Dietary Branched-chain Amino Acids Regulate Food Intake Partly

May 17, 2019 - containing 10 μL SYBR Green PCR Master Mix (Takara, Dalian, Liaoning, China) , 2. 167. μL cDNA, 0.8 μL of forward and reverse PCR pr...
1 downloads 0 Views 833KB Size
Subscriber access provided by Bethel University

Bioactive Constituents, Metabolites, and Functions

Dietary Branched-chain Amino Acids Regulate Food Intake Partly through Intestinal and Hypothalamic Amino Acid Receptors in Piglets Min Tian, Jinghui Heng, Hanqing Song, Kui Shi, Xiaofeng Lin, Fang Chen, Wutai Guan, and Shihai Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02381 • Publication Date (Web): 28 May 2019 Downloaded from http://pubs.acs.org on May 29, 2019

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 41

Journal of Agricultural and Food Chemistry

1

Dietary Branched-chain Amino Acids Regulate Food Intake Partly

2

through Intestinal and Hypothalamic Amino Acid Receptors in Piglets

3 4

Min Tian†, Jinghui Heng†, Hanqing Song†, Kui Shi†, Xiaofeng Lin†, Fang Chen†,

5

Wutai Guan*,†,‡, Shihai Zhang*,†,‡

6 7

†Guangdong

8

Animal Science, South China Agricultural University, Guangzhou, 510642, China

9

‡College

10

Province Key Laboratory of Animal Nutrition Control, College of

of Animal Science and National Engineering Research Center for Breeding

Swine Industry, South China Agricultural University, Guangzhou 510642, China

11 12

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

13

ABSTRACT

14

Strategies to increase feed intake is of great importance for producing more meat in

15

swine production. Intestinal and hypothalamic amino acid receptors are found largely

16

participated in feed intake regulation. The purpose of the current research was to study

17

the function of branched-chain amino acids (BCAAs) supplementation in the regulation

18

of feed intake through sensors which can detect amino acids in piglets. Twenty-four

19

piglets were assigned to four treatments and fed to one of the experimental diets for

20

either short period (Expt. 1) or long period (Expt. 2): normal protein diet (NP, 20.04%

21

CP), reduced-protein diet (RP, 17.05% CP), reduced-protein test diets supplemented

22

with two doses of BCAAs (BCAA1, supplemented with 0.13% L-isoleucine, 0.09% L-

23

leucine and 0.23% L-valine; BCAA2, supplemented with the 150% standardized ileal

24

digestibility BCAAs requirement as recommended by the National Research Council

25

(2012)). In Expt. 1, no differences were observed in feed intake among piglets fed with

26

different diets (P > 0.05). In Expt. 2, when compared with the RP group, feed intake of

27

piglets was significantly increased after sufficient BCAAs was supplemented in the

28

BCAA1 group, which was associated with decreased cholecystokinin (CCK) secretion

29

(P < 0.05), down-regulated expression of type 1 taste receptor 1/3 (T1R1/T1R3) in

30

intestine, as well as increased expression of pro-opiomelanocortin (POMC) and

31

activated general control nonderepressible 2 (GCN2) and eukaryotic initiation factor

32

2α (eIF2α) in the hypothalamus (P < 0.05). However, feed intake was decreased when

33

the piglets were fed with BCAAs over supplemented diet for unknown reasons. In

34

conclusion, our study confirmed that BCAAs deficit diet inhibited feed intake through 2

ACS Paragon Plus Environment

Page 2 of 41

Page 3 of 41

Journal of Agricultural and Food Chemistry

35

two potential ways: regulating amino acid T1R1/T1R3 receptor in the intestine and/or

36

activating GCN2/eIF2α pathways in the hypothalamus.

37

KEYWORDS : amino acid receptor, branched-chain amino acids, feed intake,

38

reduced-protein diet

39

3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

40

INTRODUCTION

41

Sufficient feed intake was the guarantee for more meat production and optimum feed

42

conversion ratio in swine production 1. In addition to dietary energy level, dietary amino

43

acid concentrations also play a key role in the regulation of feed intake 2. Recently, it

44

was demonstrated that pigs fed with branched-chain amino acids (BCAAs) deficient

45

diet significantly decreased feed intake, which can be reversed after sufficient BCAAs

46

were supplemented back to the diet

47

supplementation on feed intake was the most widely studied in animal models.

48

Intracerebroventricular administration of leucine activated mTOR signaling pathway in

49

the hypothalamus and subsequently decreased feed intake in rats 5. However, most of

50

the studies found that dietary supplementation of extra leucine did not affect feed intake

51

in rats 6. Similarly, extra dietary leucine supplementation did not show any beneficial

52

effect on feed intake and may even inhibit feed intake in pigs 7-9. Recently, valine was

53

also found to be participated in feed intake regulation. Pigs fed valine deficient diet

54

significantly reduced feed intake

55

supplementation

56

relationship between BCAAs and feed intake, the underlying mechanism is still largely

57

unknown.

11.

10

3-4.

Among all the BCAAs, the effect of leucine

and this effect was aggravated with extra leucine

Although large abundant evidence indicated the intimate

58

Animals detect and sense diverse dietary nutrients via intestinal enteroendocrine

59

cells (EECs) before they are absorbed into blood. EECs are distributed along the

60

gastrointestinal (GI) tract, but they are less than 1% of all the epithelial cells12. In

61

proximal gut, large numbers of I-type EECs are participated in cholecystokinin (CCK) 4

ACS Paragon Plus Environment

Page 4 of 41

Page 5 of 41

Journal of Agricultural and Food Chemistry

62

production, while in distal small intestine, there is a comparatively high density of L-

63

type EECs, which mainly secrete polypeptide YY (PYY) and glucagon‐like peptide‐1

64

(GLP-1)

65

released mainly in response to lipids, but also to carbohydrates and proteins 14. Whereas

66

GLP-1 was mainly released in response to carbohydrates and fats

67

hormones are regulated by different nutrient components indicating there are a variety

68

of nutrient sensors in EECs. In the past decade, intestinal sensors for carbohydrate, fat

69

and protein have been deciphered by scientists around the world

70

structural units of protein, amino acids are found to be sensed by different intestinal

71

receptors. The Ca-sensing receptor (CaSR) is triggered by L-aromatic, aliphatic and

72

polar amino acids 16. Type 1 taste receptor 1 (T1R1) and 3 (T1R3) belong to the T1R

73

family and their combination (T1R1/T1R3) has been demonstrated as a broad-spectrum

74

L-amino acid sensor, but not for L-tryptophan

75

(mGIuRs), originally found in the brain, are also expressed in the gut and mainly

76

activated by L-glutamate 18. While the G protein‐coupled receptor GPRC6A (GPCR,

77

Class C, group 6, subtype A) is dominantly triggered by L-cationic amino acids

78

Therefore, modifying the expression of these intestinal sensors could regulate the feed

79

intake via the gut hormone secretion.

13.

CCK secretion is largely depend on dietary fat and protein and PYY is

17.

13.

15.

Diverse gut

As the basic

Metabotropic glutamate receptors

19.

80

In recent years, it was reported that animal ingesting unbalanced amino acid diet

81

increased general control nonderepressible 2 kinase (GCN2)-mediated phosphorylation

82

of eukaryotic initiation factor 2α (eIF2α) in anterior piriform cortex (APC) and led to

83

food aversion 20-21. In addition, amino acid deprivation also led to the accumulation of 5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 41

22.

84

uncharged tRNA, which induced phosphorylation of eIF2α via GCN2

Dietary

85

BCAAs level is not only strongly related to plasma BCAAs level during the absorptive

86

phase but also post‐absorptive phase 23. However, the results regarding oral or central

87

BCAAs supplementation on feed intake are still inconsistent 6. Therefore, whether

88

BCAAs deficient diet can regulate feed intake through the activation of GCN2 in animal

89

models is still unknown. Thus, the aim of this study was to investigate whether BCAAs

90

deficient or over supplemented diet could regulate the feed intake of piglets through

91

brain and gut.

92 93

MATERIALS AND METHODS

94

Animals, Experimental diets and Sample collection. All the procedures

95

performed in animal feeding and sample harvesting during this study were approved by

96

the South China Agricultural University Animal Care and Use Committee (No.

97

20110107-1, Guangzhou, China). Four experimental diets based on maize and soybean

98

meal were listed as follows: normal protein diet (NP, positive control group, 20.04%

99

crude protein), reduced-protein negative control diet (RP, negative control group, 17.05%

100

crude protein), reduced-protein test diets supplemented with two doses of BCAAs

101

(BCAA1 and BCAA2 diets) (Table 1). Crystal BCAAs (0.13% L-isoleucine, 0.09 % L-

102

leucine and 0.23 % L-valine) were supplemented to the BCAA1 diet to meet the

103

requirement of standardized ileal digestibility amino acids (SID AAs) as recommended

104

in National Research Council (NRC) (2012). In the BCAA2 diet, BCAAs were

105

supplemented to reach 150% of SID AA requirement according to the NRC (2012). In 6

ACS Paragon Plus Environment

Page 7 of 41

Journal of Agricultural and Food Chemistry

106

order to make identical nitrogen concentrations in the experimental diets, 0.33 % L-

107

alanine was supplemented to the RP diet. The same diets were used in both Expt. 1 and

108

Expt. 2.

109

Expt. 1 was design to explore whether the bitterness of BCAA could affect the

110

feed intake of piglets at each meal (short term regulation). In this experiment, twenty-

111

four weaning piglets (Duroc × Landrace × Large White) were raised individually in

112

metabolic cages (1.40 × 0.68 × 0.90 m3) in environmental control rooms (room

113

temperature was set at 30°C). After 3-d adaptation period, 24 piglets (9.45 ± 0.60 kg)

114

were assigned to one of four treatments with a completely randomized design based on

115

initial body weight and gender and this entire experiment lasted for 3 days. All piglets

116

were fasted overnight (16:00 p.m. to 8:00 a.m.) and then individually fed with 100 g of

117

NP, RP, BCAA1 or BCAA2 diets, respectively. After the consumption of the test diets,

118

all the piglets have free access to the normal protein diet and the feed intake of piglets

119

was recorded daily.

120

In Expt. 2, twenty-four piglets (10.45 ± 0.41 kg) were classified into four treatment

121

groups according to their body weight and gender (Table 1). Piglets were given ad

122

libitum access to feed and water during the experiment. Feed intake was recorded every

123

day. To calculate average daily gain (ADG), average daily feed intake (ADFI) and feed

124

converse ratio (FCR), body weight of piglets was measured on the morning of days 7,

125

14 and 21 after overnight fasting. Blood samples were collected from the jugular vein

126

on 7 and 14 days (not fasting state). Subsequently, on the morning of day 21, blood

127

samples were collected from all piglets through the jugular vein (fasting state). Plasma 7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

128

samples were used to analyze hormones (blood samples from day 7 and 14) and AAs

129

(blood samples from day 21). All the piglets were sacrificed by electrocution at the end

130

of the experiment. Hypothalamus, duodenum (at a point 5 cm from the pylorus) and

131

jejunum were snap-frozen in liquid nitrogen and then stored at -80 °C for further

132

analysis. Part of duodenum and jejunum were fixed with 4% paraformaldehyde solution

133

and then analyzed for H&E staining.

134

Chemical Analyses. Dietary crude protein (CP) content (Table 1) was measured

135

according to the method of the Association of Official Analytical Chemists (AOAC,

136

2003). To analyze the content of dietary AAs, feed samples were acid-hydrolyzed by 6

137

N HCl for 23 h at 110 °C, before being analyzed by ion-exchange chromatography

138

(Hitachi L-8800 Amino Acid Analyzer, Tokyo, Japan) (AOAC, 2003). The level of

139

cystine and methionine was measured after performic acid oxidation before acid

140

hydrolysis and tryptophan content was determined after 16 h alkaline hydrolysis under

141

120°C (AOAC, 2003), both of which were then separated by reverse phase high-

142

performance liquid chromatography (HPLC) (Waters 2690, Waters, Milford, MA,

143

USA). Plasma AA concentrations were determined by an Ion-Exchange

144

Chromatography (S-433D Amino Acid Analyzer, Sykam, Germany). Concentrations

145

of CCK, PYY, GLP-1 were analyzed by the Pig Cholecystokinin ELISA Kit (CSB-

146

E10131p, CUSABIO, Wuhan, Hubei, China), the Pig peptide YY ELISA Kit (CSB-

147

EL019128PI, CUSABIO, Wuhan, Hubei, China), and the Pig Glucagon Like Peptide 1

148

ELISA Kit (CSB-EQ027281PI, CUSABIO, Wuhan, Hubei, China), respectively .

8

ACS Paragon Plus Environment

Page 8 of 41

Page 9 of 41

Journal of Agricultural and Food Chemistry

149

Intestinal Histology. In order to analyze the structure of small intestine, 3 cm

150

duodenum and jejunum segments were separated, washed and fixed in 4%

151

paraformaldehyde overnight. According to standard paraffin-embedding techniques,

152

the intestinal samples were embedded in paraffin, sectioned (5 μm) and then stained

153

with haematoxylin and eosin (H&E) for histopathological evaluation. The slides were

154

imaged using Nikon imaging system (Nikon YS100; Nikon Corporation, Tokyo, Japan).

155

Villus height was measured from the tip to the crypt-villous junction, and crypt depth

156

was measured from the crypt neck to the crypt base 24. The ratio of villus height to crypt

157

base (V:C) was calculated based on villus height and crypt depth.

158

RNA Extraction and Relative qualification of mRNA. RNAiso Plus (Takara,

159

Dalian, Liaoning, China) was used to isolate total RNA from the duodenum, jejunum

160

and hypothalamus of piglets according to the manufacturer’s instructions. The quality

161

and quantity of the RNA were measured by NanoDrop spectrophotometer (NanoDrop

162

Technologies, Wilmington, DE, USA). Complementary cDNA was synthesized using

163

a PrimeScript first Strand cDNA Synthesis Kit (Takara, Dalian, Liaoning, China). The

164

20 μL reaction volume of real-time PCR was conducted using an ABI Prism 7500

165

Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA), which

166

containing 10 μL SYBR Green PCR Master Mix (Takara, Dalian, Liaoning, China) , 2

167

μL cDNA, 0.8 μL of forward and reverse PCR primers (10 μM , as shown in Table 2),

168

0.4 μL ROX , and 6 μL dd water. Cycling conditions that we used for PCR program

169

were as follows: (1) denaturation at 94 °C for 5 min; (2) repeated 40 cycles of (94°C

9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

170

for 30s, 60°C for 30s, and 72°C for 30s). The 2-ΔΔCt method was used to analyzed the

171

expression of targeted genes25.

172

Immunoblotting Analysis. Total proteins of duodenum, jejunum and

173

hypothalamus tissues were extracted using RIPA Lysis Buffer (Beyotime, Shanghai,

174

China). Protease inhibitor PMSF and phosphatase inhibitor (Beyotime, Shanghai,

175

China) were added to RIPA prior to use. Protein concentration was determined using a

176

BCA protein assay kit (Beyotime, Shanghai, China). A total of 20 μg protein from each

177

sample was resolved on 8% SDS-PAGE gel, transferred to nitrocellulose membranes

178

(Millipore, Bedford, MA, USA), and blocked with 5% skimmed milk for 2 h at room

179

temperature. After rinsing with TBST buffer 5 times, blots were incubated with primary

180

antibody at 4oC overnight. Antibody against β-actin (bs-0061R) was bought from Bioss

181

(Beijing, China). Other antibodies against T1R1 (ab230788), T1R3 (ab150525), p-

182

eIF2α (ab32157), p-GCN2(ab75836) are obtain from Abcam (Cambridge, MA, USA).

183

After washing, all the membranes were incubated with corresponding secondary

184

antibodies at room temperature for 1.5 h. After rinsing, blots were detected using the

185

ECL Plus chemiluminescence detection kit (Applygen Technologies Inc., Beijing,

186

China) performed on a FluorChem M system (ProteinSimple, Santa Clara, CA, USA).

187

The final results of bands were analyzed with Image Processing Software (Image Pro

188

Plus 6.0) (Rockville, MD, USA).

189

Statistical Analysis. Statistical analysis of all data was performed using the SAS

190

9.4 (SAS institute., Cary, NC). Data were analyzed by one-way ANOVA using the

10

ACS Paragon Plus Environment

Page 10 of 41

Page 11 of 41

Journal of Agricultural and Food Chemistry

191

Mixed procedure of SAS. The statistical model included replicate of pigs as the random

192

effect and diet as the fixed effect. The significant difference between different dietary

193

treatments were separated by Student–Newman–Keuls (SNK) test. P ≤ 0.05 was

194

considered as significant and 0.05

0.10) among

199

piglets fed with different diets. In Expt. 2, the growth performance of piglets is listed

200

in Table 4. During the 1st week, no differences (P > 0.10) were observed in growth

201

performance among NP, RP, BCAA1 and BCAA2 groups. Compared with the NP

202

group, ADG and ADFI in the RP group were significantly decreased (P < 0.05) in the

203

2nd and 3rd weeks. Interestingly, the BCAA1 group showed an increase (P < 0.05) in

204

ADG and ADFI compared with the RP group, demonstrating that supplementing

205

sufficient amount of BCAAs can rescue the growth deficiency in the RP group.

206

However, extra BCAAs supplementation in the BCAA2 group was not able to promote

207

growth performance as showed in the BCAA1 group. In the 2nd and 3rd weeks as well

208

as the whole experimental period, G: F ratios were significantly reduced in the RP group

209

compared with the NP group, which were reversed when BCAAs were supplemented

210

in BCAA1 and BCAA2 groups (P < 0.05).

211

Plasma Free Amino Acid Concentrations. Concentrations of plasma free amino

11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

212

acid are shown in table 5. Compared with the NP group, the plasma urea concentration

213

was significantly declined in the BCAA1 group (P < 0.05). Piglets fed with the RP,

214

BCAA1 and BCAA2 diets had a higher (P < 0.05) plasma concentrations of lysine than

215

pigs fed the NP diet. While pigs in the NP, RP and BCAA1 groups had a lower (P