Akt-mTOR

7 days ago - College of Animal Science and Technology, Northwest A&F ... through promoting the PI3K/Akt-mTOR pathway and reducing the GCN2 pathway ...
1 downloads 3 Views 3MB Size
Subscriber access provided by UCL Library Services

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

Journal of Agricultural and Food Chemistry 1

1

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

2

pathways in pancreatic acinar cells of dairy calves

3

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

4

Yao*

5

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

6

712100, China

7 8

*Corresponding author: Junhu Yao

9

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

10

Yangling Shaanxi, PR China

11

Tel: +86-029-87092102

12

Fax: +86-029-87092164

13

E-mail: [email protected]

14

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 37 2

15

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

16

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

17

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

18

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

19

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

20

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

21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

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

ACS Paragon Plus Environment

Page 3 of 37

Journal of Agricultural and Food Chemistry 3

37

α-amylase synthesis

38

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 37 4

39

INTRODUCTION

40

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

41

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

42

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

43

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

44

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

45

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

46

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

47

have been reported more, especially leucine,2,

48

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

49

synthesis have not been elaborated.

3

isoleucine4 and phenylalanine.5,

6

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

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

60

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

ACS Paragon Plus Environment

Page 5 of 37

Journal of Agricultural and Food Chemistry 5

61

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

62

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

63

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

64

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

65

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

66

of the protein translation process.

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

α-amylase synthesis in PA cells of dairy calves.

75

MATERIALS AND METHODS

76

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

77

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

78

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

79

University (protocol number NWAFAC1008).

80

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

81

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

82

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 37 6

83

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

84

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

85

bovine

86

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

87

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

88

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

89

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

90

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

91

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

92

incubated at 37℃ with 5% CO2.

serum

(FBS,

Gibco

Laboratories,

Gaithersburg,

MD,

USA),

93

Preparation of inhibitors. The specific mTOR inhibitor rapamycin (Selleck

94

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

95

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

96

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

97

working concentrations.

98

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

99

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

100

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

101

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

102

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

103

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

104

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

ACS Paragon Plus Environment

Page 7 of 37

Journal of Agricultural and Food Chemistry 7

105

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

106

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

107

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

108

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

109

mM L-tryptophan and 0.21 mM L-tyrosine.

110

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

111

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

112

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

113

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

114

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

115

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

116

+ 10 nM rapamycin).

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

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

125

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

126

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 37 8

127

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

128

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

129

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

130

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

131

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

132

Zymogen granule observation. Transmission electron microscope (HT7700,

133

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

134

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

135

infiltration, embedding, slicing, and dyeing.

136

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

137

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

138

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

139

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

140

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

141

culturing minus that after culturing the cells.

142

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

143

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

144

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

145

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

146

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

147

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

148

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

ACS Paragon Plus Environment

Page 9 of 37

Journal of Agricultural and Food Chemistry 9

149

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

150

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

151

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

152

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

153

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

154

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

155

9611, respectively).

156

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

157

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

158

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

159

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

160

blots were developed and quantified using Image J software.

161

Statistical analysis. Each experiment included three biological replicates and

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 10 of 37 10

171

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

172

RESULTS

173

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

174

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

175

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

176

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

177

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

178

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

179

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

180

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

181

Leucine affects isoleucine consumption in cultured media.

182

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

183

isoleucine consumption without affecting other amino acids depletion. Leucine

184

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

185

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

197

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

198

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

199

= 0.07).

200 201

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

202

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

203

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

204

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

205

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

206

phosphorylation

207

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

208

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

209

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

210

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

211

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

212

expression of GCN2 (P < 0.05), respectively.

213 214

(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.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 12 of 37 12

215

Compared to leucine treatment, leucine and rapamycin mixed treatment

216

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

217

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

218

DISCUSSION

219

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

220

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

221

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

222

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

223

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

224

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

225

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

226

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

227

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

228

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

229

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

230

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

231

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

232

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

233

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

234

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

235

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

236

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

ACS Paragon Plus Environment

Page 13 of 37

Journal of Agricultural and Food Chemistry 13

237

enzymes activity or the kits sensitivity.

238

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

239

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

240

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

241

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

242

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

243

present study, the 0.45 mM leucine treatment significantly increased the

244

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

245

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

246

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

247

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

248

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

249

the cultural media contained 0.1 mg insulin.

250

mTOR signaling pathway control the main protein synthesis process in

251

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

252

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

253

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

254

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

255

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

256

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

257

significantly decreased the leucine-mediated increased of mTOR phosphorylation and

258

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 14 of 37 14

259

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

260

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

261

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

262

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

263

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

264

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

265

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

266

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

267

al found that isoleucine regulates mTOR signaling and protein synthesis independent

268

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

269

PI3K36 signal pathway.

270

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

271

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

272

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

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

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

ACS Paragon Plus Environment

Page 15 of 37

Journal of Agricultural and Food Chemistry 15

281

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

282

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

283

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

284

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

285

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

286

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

287

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

288

leucine consumption decreased and thus increased intracellular uncharged tRNA

289

levels. This aspect needed more in-depth research.

290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

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

298

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

299

of dairy calves (Figure 7).

300

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

301

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

302

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 16 of 37 16

303

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

304

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

305

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

306

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

307

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

308

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

309

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

310

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

311

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

312

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

313

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

314

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

315

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

316

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

317

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

319

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

320

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

321

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

322

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

323

used as feed additives, otherwise it will be counterproductive.

324

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

ACS Paragon Plus Environment

Page 17 of 37

Journal of Agricultural and Food Chemistry 17

325

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

ACS Paragon Plus Environment

E-mail:

Journal of Agricultural and Food Chemistry

Page 18 of 37 18

340 341 342

REFERENCES (1) Harmon, D. L., Impact of nutrition on pancreatic exocrine and endocrine secretion in ruminants: a review. J. Anim. Sci. 1992, 70, 1290-1301.

343

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

344

Relationships between leucine and the pancreatic exocrine function for improving

345

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

346

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

347

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

348

98, 169-177.

349

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

350

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

351

2018, 72, 31-41

352

(5) Yu, Z. P., Xu, M., Yao, J. H., Liu, K., Li, F., Liu, Y., Wang, F., Sun, F. F., Liu, N.

353

N., Regulation of pancreatic exocrine secretion in goats: differential effects of short-

354

and long-term duodenal phenylalanine treatment. J. Anim. Physiol. Anim. Nutr. 2013,

355

97, 431-438.

356

(6) Guo, L., Tian, H. B., Shen, J., Zheng, C., Liu, S. M., Cao, Y. C., Cai, C. J., Yao, J.

357

H., Phenylalanine regulates initiation of digestive enzyme mRNA translation in

358

pancreatic acinar cells and tissue segments in dairy calves. Biosci. Rep. 2017, 20,

359

1-13.

360 361

(7) Potier, M., Darcel, N., Tomé, D., Protein, amino acids and the control of food intake. Curr. Opin. Clin. Nutr. Metab. Care. 2009, 12, 54-58.

ACS Paragon Plus Environment

Page 19 of 37

Journal of Agricultural and Food Chemistry 19

362

(8) Panten, U., Christians, J., Von Kriegstein, E., Poser, W., Hasselblatt, A., Studies

363

on the mechanism of L-leucine-and alpha-ketoisocaproic acid-induced insulin release

364

from perifused isolated pancreatic islets. Diabetologia. 1974, 10, 149-154.

365

(9) Greiwe, J. S., Kwon, G., McDaniel, M. L., Semenkovich, C. F., Leucine and

366

insulin activate p70 S6 kinase through different pathways in human skeletal muscle.

367

Am. J. Physiol. Endocrinol. Metab. 2001, 281, 466-471.

368 369

(10) Nair, K. S., Schwartz, R. G., Welle, S., Leucine as a regulator of whole body and skeletal muscle protein metabolism in humans. Am. J. Physiol. 1992, 263, 928.

370

(11) Fox, H. L., Pham, P. T., Kimball, S. R., Jefferson, L. S., Lynch, C. J., Amino

371

acid effects on translational repressor 4E-BP1 are mediated primarily by L-leucine in

372

isolated adipocytes. Am. J. Physiol. 1998, 275, 1232-1238.

373

(12) Lynch, C. J., Fox, H. L., Vary, T. C., Jefferson, L. S., Kimball, S. R., Regulation

374

of amino acid-sensitive TOR signaling by leucine analogues in adipocytes. J. Cell.

375

Biochem. 2000, 77, 234-251.

376 377 378 379

(13) Ma, X. M., Blenis, J., Molecular mechanisms of mTOR-mediated translational control. Nat. Rev. Mol. Cell. Biol. 2009, 10, 307-318. (14) Kimball, S. R., Regulation of global and specific mRNA translation by amino acids. J. Nutr. 2002, 132, 883-886.

380

(15) Gallinetti, J., Harputlugil, E., Mitchell, J. R., Amino acid sensing in

381

dietary-restriction-mediated longevity: roles of signal-transducing kinases GCN2 and

382

TOR. Biochem. J. 2013, 449, 1-10.

383

(16) Mackle, T. R., Dwyer, D. A., Ingvartsen, K. L., Chouinard, P. Y., Ross, D. A.,

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 20 of 37 20

384

Bauman, D. E., Effects of insulin and postruminal supply of protein on use of amino

385

acids by the mammary gland for milk protein synthesis. J. Dairy. Sci. 2000, 83,

386

93-105.

387 388

(17) Chandra, R., Liddle, R.A., Modulation of pancreatic exocrine and endocrine secretion. Curr. Opin. Gastroenterol. 2013, 29, 517-522

389

(18) Sans, M. D., Crozier, S. J., Vogel, N. L., Williams, J. A., Dietary protein and

390

amino acids regulate the synthesis of pancreatic digestive enzymes. Gastroenterology.

391

2008, 134, 723.

392 393 394 395

(19) Franke, T. F., Kaplan, D. R., Cantley, L. C., PI3K: downstream AKTion blocks apoptosis. Cell. 1997, 88, 435-437. (20)

Burgering,

B.

M.,

Coffer,

P.

J.,

Protein

kinase

B

(c-Akt)

in

phosphatidylinositol-3-OH kinase signal transduction. Nature. 1995, 376, 599-602.

396

(21) Zhang, S., Ren, M., Zeng, X., He, P., Ma, X., Qiao, S., Leucine stimulates

397

ASCT2 amino acid transporter expression in porcine jejunal epithelial cell line

398

(IPEC-J2) through PI3K/Akt/mTOR and ERK signaling pathways. Amino. Acids.

399

2014, 46, 2633-2642.

400

(22) Pérez de Obanos, M. P., López Zabalza, M. J., Prieto, J., Herraiz, M. T., Iraburu,

401

M. J., Leucine stimulates procollagen alpha1(I) translation on hepatic stellate cells

402

through ERK and PI3K/Akt/mTOR activation. J. Cell. Physiol. 2006, 209, 580-586.

403

(23) Wu, C., You, J., Fu, J., Wang, X., Zhang, Y., Phosphatidylinositol 3-kinase/Akt

404

mediates integrin signaling to control RNA polymerase I transcriptional activity. Mol.

405

Cell. Biol. 2016, 36, 1555-1568.

ACS Paragon Plus Environment

Page 21 of 37

Journal of Agricultural and Food Chemistry 21

406

(24) Balage, M., Dupont, J., Mothe-Satney, I., Tesseraud, S., Mosoni, L., Dardevet,

407

D., Leucine supplementation in rats induced a delay in muscle IR/PI3K signaling

408

pathway associated with overall impaired glucose tolerance. J. Nutr. Biochem. 2011,

409

22, 219-226.

410

(25) Filiputti, E., Rafacho, A., Araújo, E. P., Silveira, L. R., Trevisan, A., Batista, T.

411

M., et al. Augmentation of insulin secretion by leucine supplementation in

412

malnourished rats: possible involvement of the phosphatidylinositol 3-phosphate

413

kinase/mammalian target protein of rapamycin pathway. Metabolism. 2010, 59,

414

635-644.

415

(26) Tischler, M. E., Desautels, M., Goldberg, A. L., Does leucine, leucyl-tRNA, or

416

some metabolite of leucine regulate protein synthesis and degradation in skeletal and

417

cardiac muscle? J. Biol. Chem. 1982, 257, 1613-1621.

418 419 420 421 422 423 424 425

(27) Wang, X., Proud, C. G., The mTOR pathway in the control of protein synthesis. Physiology. 2006, 21, 362-369. (28) Stipanuk, M. H., Leucine and protein synthesis: mTOR and beyond. Nutr. Rev. 2007, 65, 122-129. (29) Bjornsti, M. A., Houghton, P. J., The TOR pathway: a target for cancer therapy. Nat. Rev. Cancer. 2004, 4, 335-348. (30) Raman, N., Nayak, A., Muller, S., mTOR signaling regulates nucleolar targeting of the SUMO-specific isopeptidase SENP3. Mol. Cell. Biol. 2014, 34, 4474-4484.

426

(31) Guba, M., von Breitenbuch, P., Steinbauer, M., Koehl, G., Flegel, S., Hornung,

427

M., et al. Rapamycin inhibits primary and metastatic tumor growth by

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 22 of 37 22

428

antiangiogenesis: involvement of vascular endothelial growth factor. Nat. Med. 2002,

429

8, 128-135.

430

(32) Appuhamy, J. A., Knoebel, N. A., Nayananjalie, W. A., Escobar, J., Hanigan, M.

431

D., Isoleucine and leucine independently regulate mTOR signaling and protein

432

synthesis in MAC-T cells and bovine mammary tissue slices. J. Nutr. 2012, 142,

433

484-491.

434

(33) Arriola Apelo, S. I., Singer, L. M., Lin, X. Y., McGilliard, M. L., St-Pierre, N.

435

R., Hanigan, M. D., Isoleucine, leucine, methionine, and threonine effects on

436

mammalian target of rapamycin signaling in mammary tissue. J. Dairy. Sci. 2014, 97,

437

1047-1056.

438

(34) Escobar, J., Frank, J. W., Suryawan, A., Nguyen, H. V., Kimball, S. R., Jefferson,

439

L. S., Davis, T. A. Regulation of cardiac and skeletal muscle protein synthesis by

440

individual branched-chain amino acids in neonatal pigs. Am. J. Physiol. Endocrinol.

441

Metab. 2006, 290, 612-621.

442

(35) Kitsy, A., Carney, S., Vivar, J. C., Knight, M. S., Pointer, M. A., Gwathmey, J.

443

K., Ghosh, S., Effects of leucine supplementation and serum withdrawal on

444

branched-chain amino acid pathway gene and protein expression in mouse adipocytes.

445

Plos. One. 2014, 9, e102615.

446

(36) Doi, M., Yamaoka, I., Fukunaga, T., Nakayama, M., Isoleucine, a potent plasma

447

glucose-lowering amino acid, stimulates glucose uptake in C2C12 myotubes.

448

Biochem. Biophys. Res. Commun. 2003, 312, 1111-1117.

449

(37) Xiao, F., Huang, Z., Li, H., Yu, J., Wang, C., Chen, S., et al. Leucine deprivation

ACS Paragon Plus Environment

Page 23 of 37

Journal of Agricultural and Food Chemistry 23

450

increases hepatic insulin sensitivity via GCN2/mTOR/S6K1 and AMPK pathways.

451

Diabetes. 2011, 60, 746.

452

(38) Dong, J., Qiu, H., Garcia-Barrio, M., Anderson, J., Hinnebusch, A. G.,

453

Uncharged tRNA activates GCN2 by displacing the protein kinase moiety from a

454

bipartite tRNA-binding domain. Mol. Cell. 2000, 6, 269-279.

455

(39) Berlanga, J. J., Santoyo, J., De Haro, C., Characterization of a mammalian

456

homolog of the GCN2 eukaryotic initiation factor 2alpha kinase. Eur. J. Biochem.

457

1999, 265, 754-762.

458

(40) Lopez, A. B., Wang, C., Huang, C. C., Yaman, I., Li, Y., Chakravarty, K., et al.

459

A feedback transcriptional mechanism controls the level of the arginine/lysine

460

transporter cat-1 during amino acid starvation. Biochem. J. 2007, 402, 163-173.

461

(41) Siu, F., Bain, P. J., LeBlanc-Chaffin, R., Chen, H., Kilberg, M. S., ATF4 is a

462

mediator of the nutrient-sensing response pathway that activates the human

463

asparagine synthetase gene. J. Biol. Chem. 2002, 277, 24120-24127

464

(42) Averous, J., Lambert-Langlais, S., Mesclon, F., Carraro, V., Parry, L., Jousse, C.,

465

et al. GCN2 contributes to mTORC1 inhibition by leucine deprivation through an

466

ATF4 independent mechanism. Sci. Rep. 2016, 6, 27698.

467 468 469 470 471

(43) Listed, N., Adaptive responses of amino acid degrading enzymes to variation of amino acid and protein intake. Nutr. Rev. 1976, 34, 343-345. (44) Harper, A. E., Miller, R. H., Block, K. P., Branched-chain amino acid metabolism. Annu. Rev. Nutr. 1984, 4, 409-454. (45) Harper, A. E., Benton, D. A., Elvehjem, C. A., L -Leucine, an isoleucine

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 24 of 37 24

472

antagonist in the rat. Arch. Biochem. Biophys. 1955, 57, 1-12.

473

(46) Tannous, R. I., Rogers, Q. R., Harper, A. E., Effect of leucine-isoleucine

474

antagonism on the amino acid pattern of plasma and tissue of the rat. Arch. Biochem.

475

Biophys. 1966, 113, 356

476 477

(47) Clark, A. J., Peng, Y., Swendseid, M. E., Effect of different essential amino acid deficiencies on amino acid pools in rats. J. Nutr. 1966, 90, 228-234.

478

(48) Hambraeus, L., Bilmazes, C., Dippel, C., Scrimshaw, N., Young, V. R.,

479

Regulatory role of dietary leucine on plasma branched-chain amino acid levels in

480

young men. J. Nutr. 1976, 106, 230-240.

481 482

(49) Nair, K. S., Short, K. R., Hormonal and signaling role of branched-chain amino acids. J. Nutr. 2005, 135, 1547-1552.

483

(50) Wolfson, R. L., Chantranupong, L., Saxton, R. A., Shen, K., Scaria, S. M.,

484

Cantor, J. R., Sabatini, D. M., Sestrin2 is a leucine sensor for the mTORC1 pathway.

485

Science. 2016, 351, 43-48.

486 487

ACS Paragon Plus Environment

Page 25 of 37

Journal of Agricultural and Food Chemistry 25

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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

ACS Paragon Plus Environment

Page 27 of 37

Journal of Agricultural and Food Chemistry 27

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