Effects of reducing dietary protein on the expression of nutrition sensing genes (amino acid transporters) in weaned piglets

Effects of reducing dietary protein on the expression of nutrition sensing genes (amino acid transporters) in weaned piglets 降低日粮粗蛋白水平对断奶仔猪营养感受体基因（氨基酸转运载体）表达的影响 first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:tjli@isa.ac.cn †E-mail:tjli@isa.ac.cn tjli@isa.ac.cn Research Center of Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Science, Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China Research Center of Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Science, Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China The effects of crude protein (CP) levels in the diet on the mRNA expression of amino acid (AA) transporters were studied in a 45-d trial. Eighteen piglets with an initial body weight (BW) of 9.57 kg were assigned to three groups (14%, 17%, and 20% CP in the diet) in a completely randomized design (six replicates per treatment). Diets were supplemented with crystalline AA to achieve equal standardized ileal digestible contents of Lys, Met plus Cys, Thr, and Trp, and were provided ad libitum . After 45 d, all piglets were slaughtered to collect small intestine samples. Compared with the values in the 14% CP group, the expressions of ASCT2 , 4F2hc , and ATB 0 mRNA in the jejunum were increased by 23.00%, 12.00%, 6.00% and 48.00%, 47.00%, 56.00% in the 17% and 20% CP groups, respectively. These results indicate that a 14% CP diet supplemented with crystalline AA may not transport enough AA into the body and maintain growth performance of piglets. However, a reduction of dietary 17% CP may reduce the excretion of nitrogen into the environment while supporting the development of piglets. Therefore, the 17% CP level is more suitable than 14% CP level. 目的 2 验证日粮中粗蛋白质浓度对断奶仔猪氨基酸转运载体的影响效应。 创新点 2 从营养物质感应体的角度分析采用低氮日粮的可行性，并探索营养限制对营养转运体的影响及它们间的相互作用。 方法 2 十八头初始体重9.57公斤的断奶仔猪被随机分为三组（每组6个重复），分别饲喂含有14%、17%和20%的粗蛋白日粮45天。按照理想蛋白质模型，日粮分别添加赖氨酸、蛋氨酸+半胱氨酸、苏氨酸和色氨酸来满足断奶仔猪的需要，整个试验期自由采食。试验结束后，屠宰仔猪并采集小肠样品。与14%粗蛋白组相比，空肠中 ASCT2 、 4F2hc 和 ATB 0 mRNA表达在17%和20%粗蛋白水平组分别上升23.00%、12.00%、6.00%和48.00%、47.00%、56.00%。 结论 2 结果表明，14%粗蛋白水平组外源添加合成氨基酸并不能促进氨基酸转运载体增加氨基酸的吸收来满足断奶仔猪的生长性能。然而，17%粗蛋白水平组可以减少氮排放到环境中，同时又能满足此阶段断奶仔猪的生长发育。因此，17%粗蛋白水平组对这个阶段断奶仔猪最合适。 Crude protein Amino acid balance Amino acid transporters 粗蛋白 氨基酸平衡 氨基酸转运载体 Introduction 1 It has been well documented that low crude protein (CP), amino acid (AA)-supplemented diets may reduce feed costs and nitrogen excretion (Kerr et al., 2003 2003 ; Zarate et al., 2003 2003 ; Fairbrother et al., 2005 2005 ; Deng et al., 2007a 2007a ; 2007b 2007b ; Lallès et al., 2007 2007 ; Hou et al., 2008 2008 ; Kang et al., 2008 2008 ; Yue and Qiao, 2008 2008 ; Deng et al., 2009 2009 ; Opapeju et al., 2009 2009 ; Gallo et al., 2014 2014 ; Gloaguen et al., 2014 2014 ; Liu et al., 2014 2014 ; Recktenwald et al., 2014 2014 ). Therefore, the CP level in the diet of weaned pigs has been suggested to be one of the main factors that affect their growth performance (Gallo et al., 2014 2014 ), feed efficiency, and gastrointestinal health (Yin and Tan, 2010 2010 ; Acciaioli et al., 2011 2011 ). An increase in the entry of nutrients (e.g. dipeptides, tripeptides, arginine, lysine, and histidine) from the lumen of the small intestine into the enterocyte can enhance tissue protein synthesis and improve the efficiency of utilization of dietary nutrients, and it can selectively modulate the gene expression of AA transporters in intestinal cells (Wang et al., 2009 2009 ). Some studies have reported that improving the efficiency of protein use and maintaining performance can be achieved by reducing the CP content of the diet while ensuring the AA balance (Kerr and Easter, 1995 1995 ; Le Bellego and Noblet, 2002 2002 ; Kerr et al., 2003 2003 ; Opapeju et al., 2008 2008 ; Gloaguen et al., 2014 2014 ). However, Nyachoti et al. ( 2006 2006 ) demonstrated that piglet performance may be impaired when dietary CP levels are reduced from 230 to 190 or 170 g/kg, as the AA deficiency may change the efficiency of AA absorption and transportation (Tuitoek et al., 1997 1997 ; Tan et al., 2009 2009 ; 2011 2011 ; Gallo et al., 2014 2014 ). The present study investigated the effects of reducing dietary CP while supplementing with essential AA on the gene expression of AA transporters in weaned piglets. Materials and methods 1 Animals and experimental treatment 2 The experimental protocol was approved by the Protocol Management and Review Committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences (CAS). Pigs were cared for according to the guidelines of the Institute of Subtropical Agriculture on Animal Care, CAS. Eighteen piglets (Duroc×Landrace×Yorkshire, female), from the experimental field of the animal observation station in southern center of China, were weaned at 28 d of age, but the experiment started after a 7-d adaptation period when the piglets were weighed to determine an initial mean body weight (BW; mean±standard deviation (SD), (9.57±0.64) kg), and randomly assigned to three different groups (six pigs per group). Each of the groups was fed diets with different levels of CP (14%, 17%, and 20%), respectively. Their ingredients are shown in Tables 1 1 and 2 2 . The dietary treatment met the National Research Council (NRC; 2012) nutrient specifications for 11 to 20 kg BW pigs. All of the animals were housed individually in cages, and had free access to feed and drinking water at all times throughout the experimental period. Diets contain 14%, 17%, and 20% CP, respectively, with appropriate crystalline AA supplementation The values are expressed as percentage (%), except for digestible energy (DE; MJ/kg), essential AA (EAA)/nonessential AA (NEAA), and recommendation rate of NRC (2012) Premix provided these amounts of vitamins and minerals per kilogram on an as-fed basis: vitamin A, 10 800 IU; vitamin D 3 , 4000 IU; vitamin E, 40 IU; vitamin K 3 , 4 mg; vitamin B 1 , 6 mg; vitamin B 2 , 12 mg; vitamin B 6 , 6 mg; vitamin B 12 , 0.05 mg; biotin, 0.2 mg; folic acid, 2 mg; niacin, 50 mg; D-calcium pantothenate, 25 mg; Fe, 100 mg as ferrous sulfate; Cu, 150 mg as copper sulphate; Mn, 40 mg as manganese oxide; Zn, 100 mg as zinc oxide; I, 0.5 mg as potassium iodide; and Se, 0.3 mg as sodium selenite 2 Component 3 Content (%) b b 14% CP 17% CP 20% CP Ingredient  Corn 71.80 66.50 63.70  Soybean meal 13.40 18.80 19.80  Whey powder 4.40 4.30 4.30  Fish meal 1.50 4.00 9.00  Soybean oil 4.10 2.60 0.80  Lys 0.88 0.62 0.38  Met 0.27 0.19 0.10  Thr 0.33 0.21 0.09  Trp 0.08 0.04 0.01  Calcium hydrophosphate 1.15 0.74 0.00  Limestone 0.79 0.70 0.52  Salt 0.30 0.30 0.30  1% premix compound c c 1.00 1.00 1.00  Total 100.00 100.00 100.00 Calculated nutrient content  DE (MJ/kg) 14.60 14.60 14.60  CP 14.00 17.00 20.00  Total Ca 0.70 0.71 0.69  Total P 0.53 0.55 0.57  Arg 0.75 0.91 1.09  His 0.34 0.40 0.46  Ile 0.49 0.60 0.70  Leu 1.15 1.32 1.49  Lys 1.23 1.23 1.23  Met+Cys 0.68 0.68 0.68  Phe 0.59 0.69 0.80  Thr 0.73 0.73 0.73  Trp 0.20 0.20 0.20  Val 0.53 0.65 0.77 EAA/NEAA 0.90 0.80 0.70 Recommendation rate of NRC (2012) 0.80 0.80 0.80 Diet contain 14%, 17%, and 20% CP, respectively, with appropriate crystalline AA supplementation The values are expressed as percentage (%), except for essential AA (EAA)/nonessential AA (NEAA) 2 Component 3 Content (%) b b 14% CP 17% CP 20% CP CP 14.14 17.32 20.27 EAA  Arg 0.71 0.93 1.09  His 0.30 0.37 0.44  Ile 0.46 0.60 0.71  Leu 1.11 1.32 1.52  Lys 1.26 1.25 1.26  Met 0.41 0.42 0.40  Met+Cys 0.63 0.65 0.62  Phe 0.56 0.70 0.81  Thr 0.76 0.75 0.76  Trp 0.20 0.20 0.20  Tyr 0.41 0.50 0.59  Val 0.54 0.64 0.72 NEAA  Ala 0.75 0.90 1.07  Asp 1.15 1.49 1.76  Cys 0.22 0.23 0.22  Glu 2.28 2.78 3.15  Gly 0.53 0.71 0.92  Pro 0.90 1.04 1.17  Ser 0.60 0.74 0.85 EAA 6.29 7.18 7.91 NEAA 6.84 8.40 9.74 EAA/NEAA 0.92 0.85 0.81 Relative quantification of mRNA expression of AA transporters 2 Primers for the selected genes (Table 3 3 ) were designed using Oligo 6.0 software (Molecular Biology Insights, CO, USA). After the quantity of complementary DNA (cDNA) was determined by a NanoDrop® ND-1000 (NanoDrop Technologies, Rockland, DE, USA), real-time quantitative polymerase chain reaction (PCR) analyses were performed on an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems, CA, USA) with a total volume of 10 μl containing 5 ng of cDNA, 5 μl SYBR Green mix, 0.2 μl ROX Reference Dye (50×), and 0.2 μl each of the forward and reverse primers (Yao et al., 2012 2012 ). The following protocol was used: (1) pre-denaturation (30 s at 95 °C); (2) amplification and quantification, repeated 40 cycles (5 s at 95 °C, 30 s at 60 °C); and (3) a melting curve program (extension at 72 °C). β-Actin was used as an internal reference gene to normalize target gene transcript levels. The efficiency of the real-time reverse-transcription PCR was determined by the amplification of a dilution series of cDNA according to the equation 10 (−1/slope) . Target mRNA and β-actin mRNA were amplified with comparable efficiencies (Wang et al., 2009 2009 ; He et al., 2013 2013 ). Negative controls, in which cDNA was replaced by water, were also tested (Liu et al., 2012 2012 ). F: forward; R: reverse Gene Primer sequence (5'→3') Length (bp) Accession No. 2   ASCT2 F: GCTTCCGAGAGCCAAGAACT 152 XM_003127238.3 R: TCCTTAACGCCTGGAAGCTG   EAAC1 F: GCTTCCTTCTTCCAGGGTCC 148 NM_001164649.1   R: CTGGCCAATGTGGCTTGTTC   B 0+ AT F: GAGAGGTTTGGTCTTACTGCG 96 XM_003353809.2   R: GCTATGACCAAGACGGAGCG   y + LAT1 F: TTTGTCTGACCGGCTCTTCC 286 XM_005666262.1   R: GAGATCTCCTGCTGTCCTGG   4F2hc F: CTCGAACCCACCAAGGAC 174 NM_001110171.1   R: GAGGTGAGACGGCACAGAG   ATB 0 F: TGGGGCATTTTGATTGCAGC 238 NM_001166042.1   R: CTCCCCAGTCAGGGTATGGA 2   β-Actin F: GGATGCAGAAGGAGATCACG 130 DQ845171 R: ATCTGCTGGAAGGTGGACAG Statistical analysis 2 All data were subjected to a one-way analysis of variance (ANOVA; SAS Version 9.2) and data were presented as mean±standard error of the mean (SEM). Differences with P values of < 0.05 were considered to be statistically significant. Results 1 Growth performance of piglets fed diets with different levels of CP (internal data) 2 The growth performance of weaned piglets fed diets with different CP levels is unpublished data. After the animals were fed different diets for 45 d, significant differences were noted in the average daily gain (ADG) among the three groups ( P < 0.01), and the 20% CP group had a significantly higher ADG than the other groups ( P < 0.01). Average daily feed intake (ADFI) showed the same trend as ADG: the 20% CP group had the highest ADFI and ADG, and 14% CP group had the lowest ADFI and ADG. However, there was no significant difference in ADFI between the 17% and 20% CP groups. While the 20% CP group had the lowest feed to gain (F/G) ratio, there were no significant differences between the 14% and 17% CP groups. mRNA expression of AA transporters 2 Changes in the abundance of ASCT2 , EAAC1 , B 0+ AT , y + LAT1 , 4F2hc , and ATB 0 mRNA in the jejunum of the piglets are shown in Table 4 4 . While the abundance of ASCT2 , EAAC1 , B 0+ AT , and ATB 0 mRNA significantly differed among the three groups ( P < 0.05), there were no differences in the expression of y + LAT1 and 4F2hc mRNA ( P > 0.05). Compared with the values in the 14% CP group, the expression of ASCT2 , 4F2hc , and ATB 0 mRNA in the jejunum was increased by 23.00%, 12.00%, 6.00% and 48.00%, 47.00%, 56.00% in the 17% and 20% CP groups, respectively. The 17% CP group showed the lowest expression of EAAC1 mRNA, and the highest expression of B 0+ AT mRNA. Furthermore, while the expression of y + LAT1 in the 17% and 20% CP groups was decreased by 13.00% and 8.00%, respectively, compared with that in the 14% CP group, there were no significant differences among the three groups ( P > 0.05). Diets contain 14%, 17%, and 20% CP, respectively, with appropriate crystalline AA supplementation 2 mRNA 3 Relative abundance 2 SEM 2 P -value 14% CP 17% CP 20% CP   ASCT2 1.00 1.23 1.48 0.14 0.0340   EAAC1 1.00 0.94 1.41 0.15 0.0270   B 0+ AT 1.00 1.49 1.44 0.16 0.0160   y + LAT1 1.00 0.87 0.92 0.04 0.7369   4F2hc 1.00 1.12 1.47 0.14 0.0846   ATB 0 1.00 1.06 1.56 0.18 0.0142 Discussion 1 AAs regulate key metabolic pathways that are crucial for the maintenance, health, and growth of animals (Wu, 1998 1998 ; Hu et al., 2008 2008 ). The absorption of AA requires many transporter systems that differ with respect to their substrate specificity and driving force (Wang et al., 2009 2009 ). It has been reported that some genes (such as ASCT2 , EAAC1 , B 0+ AT , y + LAT1 , 4F2hc , and ATB 0 ) that are involved in the control of growth or AA metabolism are regulated by AA availability (Wu, 1998 1998 ; Lallès et al., 2007 2007 ; Zhang et al., 2012 2012 ; Abdulhussein and Wallace, 2014 2014 ). In our study, we found that, with an increase in the dietary CP level, the expression of AA transporter genes ( ASCT2 , EAAC1 , B 0+ AT , 4F2hc , and ATB 0 ) tended to increase, while an opposite trend was seen related to the abundance of y + LAT1 mRNA. A possible reason was that y + LAT1 and EAAC1 have differential expression in our study because the diets with different CP levels related with absorption in piglets. The transport of AA in the small intestine is critical for the supply of AA to all tissues and the homeostasis of serum AA levels (Zhang et al., 2012 2012 ). Since dietary CP can provide an ideal AA level, the genes of AA transporters that regulate protein metabolism in piglets should be more activated. The present results indicate that the expression of ASCT2 , EAAC1 , B 0+ AT , and ATB 0 mRNA in the jejunum was influenced by dietary CP levels. Our results regarding the expression of y + LAT1 mRNA were similar to those of He et al. ( 2013 2013 ). Under conditions of nutritional stress, the expression of some AA transporters ( y + LAT1 ) may be increased to maintain normal growth of animals under adverse conditions (He et al., 2013 2013 ). Skalli et al. ( 2014 2014 ) suggest that dietary protein levels regulate the expression of AA transporter genes by a complex regulatory system, which may also affect the energy balance and endocrine system. AAs and hormones can play an important role in the regulation of gene expression. Overall, the use of low-CP (14%) AA-supplemented diets in piglets may decrease the transport capacity of AA in the small intestine. However, the absorption and transport of AA is a complex physiological process that is influenced by many factors (Wang et al., 2009 2009 ; He et al., 2013 2013 ; Zhang et al., 2013 2013 ; Chen et al., 2014 2014 ), including the dietary CP level and the AA balance. Thus, the transport of these nutrients is a key regulatory step in the use of dietary protein by weaned piglets. 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Compliance with ethics guidelines Li WU, Liu-qin HE, Zhi-jie CUI, Gang LIU, Kang YAO, Fei WU, Jun LI, and Tie-jun LI declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. 10.1631/jzus.B1400259 Project supported by the National Basic Research Program (973) of China (No. 2013CB127301), the Natural Science Foundation of Hunan Province, China (No. 12JJ2014), and the National Natural Science Foundation of China (No. 31402088) 2014-09-26 2015-04-16 2015-03-12 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2015 2015 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 16