Dietary Branched-chain Amino Acids Regulate Food Intake Partly

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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

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Journal of Agricultural and Food Chemistry

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Dietary Branched-chain Amino Acids Regulate Food Intake Partly

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through Intestinal and Hypothalamic Amino Acid Receptors in Piglets

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

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Wutai Guan*,†,‡, Shihai Zhang*,†,‡

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†Guangdong

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

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‡College

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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

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ACS Paragon Plus Environment


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ABSTRACT

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

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

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

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

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

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

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

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

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

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

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digestibility BCAAs requirement as recommended by the National Research Council

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

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

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piglets was significantly increased after sufficient BCAAs was supplemented in the

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

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

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

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

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

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

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


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

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activating GCN2/eIF2α pathways in the hypothalamus.

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

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reduced-protein diet

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INTRODUCTION

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

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

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

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

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

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were supplemented back to the diet

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

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Intracerebroventricular administration of leucine activated mTOR signaling pathway in

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

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

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

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

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

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significantly reduced feed intake

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supplementation

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

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unknown.

11.

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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

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Animals detect and sense diverse dietary nutrients via intestinal enteroendocrine

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

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

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


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

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

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(GLP-1)

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

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

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

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

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and protein have been deciphered by scientists around the world

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

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

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

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

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

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

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

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

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

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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.

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

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

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

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



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22.

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uncharged tRNA, which induced phosphorylation of eIF2α via GCN2

Dietary

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

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

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

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

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

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

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brain and gut.

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MATERIALS AND METHODS

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Animals, Experimental diets and Sample collection. All the procedures

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

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

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

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

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

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

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

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

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

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

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


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

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

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Expt. 2.

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

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

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

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

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

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

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

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

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

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

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was recorded daily.

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

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

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

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

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

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

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

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



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

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

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

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

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

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and then analyzed for H&E staining.

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

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

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

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

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

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cystine and methionine was measured after performic acid oxidation before acid

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

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

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

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

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

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

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

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

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

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

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

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paraformaldehyde overnight. According to standard paraffin-embedding techniques,

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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expression of targeted genes25.

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Immunoblotting Analysis. Total proteins of duodenum, jejunum and

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

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

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

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

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

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

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

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

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

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

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After washing, all the membranes were incubated with corresponding secondary

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

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

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

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

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Plus 6.0) (Rockville, MD, USA).

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

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

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

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

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

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considered as significant and 0.05
0.10) among

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

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

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

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

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

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

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

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

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

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

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

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in BCAA1 and BCAA2 groups (P < 0.05).

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Plasma Free Amino Acid Concentrations. Concentrations of plasma free amino

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

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

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

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

Dietary Branched-chain Amino Acids Regulate Food Intake Partly

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