dietary Leu may directly affect protein metabolism.33-37

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Given that rapid growth and development of piglets requires enhanced protein deposition

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and the important role of Leu in protein synthesis, the objective of this study was to determine

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whether Leu could affect the fetal development during late gestation and further to investigate

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how amino acid transporters and mTOR signaling pathway were regulated by supplemental

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Leu to reveal its possible underlying mechanism.

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MATERIALS AND METHODS

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Animals and groups

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The protocols used in this experiment were approved by the Institutional Animal Care

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and Use Committee at South China Agricultural University. Healthy sows (Landrace × Large

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White, n = 200) at day 70 of gestation were selected and randomly allotted to four groups (n =

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50 sows/group) considering parity (4-5) and weight (average = 260 kg). The dietary

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treatments included a corn and soybean meal-based diet group (CON) and groups

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supplemented with 0.40%, 0.80% and 1.20% L-Leu separately (purity ≥ 98.5%;

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Jizhou Huayang Chemical Co. Ltd, Jizhou City, Hebei Province, China). The basal diet

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contained 14.99% crude protein, which exceeded the nutrient requirements as recommended

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by NRC (2012). Total contents of Leu in the diets supplemented with 0.00%, 0.40%, 0.80%

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and 1.20% Leu were 1.30%, 1.70%, 2.10% and 2.50%, respectively (Tables 1 and 2). Sows

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were fed the experimental diets from day 70 of gestation until farrowing.

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Housing, feeding and management 4



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From day 70 to 106 of gestation, sows were housed in individual stalls (2.05 m × 1.08 m)

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in an enclosed building. The crates were mounted over a solid concrete floor, and manure was

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removed manually each day. Each farrowing crate contained a stainless steel feeder and a

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nipple waterer for the sow. Equivalent amounts of feed (approximately 3.0 kg/d) was

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provided to all sows. During gestation, the sows were fed twice daily (7:00 and 14:00) and

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had free access to water through the drinking nipples. Approximately 1 week (day 107 of

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gestation) before expected farrowing, the sows were moved to the environmentally controlled

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farrowing house (temperature maintained at approximately 18-20℃) and placed in individual

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farrowing crates (0.60 m × 2.10 m) with creep space (0.45 m × 2.10 m) on both sides.

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Reproductive performance

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Within the first 12 hours after birth, litter size, number born alive, and number born

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healthy were recorded. Piglets were individually weighed at birth, and the percentage of

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piglets born healthy was calculated.

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Sample collection and processing

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Blood samples (20 mL) were collected in heparin tubes from the ear vein of 6 sows per

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treatment at day of farrowing 2 hours after feeding in the morning. Two newborn pigs (just

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born, no access to colostrum or other feeds) from each litter with a body weight (BW) closest

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to the average litter BW were selected for blood sampling by anterior vena cava puncture.

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Plasma was separated by centrifugation (3,000 × g for 10 minutes at 4 ℃) and frozen

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immediately at -80℃ for later analysis. Immediately after blood sampling, the two piglets

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were sacrificed by intramuscular injection of sodium pentobarbital (50 mg/kg BW). Heart,

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liver, thymus, pancreas and small intestine were separated and weighed to calculate the

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relative organ weight38. Samples (approximately 100 g) of piglet duodenum, jejunum, ileum,

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longissimus dorsi muscle and sow placenta39 were excised, weighed, flash frozen in liquid

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nitrogen, and stored at -80°C until analysis.

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Measurement of the plasma amino acids concentration

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Plasma amino acids concentrations of sows and piglets were determined by automatic 5


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amino acid analyzer (LP-8900, Hitachi, Tokyo, Japan). Plasma samples (400 μL) were

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deproteinized with 1,200 μL of 10% sulfosalicylic acid and the supernatant was assayed for

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amino acid composition. S-(2-amino-ethyl)-L-cysteine was used as an internal standard.

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Western blot analysis

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Primary antibodies for 4F2hc (sc-31251), LAT1 (sc-34551), rBAT (sc-32930), SNAT1

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(sc-67080), and SNAT2 (sc-67081) were purchased from Santa Cruz Biotechnology

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(Delaware Ave Santa Cruz, CA, USA). Primary antibodies for mTOR (#2972), p-mTOR

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(#5536, Ser2448), 4E-BP1 (#9644), p-4E-BP1 (#2855, Thr37/46), S6 (#2708), p-S6 (#9234,

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Ser235/236), S6K1 (#9202), and p-S6K1 (#9205, Thr389) were purchased from Cell Signaling

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Technology (Danvers, MA, USA). The general procedures for Western blot analysis were

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performed as described by Sun et al.40 Briefly, a fraction of the frozen organ tissue (40 mg)

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was weighed and homogenized in liquid nitrogen. The homogenate was treated with

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radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1%

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NP-40, 0.1% SDS, 1.0 mmol/L phenylmethylsulfonyl fluoride, 1.0 mmol/L Na3VO4, 1.0

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mmol/L NaF] containing phenylmethylsulfonyl fluoride and a mixture of protease inhibitors

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(Roche Applied Science). Supernatant fluid was collected after centrifugation at 12,000 × g

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for 15 minutes at 4°C. Protein content of samples was assayed using a BCA Protein Assay Kit

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(Beyotime, Jiangsu, China). Equal amounts of protein (30 μg) were electrophoresed on SDS

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polyacrylamide gel and then electrotransferred to a PVDF membrane (Millipore, Bedford,

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MA, USA). The PVDF membranes were blotted with 5% non-fat milk at 25°C for 1 h and

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then incubated with a primary antibody overnight at 4°C. After washing, the membranes were

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incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling

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Technology, USA) for 1 h at 25°C. The protein bands were developed using an enhanced

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chemiluminescence kit (Applygen Technologies Inc., Beijing, China) with the ImageQuant

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LAS 4000 mini system (GE Healthcare). Quantification of the band density was determined

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using Quantity One software (Bio-Rad Laboratories, Inc., California, USA).

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Statistical analysis 6



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Data obtained from the present study were analyzed by one-way analysis of variance

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(ANOVA) using the SAS 8.2 software package, followed by a Duncan’s multiple-range test to

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determine treatment effects. The results were expressed as mean ± SEM and regarded to

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achieve significant different at P < 0.05, highly significant different was set at P < 0.01.

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RESULTS

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Effects of dietary Leu in late pregnancy on growth performance of newborn piglets

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The effects of dietary Leu in late pregnancy on growth performance of newborn piglets

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were presented in Table 3. Supplementing with 0.80% Leu in the late gestation sow diets

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significantly increased mean piglet birth weight compared with CON and other treatments (P

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< 0.05; Table 3); while supplementing with 0.40%, 0.80% and 1.20% Leu in the late gestation

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sow diets had no effect on litter size, number born alive, still birth and mummified piglets,

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number born healthy and litter birth weight (P > 0.05; Table 3).

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Effects of dietary Leu in late pregnancy on relative organ weight of newborn piglets

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The effects of dietary Leu in late pregnancy on the relative organ weight of newborn

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piglets were shown in Table 4. Dietary supplementation with 0.40% Leu significantly

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increased the small intestine relative weight of newborn piglets by 22.20% compared with the

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CON group (P < 0.05; Table 4), while dietary supplementation with 0.80% and 1.20% Leu

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had no significant effect (P > 0.05; Table 4). There was little difference observed in thymus,

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heart, liver and pancreas relative weight among all treatments (P > 0.05; Table 4).

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Effects of dietary Leu in late pregnancy on plasma amino acids concentrations in

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farrowing sows and newborn piglets

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The concentrations of Val, Ile and Leu in farrowing sows plasma were affected by Leu

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supplementation (Table 5). Supplementation of 0.40%, 0.80% and 1.20% Leu resulted in

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higher plasma Leu concentration in farrowing sows (P < 0.05; Table 5). Compared with the

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CON and 0.40% Leu group, supplementation of 0.80% and 1.20% Leu into sow diet resulted

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in lower Val and Ile in farrowing sows plasma (P < 0.05; Table 5). Compared with the 0.80%

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Leu group, supplementation of 1.20% Leu into sow diet resulted in lower Val and Ile in 7


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farrowing sows plasma (P < 0.05; Table 5). No differences were observed for other amino

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acids in the plasma of sows among all treatment groups (P > 0.05; Table 5).

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Leu supplementation also affected plasma concentrations of Val, Ile and Leu in newborn

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piglets (P < 0.05; Table 6). Supplementation of 0.40%, 0.80% and 1.20% Leu into sow diet

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resulted in higher plasma concentration of Leu but lower Val and Ile compared to CON in the

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newborn piglets plasma (P < 0.05; Table 6). Compared with the 0.40% Leu group,

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supplementation of 0.80% and 1.20% Leu into sow diet resulted in lower Val in the newborn

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piglets plasma (P < 0.05; Table 6). Compared with the 0.40% and 0.80% Leu groups,

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supplementation of 1.20% Leu into sow diet resulted in lower Ile in the newborn piglets

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plasma (P < 0.05; Table 6). Compared with the 0.80% Leu group, supplementation of 1.20%

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Leu into sow diet resulted in lower Leu in the newborn piglets plasma (P < 0.05; Table 6).

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Neither difference was observed for other amino acids in the plasma of newborn piglets

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among all treatment groups (P > 0.05; Table 6).

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Effects of dietary Leu in late pregnancy on gene expressions of amino acid transporters

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in sow placenta and piglet small intestine (duodenum, jejunum, ileum) and longissimus

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dorsi muscle

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Supplementation of 0.40% Leu to late gestation diets of sows increased the expressions

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of LAT1, SNAT1, SNAT2, 4F2hc and rBAT in piglet duodenum, jejunum and ileum (P < 0.05;

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Figure 1-3). Expressions of 4F2hc, LAT1, rBAT, SNAT1 and SNAT2 in piglet duodenum and

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jejunum (Figure 1 and 2), 4F2hc and rBAT in ileum (Figure 3A & C) were increased in the

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0.80% Leu group (P < 0.05). Expressions of 4F2hc in duodenum and jejunum (Figure 1-2A ),

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4F2hc and rBAT in ileum (Figure 3A & C) were enhanced in the 1.20% Leu group (P < 0.05),

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whereas expressions of LAT1 in piglet duodenum (Figure 1B), LAT1, SNAT1 and SNAT2 in

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jejunum and ileum (Figure 2-3B, D & E) were reduced in the 1.20% Leu group compared

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with 0.40% and 0.80% Leu groups (P < 0.05).

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Supplementation of 0.40% Leu to late gestation diets of sows increased the expression of

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LAT1, SNAT1, SNAT2, 4F2hc and rBAT in sow placenta (P < 0.05; Figure 4). The 8



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expressions of SNAT1 and SNAT2 in sow placenta were also increased in the 0.80% Leu

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group (P < 0.05; Figure 4D & E). The expressions of rBAT and SNAT1 in sow placenta were

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increased in the 1.20% Leu group (P < 0.05; Figure 4C & D), while the 0.80% and 1.20% Leu

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supplementation had no effect on the expression of 4F2hc and LAT1 in sow placenta (P >

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0.05; Figure 4A & B).

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Supplementation of 0.40%, 0.80% and 1.20% Leu to late gestation diets of sows

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significantly increased the expressions of LAT1, SNAT1, SNAT2, 4F2hc and rBAT in piglet

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longissimus dorsi muscle (P < 0.05; Figure 5).

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Effects of dietary Leu in late pregnancy on phosphorylation of mTOR signaling

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pathway-associated proteins in longissimus dorsi muscle

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In order to study the mechanism by which Leu supplementation regulated protein

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synthesis in longissimus dorsi muscle of newborn piglets, expressions of mTOR signaling

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pathway-associated proteins were measured (Figure 6). Expressions of p-mTOR (Ser2448)

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(Figure 6C), p-4E-BP1 (Figure 6F), and p-S6K1 (Thr389) (Figure 6L) in longissimus dorsi

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muscle of newborn piglets were enhanced in the 0.40%, 0.80% and 1.20% Leu groups

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compared with the CON group (P < 0.05). Expressions of p-S6 (Ser235/236) (Figure 6I) in

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longissimus dorsi muscle of newborn piglets were enhanced in the 0.40% and 0.80% Leu

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groups compared with the CON group (P < 0.05).

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DISCUSSION

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It is clearly documented that Leu is not only a substrate for protein synthesis, but also

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acts as a nutrient signal that regulates protein synthesis in various tissues of the body,

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including skeletal muscle.40 Studies have shown that Leu administration increases skeletal

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muscle protein synthesis in rodents41, humans42 and piglets43. Little information about the

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effect of Leu on fetal intrauterine development was known. Therefore, the objective of this

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study was to explore the effect of Leu supplementation in sows at late gestation on the fetal

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growth and the possible underlying mechanism.

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Studies have shown that in vivo supplementation of Leu could improve the growth 9


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performance of weaned piglets9, promote piglet intestinal development40 and activate muscle

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protein synthesis10. Evidence from the literature stated that 0.55% L-Leu supplementation for

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14 days increased the weight gain of weanling pigs.9 Moreover, β-hydroxy-β-methylbutyrate,

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the irreversible metabolite of Leu in the liver cytoplasm, is widely treated as an Leu

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alternative to increase muscle protein synthesis nowdays.44-46 Nissen et al.47 reported that

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supplementing 2 g/d β-hydroxy-β-methylbutyrate to sows resulted in an increase in pig

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weight at day 21 (7%, P = 0.01). In present study, 0.80% Leu treatment showed significantly

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greater mean piglet birth weight than those of the CON, 0.40% and 1.20% Leu groups (Table

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3, P < 0.05). Our results agreed with the previous studies.9,47 It implied that the increase of

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Leu concentration added in sow diets promoted growth performance of newborn piglets.

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While the decrease of mean piglet birth weight in 1.20% Leu group may be due to the lowed

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feed intake for the reduced palatability of feed with addition of Leu. It is common sense that

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relative weight of organs was associated with the maintenance requirements of organs, and

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that visceral organ weight of newborn piglets was positively correlated with the body

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weight.48 It has been shown that dietary supplementation with 1.40 g Leu/kg BW improves

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intestinal development in suckling piglets.40 Our results suggested that 0.40% Leu

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supplementation significantly increased the relative weight of the small intestine in newborn

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piglets, and this enhancement has potential to improve overall health of piglets through

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promoting intestinal development.

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In order to further investigate the possible mechanism that how dietary supplementation

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of Leu might affect the mean piglet birth weight and organ indices of newborn piglets, we

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firstly measured the concentrations of amino acids in the plasma of farrowing sows and

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newborn piglets. Furthermore, we analyzed the expressions of amino acid transporter proteins

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in sow placenta and duodenum, jejunum, ileum, longissimus dorsi muscle of newborn piglets

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and expressions of mTOR signaling pathway proteins in the longissimus dorsi muscle of

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newborn piglets.

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Ile, Leu and Val share the same enzyme complex, branched-chain keto acid 10



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dehydrogenase enzymes in liver and common transport systems for absorption,49,50 excess Leu

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may result in promoted catabolism of all BCAA and impaired absorption of Ile and Val.31 It

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has been reported previously that supplemental Leu (0.43%) increased serum Leu by

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approximately 50% but decreased serum Ile and Val to less than 50% of the pigs fed the

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control diet.31 Consistently, the plasma concentration of Leu in sows of 0.40%, 0.80% and

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1.20% Leu groups were higher, whereas the plasma concentration of Val and Ile in sows of

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0.80% and 1.20% Leu groups were lower compared with the sows of CON group in this study.

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Interestingly, the concentrations of amino acids in newborn piglets plasma were lower than

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those of farrowing sows or other research literatures9, which mainly was due to the fact that

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newborn piglets did not feed any colostrum, and the amino acids in their blood were

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consumed excessively before slaughtering, then resulted in reduced concentrations.

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Amino acid transporters may act as sensors, as well as carriers, for tissue nutrient

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supplies.30-33 Placenta is an important organ for nutrients exchange from sows to fetus.

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Nutrients requirement for the rapid growth and development of fetal pigs at late gestation are

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almost exclusively transported from the maternal placenta and umbilical cord blood to fetal

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pigs.51,52 Thus, efficient expression of the placental amino acid transporters is vital for the

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supply of amino acids in plasma of fetal or newborn piglets. Our results indicated that dietary

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supplementation 0.40% Leu significantly increased expressions of LAT1, SNAT1, SNAT2,

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4F2hc and rBAT in sow placenta compared with the CON group, while expressions of LAT1

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and SNAT2 in 1.20% Leu group were significantly lower than 0.40% and 0.80% Leu groups

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(P < 0.05). This agreed with lower plasma Leu concentration of newborn piglets and mean

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piglet birth weight in 1.20% Leu group compared with 0.80% Leu group.

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In this study, we also observed significantly increases in expressions of amino acid

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transporters in the small intestine of the newborn piglets, which indicates that

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supplementation of 0.40-0.80% Leu could increase amino acids transport efficiency through

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the intestine epithelial cells, which could provide amino acids for intestine development,

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resulting in the relative weight of small intestine. We also showed significantly increased 11


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expressions of amino acid transporters (LAT1, SNAT1, SNAT2, 4F2hc and rBAT) in the

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longissimus dorsi muscle of neonatal piglets in all Leu groups compared with the CON group,

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but the average absolute expression values of LAT1, SNAT1, SNAT2, 4F2hc and rBAT in

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1.20% Leu group was lower than 0.40% and 0.80% Leu groups (Figure 5), which could

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explain higher mean piglet birth weight in 0.80% Leu group than other groups. This might be

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due to the lower feed intake for sows in 1.20% Leu group. Up-regulation of amino acid

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transporters such as LAT1 in skeletal muscle may provide large neutral amino acids as an

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energy source, as well as an anabolic signal and substrate for enhanced protein synthesis in

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skeletal muscle.53

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Numerous studies based on piglet models have shown that Leu can stimulate protein

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synthesis through mTOR signialing pathway in skeletal muscle.41,44,54 Yin et al. 9 reported that

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dietary supplementation with 0.55% Leu significantly enhanced the phosphorylation state of

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4E-BP1 in the skeletal muscle of weaned piglets at 21 days after birth. The phosphorylation of

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mTOR, S6K1, and 4E-BP1 in the longissimus dorsi muscle of neonatal pigs was enhanced in

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the low protein + Leu and high protein groups compared with the low protein group.55,56

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Phosphorylation of S6 and S6K1 increased in skeletal muscle of neonatal pigs at 60 min and

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120 min after the start of the 400 μmol/kg/h Leu infusion compared with saline infusion.57

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Leu activated phosphorylation of mTOR and S6K1 reacted in a dose-dependent manner in

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proliferating primary preterm rat satellite cells.58 In this experiment, supplementation with

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0.40-1.20% Leu enhanced the expressions of p-mTOR, 4E-BP1, and p-S6K1 in the

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longissimus dorsi muscle of newborn piglets, which was consistent with previous study.

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Therefore, we speculated that dietary supplementation with 0.40-0.80% Leu could promote

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the growth performance of fetal pigs through mTOR signaling pathway in longissimus dorsi

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muscle.

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In summary, supplementing 0.40-0.80% Leu to sows diet during late gestation has

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beneficial effects on growth performance in fetal pigs. These responses to sow Leu

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supplementation are possibly due to greater expressions of amino acid transporters in the sow 12



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placenta, small intestine and longissimus dorsi muscle of the newborn piglets. Up-regulation

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of mTOR signaling pathway proteins in longissimus dorsi muscle of fetal pigs may also

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contribute to greater newborn piglet birth weight, by facilitating maternal-fetal functional

310

amino acids transport, and by promoting the amino acids profile and the expressions of amino

311

acid transporters in sows placenta and intestine of fetal pigs.

312

ABBREVIATIONS

313

4E-BP1, 4E binding protein 1

314

4F2hc, 4F2 heavy chain

315

BCAA, branched-chain amino acid

316

BW, body weight

317

CON, control

318

eIF4E, eukaryotic initiation factor 4E

319

LAT1, L-type amino acid transporter 1

320

mTOR, mammalian target of rapamycin

321

mTORC1, mammalian target of rapamycin complex 1

322

rBAT, related to b0,+ amino acid transporter

323

S6K1, p70-S6 kinase 1

324

SE, standard error

325

SNAT1, sodium-coupled neutral amino acid transporter 1

326

SNAT2, sodium-coupled neutral amino acid transporter 2

327

ACKNOWLEDGMENT

328 329

Special thanks for the Guangdong Changjiang Food Group Co., Ltd and Guangdong Academy of Agricultural Sciences, for their strong support. 13


Page 14 of 34

Page 15 of 34

330 331


FUNDING This work was jointly supported by funding from the National Natural Science

332

Foundation of China (31402082).

333

CONFLICTS OF INTEREST

334 335

The authors declare no conflict of financial interest. SUPPORTING INFORMATION

336

Supporting file 1. Supporting information for review only.

337

Supporting file 2. NC3Rs ARRIVE Guidelines Checklist.

338

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339

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than

leucine

in

inhibiting

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Nguyen, H. V.; Fiorotto, M. L.; El-Kadi, S.; Davis, T. A. Leucine supplementation of a

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low-protein meal increases skeletal muscle and visceral tissue protein synthesis in neonatal

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Page 22 of 34

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Metab. 2005, 288, E914-E921.

507

(58) Dai, J. M.; Yu, M. X.; Shen, Z. Y.; Guo, C. Y.; Zhuang, S. Q.; Qiu, X. S. Leucine

508

promotes proliferation and differentiation of primary preterm rat satellite cells in part through

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mTORC1 signaling pathway. Nutrients 2015, 7, 3387-3400.

510 511 512 513 514 515 516 517 518 519 520 521 522 523

FIGURE CAPTIONS Figure 1. Effects of dietary Leu in late pregnancy on expressions of amino acid transporters proteins in the duodenum of newborn piglets. Figure 2. Effects of dietary Leu in late pregnancy on expressions of amino acid transporters proteins in the jejunum of newborn piglets. Figure 3. Effects of dietary Leu in late pregnancy on expressions of amino acid transporters proteins in the ileum of newborn piglets. Figure 4. Effects of dietary Leu in late pregnancy on expressions of amino acid transporters proteins in the placenta of sows. Figure 5. Effects of dietary Leu in late pregnancy on expressions of amino acid transporters proteins in the longissimus dorsi muscle of newborn piglets. Figure 6. Effects of dietary Leu in late pregnancy on expressions of mTOR pathway proteins in the longissimus dorsi muscle of newborn piglets.

22



524

TABLES

525

Table 1. Ingredients of the experimental diets (air-dry basis)

Page 24 of 34

Added leucine level (%) Ingredients (%)

Corn Soybean meal Soybean oil Wheat bran Limestone Di-calcium phosphate Salt L-Lysine HCl (78%) DL-Methionine (100%) L-Threonine (98.5%) L-Tryptophan (98.5%) L-Valine (98.5%) L-Leucine (98.5%) Vitamin- mineral premix a Choline chloride (50.0%) Phytase Mold removal agent Mould inhibitors Antioxidant Carrier Total

CON (0.00)

0.40

0.80

1.20

66.00 18.50 1.00 9.50 1.20 0.50 0.30 0.12 0.02 0.06 0.02 0.07 0.00 0.20 0.30 0.02 0.10 0.05 0.04 2.00 100.00

66.00 18.50 1.00 9.50 1.20 0.50 0.30 0.12 0.02 0.06 0.02 0.07 0.40 0.20 0.30 0.02 0.10 0.05 0.04 1.60 100.00

66.00 18.50 1.00 9.50 1.20 0.50 0.30 0.12 0.02 0.06 0.02 0.07 0.80 0.20 0.30 0.02 0.10 0.05 0.04 1.20 100.00

66.00 18.50 1.00 9.50 1.20 0.50 0.30 0.12 0.02 0.06 0.02 0.07 1.20 0.20 0.30 0.02 0.10 0.05 0.04 0.80 100.00

526

a

527

D3, 4,000 IU; vitamin E, 60 mg; vitamin K3, 4 mg; vitamin B1, 4 mg; vitamin B2, 10 mg;

528

vitamin B6, 4.8 mg; vitamin B12, 0.034 mg; niacin, 40 mg; pantothenic acid, 20 mg; folic

529

acid, 2 mg; biotin, 0.16 mg; 80 mg of Fe (as FeSO4•H2O); 5 mg of Cu (as CuSO4•5H2O);

530

51 mg of Zn (as ZnSO4•H2O); 20.5 mg of Mn (as MnSO4•H2O); 0.14 mg of I (as

531

Ca(IO3)2); and 0.15 mg of Se (as sodium selenite).

Vitamin and mineral premix (by per kilogram of diet): vitamin A, 13,000 IU; vitamin

23


Page 25 of 34

532


Table 2. Nutritional level of the experimental diets (as-fed basis) Added leucine level (%) Nutritional level (%)

533

a

a

CON (0.00)

0.40

0.80

1.20

Digestible energy, MJ/kg

13.35

13.31

13.27

13.22

Crude protein

14.99

15.19

15.40

15.60

Calcium

0.86

0.86

0.86

0.86

Total phosphorus

0.64

0.64

0.64

0.64

Non-phytic acid phosphorus

0.39

0.39

0.39

0.39

Salt

0.36

0.36

0.36

0.36

Arginine

0.96

0.95

0.95

0.95

Lysine

0.79

0.79

0.79

0.79

Methionine

0.24

0.24

0.24

0.24

Methionine + Cysteine

0.46

0.46

0.46

0.46

Threonine

0.60

0.60

0.60

0.60

Tryptophan

0.18

0.18

0.18

0.18

Valine

0.74

0.74

0.74

0.74

Isoleucine

0.56

0.56

0.56

0.56

Leucine

1.30

1.70

2.10

2.50

Phenylalanine

0.71

0.71

0.71

0.71

Phenylalanine + Tyrosine

1.21

1.21

1.21

1.21

Calculated values according to the tables of feed composition and nutritive values in China.

24



534

Page 26 of 34

Table 3. Effects of dietary Leu in late pregnancy on reproductive performance of sows Added leucine level (%) Items

P value CON (0.00)

0.40

0.80

1.20

Mean piglet birth weight /kg

1.49 ±0.02b

1.47 ±0.02b

1.55 ±0.02a

1.46 ±0.02b

0.038

Litter birth weight /kg

15.63 ±0.59

16.40 ±0.61

15.66 ±0.73

16.00 ±0.59

0.797

Litter size

11.31 ±0.43

11.73 ±0.45

10.50 ±0.35

11.66 ±0.48

0.205

Number born alive

10.50 ±0.44

11.17 ±0.40

10.04 ±0.37

10.86 ±0.43

0.280

Still birth and mummified piglets

0.81 ±0.18

0.57 ±0.16

0.46 ±0.15

0.79 ±0.18

0.395

Number born healthy

10.09 ±0.43

10.93 ±0.40

9.81 ±0.37

10.48 ±0.42

0.248

535

Values are means ± SE, n = 6 per group. In the same row, values with different

536

lower-case superscripts are significant different (P < 0.05), with different upper-case

537

superscripts are highly significant difference (P < 0.01), and with the same superscripts

538

or without superscripts are no significant difference (P > 0.05).

539 540

Table 4. Effects of dietary Leu in late pregnancy on relative organ weight of newborn

541

piglets Added leucine level (%)

Organs

P value (g/kg BW)

CON (0.00)

0.40

0.80

1.20

Thymus

0.80 ±0.10

0.83 ±0.06

0.78 ±0.06

0.67 ±0.06

0.415

Heart

7.39 ±0.86

7.24 ±0.57

7.19 ±0.24

7.27 ±0.18

0.994

Liver

25.20 ±2.08

25.13 ±2.26

22.92 ±1.20

23.59 ±1.46

0.756

Pancreas

1.01 ±0.14

0.89 ±0.10

0.80 ±0.05

0.90 ±0.05

0.474

27.21 ±1.11b

33.26 ±2.47a

30.34 ±2.34ab

29.89 ±2.05ab

0.044

Small intestine 542


543

lower-case superscripts are significant difference (P < 0.05), with different upper-case

544


545

or without superscripts are no significant difference (P > 0.05). 25


Page 27 of 34


546

Table 5. Effects of dietary Leu in late pregnancy on plasma concentrations of amino

547

acids in farrowing sows Added leucine level (%)

Amino acids P value (μmol/L)

CON (0.00)

0.40

0.80

1.20

13.80 ±1.46

14.40 ±1.08

15.20 ±1.02

15.80 ±1.16

0.659

Threonine, Thr

175.60 ±21.94

179.40 ±20.98

185.40 ±16.04

205.00 ±13.90

0.689

Serine, Ser

136.00 ±13.74

143.33 ±11.26

148.50 ±9.04

152.33 ±9.99

0.753

Glutamate, Glu

141.20 ±10.71

147.40 ±8.85

153.00 ±9.81

155.60 ±10.68

0.751

Glycine, Gly

664.60 ±82.57

721.80 ±53.00

746.80 ±44.73

763.20 ±47.91

0.665

Alanine, Ala

458.00 ±48.48

488.17 ±33.32

498.00 ±33.60

537.67 ±54.01

0.637

Valine, Val

304.75 ±28.50A

300.80 ±9.83A

261.75 ±14.16B

210.00 ±14.67C

0.007

39.00 ±3.93

40.17 ±3.82

42.00 ±3.74

43.50 ±3.72

0.844

Isoleucine, Ile

116.75 ±3.09A

113.50 ±9.37A

99.50 ±4.44B

81.33 ±5.89C

0.003

Leucine, Leu

217.50 ±21.58B

355.67 ±24.86A

370.50 ±46.91A

305.00 ±39.08A

0.005

Tyrosine, Tyr

111.17 ±10.48

114.33 ±10.67

119.33 ±10.18

123.50 ±9.95

0.841

98.67 ±6.47

100.67 ±5.77

102.67 ±5.68

104.83 ±6.79

0.907

195.00 ±22.18

207.00 ±18.01

215.20 ±16.64

221.20 ±13.15

0.751

Histidine, His

79.40 ±6.90

82.20 ±5.08

85.20 ±3.89

86.00 ±3.96

0.791

Arginine, Arg

231.75 ±10.45

235.75 ±10.23

238.75 ±10.97

242.50 ±11.32

0.908

Proline, Pro

300.25 ±22.70

323.75 ±28.91

328.00 ±29.63

337.75 ±29.41

0.806

1292.50 ±126.17

1379.00 ±93.30

1403.50 ±83.36

1433.50 ±72.67

0.756

Aspartic acid, Asp

Methionine, Met

Phenylalanine, Phe Lysine, Lys

Glutamine, Gln 548


549

lower-case superscripts are significant difference (P < 0.05), with different upper-case

550


551

or without superscripts are no significant difference (P > 0.05).

26



Page 28 of 34

552

Table 6. Effects of dietary Leu in late pregnancy on plasma concentrations of amino

553

acids in newborn piglets Added leucine level (%)

Amino acids P value (μmol/L)

CON (0.00)

0.40

0.80

1.20

Aspartic acid, Asp

27.33 ±4.74

30.00 ±6.01

34.50 ±7.80

35.17 ±7.94

0.821

Threonine, Thr

57.50 ±9.68

60.25 ±9.17

63.75 ±9.96

69.00 ±9.82

0.850

Serine, Ser

167.50 ±28.37

184.00 ±19.85

189.00 ±18.82

211.50 ±26.72

0.638

Glutamate, Glu

241.20 ±42.75

261.80 ±33.85

272.00 ±30.89

289.80 ±34.73

0.810

Glycine, Gly

693.20 ±72.42

729.60 ±67.40

740.60 ±68.73

762.80 ±57.50

0.902

Alanine, Ala

715.00 ±73.82

740.50 ±71.61

760.50 ±67.34

785.25 ±73.05

0.912

Valine, Val

221.00 ±35.72a

177.75 ±11.51b

148.25 ±12.09c

134.75 ±5.57c

0.044

28.00 ±4.36

31.40 ±4.07

34.20 ±5.16

35.80 ±6.18

0.709

Isoleucine, Ile

69.00 ±17.61a

30.25 ±3.75b

30.25 ±1.70b

19.00 ±7.15c

0.017

Leucine, Leu

38.83 ±2.51C

63.00 ±1.26AB

70.80 ±5.23A

57.20 ±1.39B

0.05).

27


Page 29 of 34

558


FIGURE GRAPHICS

559

560 561

Figure 1. Effects of dietary Leu in late pregnancy on expressions of amino acid transporters

562

proteins in the duodenum of newborn piglets. (A) 4F2hc, 4F2 heavy chain; (B) LAT1, L-type

563

amino acid transporter 1; (C) rBAT, related to b0,+ amino acid transporter; (D) SNAT1,

564

Sodium-coupled neutral amino acid transporter 1; and (E) SNAT2, Sodium-coupled neutral

565

amino acid transporter 2. Values are means (n = 6), with their standard errors represented by

566

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

28



567 568


569

proteins in the jejunum of newborn piglets. (A) 4F2hc, 4F2 heavy chain; (B) LAT1, L-type

570


571


572


573


29


Page 30 of 34

Page 31 of 34


574 575


576

proteins in the ileum of newborn piglets. (A) 4F2hc, 4F2 heavy chain; (B) LAT1, L-type

577


578


579


580


30



581 582


583

proteins in the placenta of sows. (A) 4F2hc, 4F2 heavy chain; (B) LAT1, L-type amino acid

584

transporter 1; (C) rBAT, related to b0,+ amino acid transporter; (D) SNAT1, Sodium-coupled

585

neutral amino acid transporter 1; and (E) SNAT2, Sodium-coupled neutral amino acid

586

transporter 2. Values are means (n = 6), with their standard errors represented by vertical bars.

587

Different letters (a, b, c) indicate significant differences (P < 0.05).

31


Page 32 of 34

Page 33 of 34


588 589


590

proteins in the longissimus dorsi muscle of newborn piglets. (A) 4F2hc, 4F2 heavy chain; (B)

591

LAT1, L-type amino acid transporter 1; (C) rBAT, related to b0,+ amino acid transporter; (D)

592

SNAT1, Sodium-coupled neutral amino acid transporter 1; and (E) SNAT2, Sodium-coupled

593

neutral amino acid transporter 2. Values are means (n = 6), with their standard errors

594

represented by vertical bars. Different letters (a, b, c) indicate significant differences (P

Leucine Promotes the Growth of Fetal Pigs by Increasing Protein

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