Effects of Low-Protein Diets Supplemented with Branched-Chain

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Effects of Low-Protein Diets Supplemented with Branched-Chain Amino Acids on Lipid Metabolism in White Adipose Tissue of Piglets Yinghui Li, Hongkui Wei, Fengna Li, Yehui Duan, Qiuping Guo, and Yulong Yin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00488 • Publication Date (Web): 15 Mar 2017 Downloaded from http://pubs.acs.org on March 16, 2017

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Effects of Low-Protein Diets Supplemented with Branched-Chain Amino Acids

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on Lipid Metabolism in White Adipose Tissue of Piglets

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Yinghui Li,†,‡ Hongkui Wei,§ Fengna Li,*,†,# Yehui Duan,†,‡ Qiuping Guo,†,‡ and

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Yulong Yin*,†,⊗

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†

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in South-Central, Ministry of Agriculture, Hunan Provincial Engineering Research

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Center of Healthy Livestock, Key Laboratory of Agro-ecological Processes in

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Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of

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Sciences, Changsha, Hunan 410125, China

Scientific Observing and Experimental Station of Animal Nutrition and Feed Science

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‡

University of Chinese Academy of Sciences, Beijing 100039, China

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§

College of Animal Sciences, Huazhong Agricultural University, Wuhan, Hubei

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430070, China

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#

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Collaborative Innovation Center for Utilization of Botanical Functional Ingredients,

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Changsha, Hunan 410128, China

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⊗Laboratory

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Normal University, Changsha, Hunan 410018, China

Hunan Co-Innovation Center of Animal Production Safety, CICAPS; Hunan

of Animal Nutrition and Human Health, School of Biology, Hunan

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ABSTRACT: This study was to evaluate the effect of branched-chain amino acids

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(BCAA) supplementation in low-protein diets on lipid metabolism in dorsal

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subcutaneous adipose (DSA), abdominal subcutaneous adipose (ASA), and perirenal

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adipose (PRA) tissues. A total of 24 piglets were allotted to 4 treatments, and each

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group fed the adequate protein (AP) diet, low-protein (LP) diet, LP diet supplemented

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with BCAA (LP + B), or LP diet supplemented with twice BCAA (LP + 2B). Serum

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concentrations of leptin in the BCAA supplemented treatments were higher (P < 0.01)

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than the AP treatment, but lower (P < 0.01) than the LP treatment. In DSA, the

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mRNA and protein levels for lipogenic-related genes were highest in the LP treatment

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and lowest in the LP + 2B treatment. However in ASA and PRA, the expression

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levels for those genes were significantly elevated in the LP + 2B treatment. In

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conclusion, BCAA supplementation could alter the body fat condition, and this effect

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was likely modulated by the expression of lipid metabolic regulators in DSA, ASA,

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and PRA in a depot-specific manner.

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KEYWORDS: branched-chain amino acids, lipid metabolism, low-protein diet,

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piglet, white adipose tissue

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INTRODUCTION

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Data available in the literature have confirmed that slightly reducing the dietary crude

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protein (CP) level and using four crystalline essential amino acids (AA, lysine,

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methionine, threonine, and tryptophan) enable the maintenance of growth

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performance and maximization of AA utilization for protein accretion1-3. Some

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studies further showed that except those four essential AA, branched-chain amino

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acids (BCAA, including leucine, isoleucine and valine) supplemented in low CP diets,

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can also enhance growth performance 4. Of note, in vivo and in vitro experiments

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have indicated that the white adipose tissue (WAT) is capable of metabolizing

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substantial amounts of BCAA 5, and it is well-established that BCAA function as

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direct-acting nutrient signals involved in the regulation of protein synthesis in WAT 6,

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but whether BCAA play the unique role in lipid metabolism of WAT receives much

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less attention at present.

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The WAT is a major energy reservoir and endocrine organ that regulates the energy

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metabolism homeostasis and nutritional status of the whole body by secreting

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adipokines 7. Normally, it is divided into subcutaneous and visceral adipose depots

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according to the anatomical location, and the adipose depots are characterized by

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metabolic differences that is due to genetic regulation of pre-adipocyte differentiation

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and the local microenvironment 8. Adipose tissue accumulation involves various and

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complicated process including fatty acid uptake, oxidation and transport, lipogenesis

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and lipolysis 9, which can be influenced by the nutrition level of diets. We have

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sounded the alarm concerning the worldwide epidemic growth in obesity, especially,

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prevalence of childhood obesity has been steadily increasing over the past several

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decades, and those obese patients are advised to change their unhealthy lifestyle, like

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diet. Therefore, piglet, an optimal and well-accepted model for childhood obesity and

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adipose tissue metabolism research 10, 11, was used in the present study and which was

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expected to provide important insights into diet-induced childhood obesity.

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A previous study showed that doubling the intake of dietary leucine decreased 12

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the body weight and fat deposition of mice fed a high-fat diet

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investigators have found mice fed any kind of BCAA-deficient diets experienced a

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dramatic drop in WAT

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leucine manipulation had no effect on lipid metabolism

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discrepancy and the underlying mechanism of the feeding response to BCAA

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treatments is still unclear. The objective of this study was to investigate the effect of

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BCAA supplementation on lipid metabolism in different adipose depots of piglets fed

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the low-protein diets.

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MATERIALS AND METHODS

. In contrast, some

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. In addition, another independent study reported that 15

. The reason for this

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Animals and Experimental Design. All procedures outlined in this study were

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approved by the Animal Care and Use Committee of the Chinese Academy of

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Sciences. A total of 24 healthy crossbred barrows (Duroc × Landrace × Large White,

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8.45 ± 0.56 kg, weaned at 28 d) were allotted to 1 of 4 treatments using a completely

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randomized design. There were 6 piglets per treatment. The experimental diets were

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based on corn-soybean meal, and 4 diets were formulated, including: (1) a

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recommended adequate protein (AP) diet containing 20.0% CP; (2) a low-protein (LP)

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diet containing 17.0% CP; (3) the LP diet supplemented with BCAA (LP + B)

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containing 17.0% CP, to contain the same level as that of the AP diet; (4) the LP diet

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supplemented with twice BCAA (LP + 2B) containing 17.3% CP. All diets were

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formulated to be isoenergetic, and four limiting AA (lysine, methionine, threonine,

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and tryptophan) were kept at the same level by adding crystalline AA in the LP diet to

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meet the NRC-recommended requirements (NRC,2012), their ingredients are shown

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in Table 1. Piglets were housed individually in environmentally controlled pens. Each

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pen was equipped with a stainless steel self-feeder and a nipple drinker. Piglets had

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free access to feed and water throughout the 28 d trial period. At the beginning and

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the end of the experiment, all piglets were weighed after an overnight fast, feed intake

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and feed refusals were recorded daily to calculate average daily gain (ADG), average

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daily feed intake (ADFI), and gain:feed (G:F).

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Sample Collection. Blood samples (5 mL) from the overnight fasting piglets were

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obtained by anterior vena cava puncture using anticoagulant-free vacuum tubes, kept at

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37°C for 30 min, and centrifuged at 3000g for 15 min at 4°C. The supernatant (serum)

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was collected and stored at -20°C until analysis. After blood sampling, piglets were

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electrically stunned (250 V, 0.5 A, for 5-6 s) and bled by exsanguination in a

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slaughterhouse. The head was removed, and the carcass was split longitudinally. The

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left side of each carcass was weighed. Samples were collected from the dorsal

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subcutaneous adipose (DSA) at the 10th rib of the carcass, the abdominal

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subcutaneous adipose (ASA) at the 10th rib of the carcass, and the perirenal adipose

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(PRA) near the kidneys and inside the abdomen, rapidly frozen in liquid nitrogen and

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stored at -80°C for RNA extraction and Western blot analysis.

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Serum Analysis. The concentrations of high-density lipoprotein-cholesterol

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(HDL-C), low-density lipoprotein-cholesterol (LDL-C), and total cholesterol (TC) in

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serum were measured using the Biochemical Analytical Instrument (Beckman CX4

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Chemistry Analyzer; Beckman Coulter Inc., Brea, CA) and commercial kits

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(Sino-German Beijing Leadman Biotech Ltd., Beijing, China). The concentrations of

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leptin and adiponectin were measured using the commercial ELISA kits (Cusabio

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Biotech Co., Ltd., Wuhan, China). The concentration of non-esterified fatty acid

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(NEFA) and the activity of lipase were analyzed with corresponding commercial kits

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(Nanjing Jiancheng Biochemical Reagent Co., Nanjing, China) following the

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recommended procedures.

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Quantitative Real-time PCR Analysis. The mRNA expression of genes related to

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the lipid metabolism including: (1) lipogenesis: acetyl-CoA carboxylase α (ACC),

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fatty acid synthase (FASN), and glycerophosphate dehydrogenase (GPDH); (2)

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lipolysis: carnitine palmitoyl transferase 1B (CPT1B), hormone-sensitive lipase

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(HSL), and adipose triglyceride lipase (ATGL); (3) lipid uptake: fatty acid translocase

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(FAT/CD36), fatty acid transport protein 1 (FATP1), and fatty acid binding protein 4

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(FABP4); (4) transcription factors: CCAAT/enhancer-binding protein α (C/EBPα),

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peroxisome proliferator-activated receptor γ (PPARγ), and sterol regulatory element

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binding protein-1c (SREBP-1c) in DSA, ASA and PRA tissues were detected by

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Real-time PCR as described previously

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relative expression was expressed as a ratio of the target gene to the control gene

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using the formula 2–∆∆Ct, where ∆∆Ct = (Cttarget – Ctβ-actin)treatment – (Cttarget –

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Ctβ-actin)control 18.

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. The primers are listed in Table 2. The

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Western Blot Analysis. Relative protein levels for FABP4, PPARγ, and

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extracellular signal-regulated protein kinases 1/2 (ERK1/2) in DSA, ASA, and PRA

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tissues were determined by Western blot technique as we described previously

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The primary antibodies used in this study are FABP4 (catalog number Sc-18661;

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Santa Cruz Biotechnology Inc., Santa Cruz, CA), PPARγ (catalog number 2435; Cell

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Signaling Technology Inc.), and phospho (p)- 44/42 ERK1/2 (Thr202/Tyr204, catalog

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number 9101; Cell Signaling Technology Inc., Danvers, MA) at a 1:1000 dilution.

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Mouse anti-β-actin (Santa Cruz Biotechnology Inc.) or ERK1/2 (Cell Signaling

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Technology Inc.) diluted at a 1:1000 were used as internal control.

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.

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Statistical Analysis. Data were analyzed by ANOVA using SAS 9.2 software

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(SAS Institute Inc., Cary, NC) followed by a Duncan’s multiple-comparison test.

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Effects were considered statistically significant at P < 0.05 and supposed as tendency

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when 0.05 ≤ P ≤ 0.10.

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RESULTS

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Growth Performance. Growth performance data is presented in Table 3. Piglets

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fed the LP diet had the lowest (P < 0.05) final body weight, ADG, ADFI, G:F, and

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half carcass weight among the treatments, whereas there were no differences among

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those fed the AP, LP + B, and LP + 2B diets. Particularly, G:F and half carcass weight

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tended to increase in piglets fed the LP + B and LP + 2B diets compared with those

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fed the AP diet.

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Lipid-Related Metabolite and Hormone Levels in Serum. As listed in Table 4,

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the concentrations of HDL-C, LDL-C, TC, NEFA, lipase, and adiponectin in serum

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were not affected by the dietary treatments. However, in comparison with the AP

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treatment, serum leptin concentration was increased (P < 0.01) by approximately 55%

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in the LP treatment, and the LP + B and LP + 2B treatments also exhibited an increase

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(P < 0.01) but still lower (P < 0.01) than that of the LP treatment.

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Gene Expression of Lipid Metabolic Regulators in DSA. As shown in Figure 1A

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and 1B, in DSA, piglets fed the LP diet showed greater (P < 0.05) mRNA levels for

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ACC, FASN, CD36, FABP4, C/EBPα, and PPARγ than those fed the AP diet.

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However, piglets fed the LP + B and LP + 2B diets had no significantly differences in

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the mRNA levels for the above-mentioned genes compared with those fed the AP diet.

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Notably, ACC, FASN, FABP4, and PPARγ in the LP + 2B treatment expressed

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slightly lower mRNA levels than those in the LP + B treatment.

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The Western blot analysis in DSA revealed a pronounced elevation (P < 0.05) in

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the protein levels for FABP4, P-ERK1/2, and PPARγ in piglets fed the LP diet,

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compared with those fed the other three diets. However, in comparison with the AP

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treatment, the LP + 2B treatment reduced (P < 0.05) the expression levels for these

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proteins, but there were no significant differences between the AP and LP + B

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treatments (Figure 1C).

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Gene Expression of Lipid Metabolic Regulators in ASA. Figure 2 A and 2B

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showed that in ASA, the mRNA levels for ACC, CD36, FATP1, and FABP4 in the

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LP and LP + B treatments were lower (P < 0.05) than those in the AP treatment, but

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no significant differences were observed between the LP + 2B and AP treatments.

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The mRNA levels for FASN and PPARγ in the LP + 2B treatment appeared to be

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higher (P < 0.05) than in the AP and LP treatments.

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The protein levels for FABP4 and P-ERK1/2 in the LP and LP + B treatments were

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lower (P < 0.05) than those in the AP and LP + 2B treatments, which were not

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significantly different. With the rise of mRNA level for PPARγ in the LP + 2B

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treatment, as expected, we also found that the protein level to rise in the same fashion

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(Figure 2C).

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Gene Expression of Lipid Metabolic Regulators in PRA. In PRA (Figure 3A and

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3B), the mRNA levels for ACC, FABP4 and PPARγ were not significantly different

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among the AP, LP, and LP + B treatments, but the LP + 2B treatment up-regulated (P

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< 0.05) or tended to up-regulate their mRNA levels compared with the other three

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treatments. The mRNA levels for FASN and SREBP-1c were down-regulated (P

Effects of Low-Protein Diets Supplemented with Branched-Chain

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