低蛋白日粮添加支链氨基酸对仔猪采食量和骨路肌生长的影响及调控机制研究

发布时间：2018-05-01 22:14

本文选题：仔猪 + 低蛋白日粮　；参考：《华中农业大学》2017年博士论文

【摘要】：充足的底物浓度和平衡的氨基酸模式是猪骨骼肌高效蛋白质合成的必要条件。其中,采食量是保障充足的底物浓度的关键。除作为合成蛋白质底物外,支链氨基酸(branched chain amino acids,BCAAs)(包括亮氨酸、异亮氨酸、缬氨酸)可能还发挥生物学调控作用。比如,缬氨酸能够调控猪采食量。此外,几乎所有谷氨酸和天冬氨酸以及大部分谷氨酰胺在肠道发生了分解代谢,导致不可逆的损失。骨骼肌蛋白质合成所需的这三个非必需氨基酸必须要由BCAAs代谢生成。而且BCAAs仅有部分被肠道和肝脏等器官截留。因此,通过日粮中添加BCAAs可能增加骨骼肌内自身代谢生成谷氨酸、谷氨酰胺和天冬氨酸,优化胞内氨基酸模式,促进蛋白质合成。目前已有的研究表明,日粮添加BCAAs可以提高猪生长性能,但其对采食量及骨骼肌蛋白质沉积的调控机制尚待进一步阐明。本研究围绕BCAAs提高仔猪采食及骨骼肌生长的关键问题,通过两轮仔猪饲养试验证实了低蛋白日粮添加BCAAs通过提高采食量和直接促进肌肉生长,改善仔猪生长性能,并进一步阐明了下丘脑感应BCAAs调节仔猪采食量的机制。应用血插管结合代谢组学技术,研究了日粮BCAAs对摄食状态下骨骼肌的氨基酸净利用及代谢的影响,并探究了BCAAs代谢与肌肉氨基酸净利用的关系;应用血插管结合稳定同位素示踪技术,进一步阐明了BCAAs通过自身代谢调控骨骼肌蛋白质合成的机制。最后研究了日粮BCAAs对禁食状态下骨骼肌内调节蛋白质合成与降解的信号通路活性的影响。主要研究内容及结果如下:第一部分:通过两轮仔猪饲养试验研究了低蛋白日粮添加BCAAs对仔猪生长性能、采食量及骨骼肌生长的影响。在试验一中,选取28头断奶仔猪分为4个组,即正对照组、负对照组、试验1组以及试验2组,日粮粗蛋白水平依次为19.5%、16.7%、16.7%和17.2%。其中,试验1组在负对照组基础上添加缬氨酸、异亮氨酸和亮氨酸保证日粮中三种BCAAs水平与正对照组一致,并增加一倍的添加剂量形成试验2组的日粮。自由采食。试验二为配对试验,选取21头断奶仔猪分为3个组,即负对照组、试验1组以及配对组。其中,负对照组和试验1组同试验一。配对组试猪饲喂试验1组的日粮,但采食量与负对照组保持一致。28 d的饲养试验结束后,每组随机选择6头屠宰,测定胴体性状,采集下丘脑及背最长肌样。同时,剥离背最长肌等主要的23块肌肉,分别称量肌肉块重量。主要结果如下:1.与负对照组相比,试验1组和试验2组显著提高了平均日增重、末重、采食量、前躯、中躯和后躯肌肉总重、背最长肌等大多数肌肉块重量以及氮沉积(P0.05),并能恢复到正对照组的水平。当采食量保持一致时,添加BCAAs的配对组仍能显著提高仔猪平均日增重和肉料比(P0.05),并也显著提高了冈上肌、背阔肌、背最长肌等大块肌肉重量(P0.05)。2.与负对照组相比,日粮添加BCAAs以剂量依赖的方式上调下丘脑Agrp和NPY mRNA水平,下调MC4R mRNA水平,并在试验2组达到差异显著水平(P0.05)。此外,试验1组显著下调了CART mRNA水平(P0.05)。回归分析显示,采食量随NPY mRNA水平增加呈二次曲线增加(P=0.02),而随MC4R mRNA水平增加呈线性降低(P=0.02)。表明日粮添加BCAAs对采食量的提高与其上调下丘脑NPY基因表达以及下调MC4R基因表达有关。3.与负对照组相比,试验1组和试验2组显著降低了下丘脑eIF2α磷酸化水平,以及显著提高了S6K1磷酸化水平(P0.05),并能恢复到正对照组的水平。此外,试验1组的mTOR磷酸化水平显著高于负对照组(P0.05)。表明日粮添加BCAAs对采食量的提高与其降低GAAC通路活性以及提高mTORC1通路活性有关。4.与负对照组相比,试验1组和配对组均显著提高了背最长肌S6K1和mTOR磷酸化水平(P0.05),但试验1组和配对组之间差异不显著。说明BCAAs能够激活骨骼肌mTORC1通路活性,且不依赖于采食量的变化。第二部分:利用血插管结合代谢组学技术研究了日粮添加BCAAs对仔猪肌肉氨基酸净利用和股静脉代谢产物谱的影响。试验动物同第一部分的配对试验。28 d的饲养试验结束后,对负对照组及试验1组的试猪分别在股动脉、股静脉、颈动脉和颈静脉安装插管。在术后的第5 d,每组各选择6头健康且插管通畅的试猪进行正式试验。通过股动脉持续9 h灌注对氨基马尿酸(pAH)以测定肌肉血流速度。每隔1 h饲喂以保持试猪始终处于摄食状态,连续饲喂8次。摄食前及摄食后每隔1 h分别采集颈动脉和股静脉血样以供分析pAH、游离氨基酸及支链α酮酸,连续采集8次。待血样采集完成后屠宰,采集股二头肌样用于游离氨基酸及3-甲基组氨酸分析,股静脉血样同时进行代谢组学分析。主要结果如下:1.肌肉血流速度在摄食后显著提高了(P0.05),但不受日粮处理的影响。日粮添加BCAAs显著增加了动脉和肌肉游离的异亮氨酸、亮氨酸以及缬氨酸浓度(P0.05),但显著降低了动脉游离的苯丙氨酸和丝氨酸浓度以及肌肉游离的丝氨酸浓度(P0.05)。同时,添加BCAAs显著降低了肌肉游离的3-甲基组氨酸浓度(P0.05),表明BCAAs抑制骨骼肌蛋白质动员。2.日粮添加BCAAs显著提高了总BCAAs、总必需氨基酸、总非必需氨基酸及总氨基酸的肌肉净利用量(P0.05),从而维持高效的蛋白质沉积。此外,添加BCAAs显著增加了异亮氨酸、亮氨酸、缬氨酸及其代谢产物丙氨酸、谷氨酸和谷氨酰胺的肌肉净利用量(P0.05),同时还显著增加了组氨酸、蛋氨酸以及甘氨酸、脯氨酸和丝氨酸等功能性非必需氨基酸的肌肉净利用量(P0.05)。3.日粮添加BCAAs对股静脉代谢物有显著影响,这些差异化合物与BCAAs代谢和蛋白质合成相关。例如,添加BCAAs显著提高了三种支链α酮酸、谷氨酰胺以及天冬酰胺等代谢产物含量(P0.05);同时也显著提高了二十碳五烯酸等脂质代谢相关产物含量(P0.05)。进一步对产物定量的结果显示,添加BCAAs显著提高了KIC和KMV(分别为亮氨酸和异亮氨酸的转氨产物)的肌肉净生成量(P0.05),提示添加BCAAs增加了骨骼肌内自身的代谢。4.肌肉氨基酸净利用量分别与动脉的亮氨酸、异亮氨酸及缬氨酸浓度呈显著正相关(P0.05),而与肌肉的BCAAs浓度之间无相关性。有趣的是,肌肉氨基酸净利用量分别与KIC和KMV净生成量呈显著正相关(P0.05)。这些结果暗示添加BCAAs可能通过增加动脉浓度及胞内自身代谢来提高肌肉氨基酸净利用量。第三部分:利用血插管结合稳定同位素示踪技术研究了日粮BCAAs通过调控自身代谢影响仔猪骨骼肌蛋白质合成与降解的机制。摄食状态下机制研究的试验动物同第二部分试验。在试猪第一次采食完,通过颈静脉灌注NaH~(13)CO_3,持续2 h。随后灌注[1-~(13)C]亮氨酸,持续6 h。pAH灌注以及血样采集同第二部分试验。血样用于分析亮氨酸、KIC和CO_2浓度及其同位素丰度。禁食状态下机制研究的试验动物同第一部分试验的试验一。采集负对照组和试验1组的背最长肌样。主要结果如下:1.日粮添加BCAAs显著提高亮氨酸的动脉摄入量和净利用量(P0.05),从而显著提高蛋白质合成量以及沉积量(P0.05)。添加BCAAs也显著增加净转氨量(P0.05),但不影响氧化脱羧量。有趣的是,蛋白质降解在添加BCAAs后亦显著增加了(P0.05)。进一步的代谢命运分析显示,肌肉摄取亮氨酸约有30%发生代谢,并主要经转氨作用生成KIC。2.肌肉的[1-~(13)C]亮氨酸净摄取以及[1-~(13)C]KIC净生成在日粮添加BCAAs后也显著提高了(P0.05)。相关分析显示,骨骼肌蛋白质合成分别与蛋白质降解及[1-~(13)C]KIC净生成之间呈显著正相关(P0.05),暗示日粮BCAAs可能通过增加蛋白质降解以及自身的转氨代谢,提高摄食状态下蛋白质合成。3.日粮添加BCAAs显著提高了背最长肌的Akt、mTOR及其下游靶蛋白S6K1的磷酸化水平(P0.05),提示饲喂BCAAs仔猪在禁食状态下骨骼肌具有更高的蛋白质合成能力。此外,添加BCAAs显著增加了FoxO1磷酸化水平(P0.05),相应地下调了Atrogin-1、MuRF1以及LC3-II蛋白的表达(P0.05),提示饲喂BCAAs仔猪在禁食状态下骨骼肌呈现更低水平的蛋白质降解。本研究的主要结论为:(1)低蛋白日粮添加BCAAs可以提高采食量以及直接促进肌肉生长,从而改善仔猪生长性能,并能恢复到饲喂高蛋白日粮的水平;(2)日粮BCAAs可以通过影响下丘脑的食欲调节基因表达及通路活性,提高采食量;(3)日粮BCAAs通过促进自身在肌细胞内的代谢以及蛋白质降解,进而增加氨基酸净利用量,提高摄食状态下骨骼肌蛋白质合成速率;同时,日粮BCAAs通过下调泛素-蛋白酶体以及自噬-溶酶体途径的关键蛋白表达,抑制禁食状态下骨骼肌蛋白质降解,并最终提高蛋白质沉积,从而促进骨骼肌生长。
[Abstract]:Sufficient substrate concentration and balanced amino acid patterns are essential for the synthesis of high efficient protein in porcine skeletal muscles. In addition, feed intake is the key to ensuring sufficient substrate concentration. The branched chain amino acids (BCAAs) (including leucine, isoleucine, valine) may also be produced as a synthetic protein substrate. In addition, almost all glutamic acid and aspartic acid, as well as most glutamine, are metabolized in the intestines, causing irreversible loss. The three non essential amino acids needed for the protein synthesis of skeletal muscle must be metabolized by BCAAs. And only part of the BCAAs Therefore, the addition of BCAAs to the diet may increase the metabolism of the skeletal muscle to produce glutamic acid, glutamine and aspartic acid, optimize the intracellular amino acid pattern and promote protein synthesis. Current studies have shown that dietary supplementation of BCAAs can improve the growth performance of pigs, but it is for food intake and bone. The regulation mechanism of muscle protein deposition has yet to be further elucidated. This study focuses on the key problems of improving the growth of piglets and the growth of skeletal muscle around BCAAs. Through two rounds of piglet feeding tests, it is proved that the addition of BCAAs to the low protein diet improves the growth performance of the piglets by increasing the feed intake and directly promoting the growth of the muscle, and further clarifies the hypothalamus sense. The effect of dietary BCAAs on the net use and metabolism of amino acids in skeletal muscles under feeding condition was studied by using blood intubation and metabonomics, and the relationship between the metabolism of BCAAs and the net use of amino acids in the muscle was investigated by using the blood cannula and metabonomics, and the BCAAs was further elucidated by the use of blood intubation and stable isotope tracer technology. The effect of dietary BCAAs on the signaling pathway activity of protein synthesis and degradation in skeletal muscles under fasting state was studied by self metabolism. The main contents and results were as follows: the first part was to study the growth of low protein diet supplemented with BCAAs for piglet birth through two rounds of piglet feeding tests. In the experiment one, 28 weanling piglets were divided into 4 groups, namely, the normal control group, the negative control group, the test 1 and the 2 groups, and the dietary crude protein levels were 19.5%, 16.7%, 16.7% and 17.2%. respectively. The experiment 1 added valine, isoleucine and leucine on the basis of the negative control group. The three BCAAs levels in the diet were the same as those in the normal control group, and increased the dosage form of the 2 groups to form the diet. The test two was paired test, and 21 weanling piglets were divided into 3 groups, that is, negative control group, test 1 group and paired group. Among them, the negative control group and the test 1 groups were the same test. 1 groups of paired test pig feeding test. Diet, but after the feeding test and the negative control group kept the same.28 D feeding test, each group randomly selected 6 butchers, measured the carcass traits, collected the hypothalamus and the longest muscle in the back. At the same time, the main 23 muscles, such as the longest muscle of the peeling back, were weighed respectively. The main results were as follows: 1. compared with the negative control group, 1 groups and tests were tested. The 2 groups significantly improved the average daily gain, the end weight, the feed intake, the body weight of the anterior body, the middle body and the posterior body, the most weight of the muscle mass as well as the nitrogen deposition (P0.05), and it could be restored to the level of the control group. When the feed intake remained consistent, the average daily gain and the meat ratio (P0.05) of the piglets could still be significantly increased by the addition of BCAAs. Compared with the negative control group, the weight (P0.05).2. of the supraspinatus, latissimus dorsi and the longest muscle of the dorsal muscle (P0.05) was significantly higher than that in the negative control group. The level of Agrp and NPY mRNA in the hypothalamus was up-regulated in a dose-dependent manner, and the level of MC4R mRNA was down regulated in a dose dependent manner, and the difference was significant (P0.05) in the 2 groups. Moreover, the 1 groups were significantly reduced to CART mRNA water. Regression analysis (P0.05). The regression analysis showed that the feed intake was increased two times with the increase of NPY mRNA level (P=0.02), but linearly decreased with the increase of MC4R mRNA level (P=0.02). Compared with the increase of BCAAs for the feed intake and the up-regulation of the NPY gene expression in the hypothalamus and the.3. and negative control of MC4R gene expression, 1 groups and tests were tested. The 2 groups significantly reduced the level of eIF2 alpha phosphorylation in the hypothalamus, and significantly increased the level of S6K1 phosphorylation (P0.05), and was able to restore to the level of the control group. In addition, the level of mTOR phosphorylation in the 1 groups was significantly higher than that of the negative control group (P0.05). The increase of the amount of food added to the diet by adding BCAAs to the diet and the reduction of the activity of GAAC pathway and the increase of mTORC1 in the diet. Compared with the negative control group, the pathway activity related.4. significantly increased the S6K1 and mTOR phosphorylation level (P0.05) of the longest muscle in the 1 groups and the paired groups, but there was no significant difference between the 1 groups and the paired groups. It showed that BCAAs could activate the mTORC1 pathway activity of skeletal muscle and did not depend on the change of feed intake. The second part was the use of blood intubation in combination. The effects of dietary supplementation of BCAAs on the net use of amino acids and the profile of femoral vein metabolites in the muscle of piglets were studied. After the test animals and the first part of the paired test of.28 D, the test pigs in the negative control group and the 1 groups were installed in the femoral artery, femoral vein, carotid artery and jugular vein respectively. Fifth after the operation. D, a formal test was conducted in each group of 6 healthy and unobstructed pigs. The muscle blood flow velocity was measured by continuous perfusion of 9 h in the femoral artery with aminaminic acid (pAH). Every 1 h was fed to keep the test pigs in the feeding state and feeding continuously for 8 times. The carotid and femoral vein blood samples were collected at 1 h every septum before feeding and after feeding. PAH, free amino acid and branched chain alpha ketoacid were collected for 8 consecutive times. After the collection of blood samples, the two head samples were collected for free amino acids and 3- methyl histidine analysis, and the femoral vein blood samples were analyzed simultaneously. The main results were as follows: 1. muscle blood flow velocity increased significantly after feeding (P0.05), but not in the diet. Dietary supplementation of BCAAs significantly increased the dissociative isoleucine, leucine and valine concentration (P0.05) in the arteries and muscles, but significantly reduced the concentration of phenylalanine and serine and the free serine concentration (P0.05) of the free artery (P0.05). At the same time, the addition of BCAAs significantly reduced the concentration of 3- methylhistidine in the muscle. P0.05) showed that BCAAs inhibited skeletal muscle protein mobilization by adding BCAAs to the total BCAAs, total essential amino acids, total non essential amino acids and total amino acids (P0.05), thus maintaining efficient protein deposition. In addition, the addition of BCAAs significantly increased isoleucine, leucine, valine and its metabolite C The muscle net benefit of ammonia, glutamic acid, and glutamine (P0.05), and also significantly increased the muscle net benefit of functional non essential amino acids such as methionine, glycine, proline and serine (P0.05).3. diet added BCAAs to the femoral vein metabolites significantly, these differential compounds and BCAAs metabolism and protein For example, adding BCAAs significantly increased the content of three kinds of branched chain alpha ketoacid, glutamine and asparagine (P0.05), and increased the content of lipid metabolism related products such as twenty carbon five enoic acid (P0.05). Further quantitative results showed that adding BCAAs significantly increased KIC and KMV (respectively Muscle net production (P0.05) of leucine and isoleucine (leucine and isoleucine), suggesting that adding BCAAs increases the metabolism of.4. muscle amino acids in the skeletal muscle itself and has a significant positive correlation with the arterial leucine, isoleucine and valine concentration (P0.05), but no correlation with the BCAAs concentration in the muscles. Interestingly, the muscle is the muscle. The net yield of meat amino acids was positively correlated with net production of KIC and KMV respectively (P0.05). These results suggest that adding BCAAs may increase the net benefit of muscle amino acids by increasing arterial concentration and intracellular metabolism. The third part: using blood intubation and stable isotope tracing technique to study the regulation of BCAAs by regulating self generation. The mechanism of protein synthesis and degradation in the skeletal muscle of piglets. Experimental animals studied in the feeding state were tested in second parts. After the first feeding of the pig, NaH~ (13) CO_3 was perfused through the jugular vein, [1-~ (13) C] leucine was perfused after 2 h., and the continuous 6 h.pAH perfusion and blood samples were collected with second parts of the experiment. Blood samples were used in the test. Analysis of leucine, KIC and CO_2 concentrations and their isotopic abundances. Experimental animals studied in the fasting state of the first part of the first part of the test. A negative control group and 1 groups of the longest dorsal muscle samples were collected. The main results were as follows: the addition of BCAAs to the 1. diet increased significantly the arterial intake and net benefit of leucine (P0.05). Protein synthesis and deposition (P0.05). Adding BCAAs also significantly increased the net ammonia conversion (P0.05), but did not affect the oxidation decarboxylation. Interestingly, the protein degradation was also significantly increased after the addition of BCAAs (P0.05). Further metabolic fate analysis showed that muscle uptake of leucine was about 30% metabolism, and mainly through the action of ammonia to produce KIC The net uptake of [1-~ (13) C] leucine and net production of [1-~ (13) C]KIC in.2. muscles also increased significantly (P0.05) after dietary supplementation with BCAAs. The correlation analysis showed that the protein synthesis in skeletal muscle was positively correlated with protein degradation and [1-~ (13) C]KIC net formation respectively (P0.05), suggesting that dietary BCAAs may be degraded by protein degradation and The addition of BCAAs to the dietary protein synthesis.3. diet enhanced the Akt, mTOR and its downstream target protein S6K1 phosphorylation level (P0.05), suggesting that the BCAAs piglets had a higher protein binding ability in the fasting state of the skeletal muscle. In addition, the addition of BCAAs significantly increased FoxO1 phosphoric acid. The expression of Atrogin-1, MuRF1 and LC3-II protein (P0.05) was adjusted accordingly, suggesting that the skeletal muscles of BCAAs piglets were reduced to a lower level of protein degradation in the fasting state. The main conclusions of this study were: (1) the addition of BCAAs to low protein diet could improve the feed intake and direct the growth of muscle, thus improving the piglets. Growth performance, and can be restored to the level of feeding high protein diet; (2) diet BCAAs can increase feed intake by affecting the appetite regulating gene expression and pathway activity in the hypothalamus, and increase feed intake. (3) dietary metabolism and protein degradation in the muscle cells, and then increase the net benefit of amino acids, and increase the skeleton of the feeding state. The rate of muscle protein synthesis; at the same time, dietary BCAAs can inhibit the protein degradation of skeletal muscle in the fasting state by down regulation of the key protein expression of ubiquitin proteasome and autophagy lysosome pathway, and ultimately improve protein deposition, thus promoting skeletal muscle growth.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S828.5

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