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Bioactive Constituents, Metabolites, and Functions

Leucine affects #-amylase synthesis through PI3K/Akt-mTOR signaling pathways in pancreatic acinar cells of dairy calves Long Guo, Ziqi Liang, Chen Zheng, Baolong Liu, Qingyan Yin, Yangchun Cao, and J. H. Yao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01111 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 9, 2018

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Journal of Agricultural and Food Chemistry 1

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Leucine affects α-amylase synthesis through PI3K/Akt-mTOR signaling

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pathways in pancreatic acinar cells of dairy calves

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Long Guo, Ziqi Liang, Chen Zheng, Baolong Liu, Qingyan Yin, Yangchun Cao, Junhu

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Yao*

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College of Animal Science and Technology, Northwest A&F University, Yangling,

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712100, China

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*Corresponding author: Junhu Yao

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Full Address: College of Animal Science and Technology, Northwest A&F University,

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Yangling Shaanxi, PR China

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Tel: +86-029-87092102

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Fax: +86-029-87092164

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E-mail: [email protected]

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ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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ABSTRACT: Dietary nutrients utilization, particularly starch, is potentially limited

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by digestion in dairy cow small intestine because of shortage of α-amylase. Leucine

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acts as an effective signal molecular in the mTOR signaling pathway, which regulates

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a series of biological processes, especially protein synthesis. It has been reported that

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leucine could affect α-amylase synthesis and secretion in ruminant pancreas, but

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mechanisms have not been elaborated. In this study, pancreatic acinar (PA) cells were

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used as a model to determine the cellular signal of leucine influence on α-amylase

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synthesis. PA cells were isolated from new born Holstein dairy bull calves and

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cultured in DMEM/F12 (Dulbecco's Modifed Eagle's Medium/Nutrient Mixture F12)

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Liquid media containing four leucine treatments (0, 0.23, 0.45 and 0.90 mM,

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respectively), following α-amylase activity, zymogen granule and signal pathway

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factors expression detection. Rapamycin, a specific inhibitor of mTOR, was also

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applied to PA cells. Results showed that leucine increased (P < 0.05) synthesis of

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α-amylase as well as phosphorylation of PI3K, Akt, mTOR, and S6K1, while reduced

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(P < 0.05) GCN2 expression. Inhibition of mTOR signaling down-regulated the

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α-amylase synthesis. In addition, the extracellular leucine dosage significantly

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influenced intracellular metabolism of isoleucine (P < 0.05). Overall, leucine

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regulates α-amylase synthesis through promoting PI3K/Akt-mTOR pathway and

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reducing GCN2 pathway in PA cells of dairy calves. These pathways form the

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signaling network which controls the protein synthesis and metabolism. It would be of

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great interest in future studies to explore the function of leucine in ruminant nutrition.

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KEYWORDS: leucine, mTOR signaling, GCN2 signaling, PI3K/Akt signaling,


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α-amylase synthesis

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INTRODUCTION

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Utilization of dietary nutrients is potentially limited by digestion in the small intestine

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of dairy cow, especially carbohydrates. 1 Pancreatic enzymes digest nutrients flowing

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to the small intestine, but it is hard to regulate the pancreas to produce more digestive

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enzymes in ruminant. It is postulated that, because of the increasing demands placed

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on dairy production, pancreatic secretion may be a point at which productivity can be

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enhanced and provide great benefit, such as relieving negative energy balance during

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perinatal period. The effect of amino acids on the secretory processes as regulators

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have been reported more, especially leucine,2,

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However, the regulatory mechanisms of leucine in dairy cow pancreatic enzymes

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synthesis have not been elaborated.

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isoleucine4 and phenylalanine.5,

6

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It has long been appreciated that in addition to being a proteogenic amino acid,

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leucine is a signaling molecule that directly regulates animal physiology, including

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satiety,7 insulin secretion,8 and skeletal muscle anabolism.9, 10 Relatively more were

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known about the role of leucine in regulation of protein translation. A key mediator of

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effects of leucine is the mammalian target of rapamycin complex1 (mTORC1) protein

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kinase,11, 12 which regulates growth by controlling processes like protein synthesis.

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The mechanistic mTORC1 is a major regulator of eukaryotic growth that coordinates

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anabolic and catabolic cellular processes with outputs such as ribosomal protein S6

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kinase 1 (S6K1), and the eukaryotic initiation factor 4E-binding protein (4EBP1).13

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The phosphatidylinositide 3 kinases (PI3K) signaling pathway is upstream signaling

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pathway of mTOR signaling pathway and also regulates the activation of mTOR.14


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Besides, general amino acid control non-derepressible 2 (GCN2) signaling pathway is

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another signal pathway which regulates the body’s protein translation. The eukaryotic

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initiation factor 2α (eIF2α) kinase GCN2 senses the absence of one or more amino

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acids (leucine) by virtue of direct binding to uncharged cognate transport RNAs

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(tRNAs).15 These signaling pathways form a network involved in leucine regulation

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of the protein translation process.

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The effect of cell signaling on digestive enzymes synthesis in pancreatic acinar

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(PA) cells of dairy calves have been explored,6 however, influence of leucine on cell

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signaling and the association of the resulting cell signaling patterns with digestive

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enzymes synthesis have not been examined. We hypothesized that leucine affects the

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potential cellular signals to regulate α-amylase synthesis. The objective of the present

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study was to investigate the effects of leucine on PI3K/Akt-mTOR and GCN2

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signaling pathways and to evaluate the associations of these signaling activities with

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α-amylase synthesis in PA cells of dairy calves.

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MATERIALS AND METHODS

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Ethics statement. In this study, animal experiment was approved by the

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Institutional Animal Care and Use Committee and carried out strictly in comply with

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the guidelines for the care and use of experimental animals at Northwest A&F

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University (protocol number NWAFAC1008).

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Cell isolation and culture. The protocols of PA cells isolation and culture

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referred to the research of Guo et al.6 Whole pancreas was harvested and digested in

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Kreb-Ringer bicarbonate (KRB) buffer containing collagenase Ⅲ (1 mg/mL) and 5%



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BSA at 37℃. The incubation last for 15 min with constant shaking. Following

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multiple washes with Hanks balanced salt solution supplemented with 5% fetal

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bovine

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collagenase-digested pancreatic tissue was sequentially filtered through 500 µm and

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154 µm polypropylene mesh (Solarbio, Beijing, China). The filtrate was passed

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through a 30% FBS cushion at 500× g for 30 s. The cellular pellet obtained was

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washed twice followed by centrifugation. Cells were cultured in suspension or in

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monolayer in Dulbecco's Modifed Eagle's Medium/Nutrient Mixture F12 Ham's

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Liquid (DMEM/F12) media (HyClone, Thermo scientific, Logan, Utah, USA) and

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incubated at 37℃ with 5% CO2.

serum

(FBS,

Gibco

Laboratories,

Gaithersburg,

MD,

USA),

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Preparation of inhibitors. The specific mTOR inhibitor rapamycin (Selleck

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Chemicals, Houston, TX, USA) was dissolved in dimethylsulfoxide (DMSO,

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Sigma-Aldrich Inc., St. Louis, MO, USA) to produce 10 mM stock solutions that were

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stored at -80℃. Prior to applications in cell treatments, stock solutions were diluted to

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working concentrations.

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Experiment design. The isolated PA cells were cultured in 6-well cell culture

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plate and each well had 1×106 cells and 2 mL culture media. All treatment media

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(complete DMEM/F12 media) were adjusted to a pH of 7.4, were serum-free, and

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contained 17.5 mmol D-glucose, 50 mg insulin, 1nmol Epidermal growth factor, 0.02

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mmol phenol red, 0.50 mmol sodium pyruvate, 10 kU penicillin/streptomycin, 2.5 g

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soybean trypsin inhibitor and 14.0 mmol sodium bicarbonate per liter. The amino acid

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concentration in each treatment was shown below: 0.05 mM L-alanine, 0.70 mM


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L-argnine, 0.05 mM L-aspartic acid, 0.10 mM L-cystine, 0.05 mM L-glutamic acid,

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2.50 mM L-glutamine, 0.25 mM glycine, 0.15 mM L-histidine, 0.42 mM L-isoleucine,

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0.45 mM L-valine, 0.50 mM L-lysine, 0.12 mM L-methionine, 0.21 mM

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L-phenylalanine, 0.15 mM L-proline, 0.25 mM L-serine, 0.45 mM L-threonine, 0.04

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mM L-tryptophan and 0.21 mM L-tyrosine.

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There were four leucine treatments: the medium containing 0 mM leucine

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(custom media from HyClone, Thermo scientific, Logan, UT, USA), 0.23 mM leucine

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(the normal plasm leucine concentration of dairy cow),16 0.45 mM leucine and 0.90

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mM leucine. For rapamycin inhibition assays, cells were treated with leucine or

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rapamycin (10 nM). There were four treatments including control (0 mM leucine),

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Rapa (10 nM rapamycin), Leu (0.45 mM leucine), and Leu + Rapa (0.45 mM leucine

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+ 10 nM rapamycin).

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After incubation for 60 min, cells were harvested by scraping in the presence of

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ice-cold lysis buffer containing 1% (v:v) of protease and phosphatase inhibitors

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cocktail (Roche, Mannheim, Baden-Württemberg, Germany). Cell lysates from a

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6-well plate of each medium were combined. The culture media were also collected

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for subsequent analysis of enzymes activity and amino acid composition. The cell

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culturing was repeated for three times. On each time, cells from a calf were cultured

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in four 6-well culture plates with four kinds of media, respectively. Therefore, each

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experiment had a total of 3 replicates from three calves (n = 3).

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Analysis of enzyme synthesis and secretion. The intracellular α-amylase was

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extracted by Ultrasonic cell disruptor (Sonics, Newtown, CT, USA). The activity of



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α-amylase in culture medium and acinar cells was determined using a commercial kit

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(Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China), according to

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the manufacturer’s instructions. The enzyme activity was expressed in units per

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milliter (in culture media) or units per milligram protein (intracellular). One unit was

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defined as 1 µmol product released per minute at 39℃.

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Zymogen granule observation. Transmission electron microscope (HT7700,

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Hitachi, Tokyo, Japan) was used to photograph the acinar cell zymogen granules after

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cultured. The procedure involved the cell fixation, post-fixation, dehydration,

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infiltration, embedding, slicing, and dyeing.

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Amino acids analysis in culture media. The kind and concentration of amino

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acids in culture media were measured with amino acids analyzer (A300 Advanced,

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MembraPure GmbH, Berlin, Germany). Briefly, the procedure involved the sulfonyl

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salicylic acid reaction, centrifuge, the sample dilution and filtration. The consumption

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of amino acids was calculated as the amino acid amount of the culture media before

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culturing minus that after culturing the cells.

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Protein preparation and western blot. Protein concentration in cell lysate was

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determined using a PierceTM BCA assay kit (Thermo fisher, Rockford, IL, USA). The

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protein samples were boiled at 100℃ for 5 minutes in 5 × sample buffer (Beijing

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CoWin Biotech Co., Ltd., Beijing, China). The protein extracts (30 µg protein each)

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were electrophoresed in 6-15% SDS-polyacrylamide gels. The separated proteins

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were then transferred onto a nitrocellulose membrane (Pall Corp., Port Washing-ton,

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NY, USA) in Tris-glycine buffer containing 20% methanol. The membranes were


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blocked and immunoblotted with a 1:1,000 dilution of a primary antibody including

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anti-β-actin (Beijing CoWin Biotech Co., Ltd., Beijing, China, Catalog Nos.

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CW0096M), anti-mTOR, anti-P-mTOR, anti-p70S6K, anti-P-p70S6K, anti-4EPB1,

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anti-P-4EBP1, anti-GCN2, anti-eIF2α, anti-P-eIF2a, anti-PI3Kp85, anti-P-PI3Kp85,

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anti-Akt and anti-P-Akt (Cell Signaling Technology, Danvers, MA, USA, Catalog

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Nos. 2972, 2971, 9452, 9459, 9202, 9205, 3302, 9722, 9721, 4292, 4228, 9272, and

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9611, respectively).

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The proteins were detected using either goat anti-rabbit IgG (H+L)-HRP

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conjugated secondary antibody (1:3,000) or goat anti-mouse IgG (H+L) secondary

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antibody (1:3,000) with chemiluminescence (ECL) western blot detection reagents

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(Bio-Rad, Hercules, CA, USA). The β-actin was used as an internal control. Western

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blots were developed and quantified using Image J software.

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Statistical analysis. Each experiment included three biological replicates and

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results were expressed as means ± standard error of means. Statistical significance

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was analyzed by SPSS 20.0 (SPSS Inc., Chicago, IL, USA). The data of α-amylase

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activity, amino acid concentration, and proteins expression were analyzed by one-way

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analysis of variance (ANOVA) using the general linear model procedure, leucine

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treatment as factor and calves as blocks. Pearson’s correlation analysis test (SPSS

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20.0 software) was used to analyze the relationship among α-amylase synthesis,

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signaling protein phosphorylation or expression, and amino acids consumption. The

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total protein expression levels were calculated as the ratio of the band intensity of

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β-actin. The protein phosphorylation levels were calculated as the ratio of the



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phosphorylation form to total form. Significant differences was declared at P < 0.05.

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RESULTS

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Leucine regulates α-amylase synthesis and secretion in PA cells.

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As leucine concentration increased (P < 0.05) from low to high, α-amylase

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synthesis gradually increased and peaked at the leucine concentration of 0.45 mM,

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followed by a decrease (P < 0.05) (Figure 1). The release of α-amylase also showed

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the same trend but with some difference that it was not decreased (P > 0.05) after

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peak (Figure 2). Images of zymogen granules showed that higher concentration of

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leucine (0.45 and 0.90 mM) had more quantity and volume zymogen granules than

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that of the lower concentration of leucine (0 and 0.23 mM) (Figure 3).

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Leucine affects isoleucine consumption in cultured media.

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The results (Table 1) showed that leucine concentration in culture media affected

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isoleucine consumption without affecting other amino acids depletion. Leucine

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consumption was highest at the 0.45 mM leucine group, while leucine depletion has

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dropped to lower level when the leucine concentration increased to 0.90 mM (P
0.05). The abundance

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of total GCN2 (Figure 5B) was decreased (P < 0.05) by leucine at 0.23 mM and 0.45

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mM. The phosphorylation of eIF2α (Figure 5C) was not affected by all treatments (P

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= 0.07).

200 201

Relationships among α-amylase synthesis, signaling protein phosphorylation or expression, and leucine consumption.

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The results (Table 2) showed that α-amylase synthesis was positively correlated

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with PI3K phosphorylation (P < 0.05), Akt phosphorylation (P < 0.05), mTOR

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phosphorylation (P < 0.01), S6K1 phosphorylation (P < 0.01), and leucine

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consumption (P < 0.01). Phosphorylation of PI3K was positively correlated with Akt

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phosphorylation

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phosphorylation (P < 0.05), and leucine consumption (P < 0.05). Phosphorylation of

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Akt was negatively and positively associated with GCN2 expression (P < 0.05) and

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leucine consumption (P < 0.05), respectively. mTOR phosphorylation was positively

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correlated with S6K1 phosphorylation (P < 0.01). Leucine consumption was

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positively and negatively associated with phosphorylation of S6K1 (P < 0.05) and

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expression of GCN2 (P < 0.05), respectively.

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(P < 0.05), mTOR phosphorylation

(P < 0.05),

S6K1

Inhibition of mTORC1 downregulated the leucine-mediated mTOR phosphorylation and α-amylase synthesis in PA cells.



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Compared to leucine treatment, leucine and rapamycin mixed treatment

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significantly decreased the phosphorylation of mTOR (P < 0.05) (Figure 6A and 6B)

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and α-amylase synthesis (P < 0.05) (Figure 6C).

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DISCUSSION

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PA cell is typical exocrine glandular cells that synthesis and secretion digestive

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enzymes which eventually flow through the pancreatic duct into the duodenum.17

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These digestive enzymes including pancreatic amylase, trypsin, and lipase primarily

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influence the digestion process of animals. Proteins and amino acids are necessary as

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a signal as well as a substrate for pancreatic digestive enzyme synthesis after meal.18

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On account of the unique features of digestive system, the regulation of pancreatic

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digestive enzymes synthesis became a big challenge in ruminant. Our recent study

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demonstrated that duodenal infusions of 3, 6, or 9 g/d leucine in dairy goats for a

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period of 10 h affected the secretion of pancreatic fluid with a quadratic dose-response

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curve, and the greatest effect was obtained with 3 g/d leucine infusion.3 A duodenal

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infusion of 10 g/d of leucine in dairy heifers increased concentration (U / mL) and

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secretion rate (U / h) of α-amylase in the pancreatic fluid.2 These studies were all in

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vivo experiments, and all experimental animals underwent duodenal intubation. So the

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results needed to be validated at the cellular level and the mechanisms needed to be

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studied at the molecular level of proteins. PA cells were used as the model in the

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present study, and we found that leucine could improve the α-amylase synthesis and

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secretion after culturing for 60 min. We did not measure trypsin and lipase

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successfully, because the presence of trypsin inhibitor as well as the low levels


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enzymes activity or the kits sensitivity.

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The role of PI3K in intracellular signaling has been underscored by its

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implication in a plethora of biological responses.19 A signaling pathway from PI3K to

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the serine/threonine protein kinase PKB/Akt may mediate some cellular responses of

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PI3K.20 One of aspects involved that leucine could increase the specific protein

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production in some cells via the PI3K-Akt-mTOR signaling pathway.21-23 In the

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present study, the 0.45 mM leucine treatment significantly increased the

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phosphorylation of PI3K and Akt, as well as the synthesis level of α-amylase. These

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results indicated that leucine raised the protein synthesis in PA cells, which was

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related to activation of the PI3K/Akt signal pathway. Some studies showed that

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insulin plays a role in leucine-activated PI3K/Akt pathway activation in rat.24, 25 In the

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present study, it was impossible to determine the effects of insulin in PA cells because

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the cultural media contained 0.1 mg insulin.

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mTOR signaling pathway control the main protein synthesis process in

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mammalian cells.26, 27 Leucine is the effective amino acid signal molecular for mTOR

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protein.28 Rapamycin is a highly specific inhibitor of mTOR protein.29 The previous

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studies have shown that rapamycin could inhibit the phosphorylation of mTOR.30, 31

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In the present study, when leucine concentration increased from 0.23 mM to 0.45 mM,

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the phosphorylation of PI3K, mTOR and S6K1 raised significantly, at the same time

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α-amylase synthesis also showed the same trend. However, rapamycin was

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significantly decreased the leucine-mediated increased of mTOR phosphorylation and

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α-amylase synthesis. In addition, omission of leucine decreased α-amylase synthesis



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and release, as well as the phosphorylation of S6K1 and Akt. These data indicated that

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the mTOR signal pathway control the synthesis of α-amylase in PA cells. The similar

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results were shown in research of leucine regulate protein synthesis in MAC-T cells

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and bovine mammary tissue slices,32, 33 skeletal muscle,34 and adipocytes.35 Interesting

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results also be found that omission of leucine did not decrease the phosphorylation of

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PI3K and mTOR. The main reason must be that isoleucine replaced leucine and acted

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as a signaling molecule. This was confirmed by the highest consumption of isoleucine

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in 0 mM leucine treatment. In MAC-T cells and mammary tissue slices, Appuhamy et

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al found that isoleucine regulates mTOR signaling and protein synthesis independent

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of leucine.32 Some researchers also found that isoleucine could stimulate mTOR33 and

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PI3K36 signal pathway.

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Besides its action on mTORC1 signaling, it is well established that leucine is a

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potent regulator of the kinase GCN2.37 GNC2 signal pathway is sensitive of amino

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acids starvation in mammalian cells, because uncharged tRNA could activate of

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GCN2.38 The eIF2α is a downstream factor of GCN2. GCN2 is activated during

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scarcity of an essential amino acid and phosphorylates the α-subunit of eIF2α.39 This

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leads to the general inhibition of protein synthesis. However, the phosphorylation of

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eIF2α actually related to the expression of amino acid transporters,40 enzymes

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involved in amino acid metabolism.41 In the present study, the expression of GCN2

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was higher in 0 mM leucine treatment than that in 0.23 mM and 0.45 mM leucine

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treatments. The phosphorylation of eIF2α was not significantly expressed, but there

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was a similar trend (P = 0.07). While, the activation of mTOR signal pathway factors


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was the opposite. The most important point was lower α-amylase synthesis in 0 mM

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leucine treatment when compared with the control. In addition, GCN2 pathway

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proteins showed the negatively correlated with leucine consumption and α-amylase

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synthesis. These results indicated that when absence of leucine, the activation of

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GCN2 signal could reduce the protein synthesis through the mTOR inhibition, which

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consistent with the results of Julien et al.42 About the expression of GCN2 increased

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in 0.90 mM leucine treatment, the possible reason was that excess of leucine induced

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leucine consumption decreased and thus increased intracellular uncharged tRNA

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levels. This aspect needed more in-depth research.

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Phosphorylation of PI3K, Akt, mTOR, S6K1 were all individually correlated

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with α-amylase synthesis, but the correlation coefficient and P value were different.

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Given the higher correlation coefficient of the mTOR pathway than the PI3K pathway,

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we can concluded that the mTOR signaling pathway is more strongly linked with

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α-amylase synthesis than the PI3K/Akt pathway. Moreover, GCN2 pathway proteins

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showed the negatively correlated with leucine consumption and α-amylase synthesis.

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The current experimental results confirmed the previous findings at cellular level and

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further investigated molecular mechanisms. Based on the results of this study, we

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proposed a model to illustrate how leucine regulates α-amylase synthesis in PA cells

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of dairy calves (Figure 7).

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The synthesis of enzyme required a lot of amino acids intake.43 Amino acids

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consumption in culture medium can reflect PA cell amino acid utilization. In the

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present study, the amino acids consumption was detected. As the leucine



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concentration increased, the consumption of leucine also increased, but in 0.90 mM

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leucine treatment, the consumption of leucine was decreased. At same time, the

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synthesis of α-amylase also decreased. The possible reason was that high

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concentration of leucine induced the amino acids unbalance or branch-chain amino

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acids (BCAA) antagonism. Early study found that only the leucine-induced branch

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chain amino acid antagonism can be demonstrated without careful manipulation of the

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amino acid composition of the basal diet.44 The detail results were shown that high

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intakes of leucine by human subjects or animals depress valine and isoleucine

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concentrations in blood and muscle. In addition, the effects of excess leucine on the

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growth of the rat were assumed to be the result of leucine acting as an antimetabolite

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of isoleucine and valine, while addition of excess isoleucine or valine to the

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low-protein diet resulted in only slight depression in growth of the rat.45 It was

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suggested that excess leucine results in changes in the quantities of isoleucine and

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valine available for protein synthesis.46 We also found that omission of leucine

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increased the isoleucine consumption. The results were consistent with the previous

318

studies that when diets deficient in leucine were fed to rats47 and humans,48 plasma

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and tissue concentrations of isoleucine was elevated. Apart from this, isoleucine was

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also a signaling molecule with a similar effect as leucine to participate the protein

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synthesis, as mentioned earlier. Together, these results indicated that more attention

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should be paid to focus on the balance of amino acids when single amino acid was

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used as feed additives, otherwise it will be counterproductive.

324

In summary, the present study suggests that leucine regulates synthesis of


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α-amylase through promoting the PI3K/Akt-mTOR pathway while reducing the

326

GCN2 pathway in PA cells of dairy calves. These pathways formed the signaling

327

network which controls the protein synthesis and metabolism in dairy calves. It would

328

be of great interest in future studies to explore the function of leucine in ruminant

329

nutrition.

330

AUTHOR INFORMATION

331

Corresponding author

332

*

333

[email protected]. (J H Yao)

334

Funding

335

The work was partially supported by awards of the National Natural Science

336

Foundation of China (award No.: 31472122 and 31672451).

337

Notes

338

The authors declare no competing financial interest.

Tel:

+86-029-87092102.

Fax:

+86-029-87092164.

339


E-mail:


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(2) Liu, K., Liu, Y., Liu, S. M., Xu, M., Yu, Z. P., Wang, X., Cao, Y. C., Yao, J. H.,

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Relationships between leucine and the pancreatic exocrine function for improving

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starch digestibility in ruminants. J. Dairy. Sci. 2015, 98, 2576-2582.

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(3) Yu, Z. P., Xu, M., Liu, K., Yao, J. H., Yu, H. X., Wang, F., Leucine markedly

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regulates pancreatic exocrine secretion in goats. J. Anim. Physiol. Anim. Nutr. 2014,

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(4) Liu, K., Shen, J., Cao, Y. C., Cai, C. J., Yao, J, H., Duodenal infusions of

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isoleucine influence pancreatic exocrine function in dairy heifers. Arch. Anim. Nutr.

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2018, 72, 31-41

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485

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486 487


Page 25 of 37


488

Table 1. The consumption of amino acid in cell culture media Amino acids

leucine concentration, mM

SEM1

P value

0.00

0.23

0.45

0.90

Leucine, µg

0.00a

24.01c

37.54d

10.42b

4.46

0.000

Isoleucine,

50.00cd

36.00ab

43.72bc

28.18a

2.74

0.003

µg 489

a-d

490

1

Means within a row with different superscripts differ (P < 0.05).

Pooled standard error of the means, n = 3.

491



Page 26 of 37 26

492

Table 2. Correlations among α-amylase synthesis, signaling proteins phosphorylation

493

or expression, and leucine consumption. Dependent variable

Independent variable

Pearson

P-value

Correlation PI3K

0.592*

0.04

Akt

0.619*

0.03

mTOR

0.722**

0.01

S6K1

0.826**

0.00

GCN2

-0.547

0.07

Leucine consumption

0.890**

< 0.01

Akt

0.654*

0.02

mTOR

0.612*

0.03

S6K1

0.615*

0.03

Leucine consumption

0.582*

0.05

GCN2

-0.617*

0.03

Leucine consumption

0.686

0.01

mTOR

S6K1

0.889**

< 0.01

S6K

Leucine consumption

0.679*

0.02

GCN2

Leucine consumption

-0.757**

0.00

α-Amylase synthesis

PI3K

Akt

494

** Correlation is significant at the 0.01 level (2-tailed).

495

* Correlation is significant at the 0.05 level (2-tailed).

496


Page 27 of 37


497

Figure legends

498

Figure 1. Effect of leucine treatment on α-amylase synthesis of pancreatic acinar cells

499

at 60 min. Data are expressed as means ± S.E.M., n = 3. Different letters mean

500

significantly different (P < 0.05).

501

Figure 2. Effect of leucine treatment on α-amylase release of pancreatic acinar cells at

502

60 min. Data are expressed as means ± S.E.M., n = 3. Different letters mean

503

significantly different (P < 0.05).

504

Figure 3. Transmission electron microscope of zymogen granule after 60 min of

505

incubation in pancreatic acinar cells of dairy calves. Panel A-D showed the scanning

506

electron micrograph of the zymogen granule in 0 mM, 0.23 mM, 0.45 mM, and 0.90

507

mM leucine treatments of pancreatic acinar cell, respectively. Bar = 2 µm. The red

508

arrow indicates the zymogen granule.

509

Figure 4. The ratio of phosphorylated to total PI3K-Akt-mTOR signaling pathway

510

factors in pancreatic acinar cells of dairy calves cultured at 60 min in the presence of

511

0, 0.23, 0.45, 0.90 mM leucine. A: Represents the immunoblots of phosphorylation

512

forms and total of PI3K, Akt, mTOR, S6K, 4EBP1 and β-actin. B-F: Represent the

513

ratio of the phosphorylated to total PI3K, Akt, mTOR, S6K, 4EBP1, respectively.

514

Error bar represent SEM, n = 3. Different letters mean significantly different (P

Akt-mTOR

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