缬氨酸缺乏对小鼠摄食行为和采食量的影响及机制研究

发布时间：2018-05-16 14:14

本文选题：必需氨基酸缺乏 + 缬氨酸　；参考：《华中农业大学》2017年硕士论文

【摘要】：断奶仔猪阶段,以玉米-豆粕型为基础的日粮中,Val是继Lys、Met、Thr、Trp后又一限制性氨基酸。研究还发现,Val缺乏能造成更为显著的采食量和体重下降,但Val缺乏引起采食量下降的机制目前还不清楚。因此,研究Val缺乏对动物采食量的调控机制有着重要意义。采食量按照调控部位分为外周调控和中枢调控。外周调控中胃肠激素是调控动物采食量的主要方式。最新的研究表明,肝脏分泌的FGF21也参与到营养物质的摄入调控。中枢系统中下丘脑是重要的调控部位,一方面,下丘脑通过调节食欲肽(NPY/Agrp)和厌食欲肽(POMC/CART)的表达调控采食量。另外,下丘脑mTOR、AMPK信号通路也发挥着重要作用。中枢前梨状皮层还能感应必需氨基酸缺乏,激活GCN2通路抑制采食。本试验通过行为学分析、检测外周和中枢食欲相关基因、蛋白表达和信号通路活性,以及血液中氨基酸变化,阐明Val缺乏对动物采食量的调控机制。主要内容及结果如下:1.日粮新奇性测试:为了排除日粮类型的影响,选择12只体重相近的雄性昆明小鼠,饲喂普通日粮5天后,随机分配到Val缺乏组和对照组中,记录第6、7天采食量。试验结果显示,与对照组和更换日粮前的采食量相比,Val缺乏组小鼠的采食量极显著降低(P0.01)。第6天分段累计采食量表明,在第1小时Val缺乏组采食量显著低于对照组(P0.05),表明小鼠能够快速感应和拒绝Val缺乏的日粮。2.Val缺乏对短期采食量和摄食行为的影响:将16只野生和14只FGF21杂合雄性小鼠随机分配到对照组和Val缺乏组中,禁食24小时后,观察小鼠的摄食行为,并分段记录各阶段采食量。结果显示进食Val缺乏的野生小鼠第2小时采食量显著低于对照组(P0.05),FGF21杂合小鼠则延长到第12小时。无论是野生型小鼠,还是FGF21杂合小鼠,Val缺乏对禁食后第一次摄食行为影响均不显著。餐模式结果表明,Val缺乏显著降低了(P0.05)每餐采食量、每餐采食量以及全天和夜间每餐进食时间。Val缺乏显著降低(P0.05)全天和夜间摄食时间和进食间隔。与野生型小鼠相比,FGF21杂合小鼠显著降低了(P0.05)全天进食次数,但FGF21杂合小鼠显著增加了(P0.05)平均每餐摄食时间。3.Val缺乏对小鼠长期采食量的影响:摄食行为试验结束后,接着饲喂4周,记录每天采食量变化,每两天称一次体重。试验结果表明,Val缺乏显著降低了小鼠(P0.05)每周以及全期平均日采食量、体重和屠宰性能。4.对比试验:选择8只体重相近的雄性野生昆明小鼠,前10天饲喂Val缺乏日粮,后10天饲喂对照日粮。结果发现,在进食Val缺乏阶段显著降低小鼠的采食量(P0.05),并逐渐降低体重。当进食对照日粮后,小鼠的采食量和体重分别在第1天和第2天恢复到正常水平。5.Val缺乏对小鼠外周胃肠激素及肝脏FGF21表达的影响:尽管Val缺乏对小肠GLP-1、PYY、Ghrelin mRNA水平无显著差异,但Val缺乏显著增加了(P0.05)小肠中CCK mRNA水平。相反的是,Val缺乏极显著地降低胃中leptin mRNA水平(P0.01)。无论是野生小鼠还是FGF21杂合小鼠,Val缺乏组都显著增加了(P0.05)肝脏FGF21、ATF4的mRNA表达水平、p-eIFα水平以及血液中FGF21浓度。重要的是,肝脏FGF21表达水平与采食量之间有一定的线性(R2=0.515,P=0.0003)和二次回归关系(R2=0.60,P=0.0003),并且血液中FGF21与采食量之间存在负相关(R=-0.502,P=0.0047)。6.Val缺乏对血液中氨基酸模式的影响:无论是野生小鼠还是FGF21杂合小鼠中,Val缺乏显著降低了(P0.05)小鼠血液中游离Val、Met浓度,极显著提高了(P0.01)His浓度。血液中Val浓度与采食量有良好的线性(R2=0.79,P0.0001)和二次回归关系(R2=0.794,P0.0001)。相似的是血液中Val浓度与肝脏中FGF21mRNA表达水平也有一定的线性(R2=0.56,P0.0001)和二次回归关系(R2=0.615,P=0.0002)。血液Val浓度还与血液中FGF21浓度呈极显著的负相关(R=-0.47,P=0.008)。另外,血液中His浓度与采食量同样具有一定的线性关系(R2=0.58,P0.0001)。7.Val缺乏对中枢食欲肽NPY和信号通路活性的影响:Val缺乏对下丘脑NPY阳性细胞率无显著影响(P0.05),相似的是Val缺乏对下丘脑弓状核中p-mTOR阳性细胞数也无显著影响(P0.05),但显著地增加(P0.05)前梨状皮层中p-eIF2α阳性细胞数。综上所述,小鼠能够快速感应日粮中Val缺乏,并改变摄食行为。主要通过减少进食时间和每餐采食量降低小鼠短期采食量。Val长期缺乏时,促进了小肠CCK的表达,增加了血液中His浓度,降低了血液中Val浓度并刺激肝脏FGF21的表达,同时增加中枢前梨状皮层p-eIF2α阳性细胞数量,抑制了小鼠长期的采食量,并减轻体重。
[Abstract]:In the weaned piglet stage, Val is another limiting amino acid following Lys, Met, Thr and Trp in the diet based on the corn soybean meal type. It is also found that Val deficiency can cause more significant feed intake and weight loss, but the mechanism of Val deficiency causing the decrease of feed intake is unclear. Therefore, the regulation machine for Val deficiency on animal feed intake is studied. Food intake is of great significance. The intake of food is divided into peripheral control and central control according to the regulatory parts. Gastrointestinal hormones are the main ways to regulate animal feed intake in peripheral control. The latest research shows that the FGF21 secreted by the liver is also involved in the regulation of nutrient intake. The central nervous system is an important regulatory part of the lower thalamus, on the one hand, the lower colliculus. The brain regulates the intake of food by regulating the expression of orexin (NPY/Agrp) and anorexin (POMC/CART). In addition, the mTOR, AMPK signaling pathway in the hypothalamus also plays an important role. The central pyriform cortex can also induce essential amino acid deficiency and activate the GCN2 pathway to inhibit eating. This test was conducted by behavioral analysis to detect the peripheral and central appetite related groups. Due to protein expression, signaling pathway activity and changes in amino acids in the blood, the regulation mechanism of Val deficiency on animal feed intake was clarified. The main contents and results were as follows: 1. diet novelty test: to exclude the influence of diet type, 12 male Kunming mice with similar weight were selected and randomly assigned to Val deficiency for 5 days. In the group and the control group, the feed intake was recorded on day 6,7. The test results showed that the intake of mice in the Val deficiency group was significantly lower than the control group and the food intake before the replacement of the diet (P0.01). The cumulative feed intake of the sixth day subsection showed that the intake of the Val deficiency group was significantly lower than the control group (P0.05) in the first hour period, indicating that the mice could be quickly induced and refused. The effect of Val deficiency.2.Val deficiency on short-term feed intake and feeding behavior: 16 wild and 14 FGF21 heterozygous male mice were randomly assigned to the control group and the Val deficiency group. After 24 hours of fasting, the feeding behavior of the mice was observed and the feed intake at each stage was recorded in segments. The fruit showed that the wild mice fed with Val deficiency were collected for second hours. The food intake was significantly lower than that of the control group (P0.05), and the FGF21 heterozygous mice were prolonged to twelfth hours. Both the wild and FGF21 heterozygous mice, the Val deficiency had no significant influence on the first feeding behavior after the fasting. The meal pattern showed that Val deficiency significantly reduced the intake of (P0.05) per meal, food intake per meal, and every meal at night and night. Feeding time.Val was not significantly reduced (P0.05) all day and night feeding time and feeding interval. Compared with wild type mice, FGF21 heterozygous mice significantly decreased (P0.05) eating times of all days, but FGF21 heterozygous mice significantly increased (P0.05) the average feeding time of.3.Val deficiency on the long-term intake of mice: feeding behavior test After 4 weeks, the daily feed intake was recorded for 4 weeks and the weight was recorded every two days. The results showed that the Val deficiency significantly reduced the daily and full average daily feed intake, weight and slaughter performance of the mice (P0.05): 8 similar male wild Kunming mice were selected, the first 10 days were fed with Val diet, and 10 days later. It was found that the feed intake (P0.05) of mice was reduced significantly and the weight was gradually reduced at the stage of Val deficiency. After eating control diet, the intake and weight of mice recovered to the normal level in first days and second days, respectively, to the normal level of.5.Val deficiency on the expression of gastric intestinal hormone and liver FGF21 in mice, although Val deficiency was lacking. There was no significant difference in GLP-1, PYY, and Ghrelin mRNA levels in the small intestine, but Val deficiency significantly increased the level of CCK mRNA in the small intestine. On the contrary, Val deficiency significantly reduced leptin mRNA in the stomach (P0.01). The level of p-eIF alpha and the concentration of FGF21 in the blood. It is important that there is a certain linear (R2=0.515, P=0.0003) and two regression relationship between the FGF21 expression level and the intake of the liver (R2=0.60, P=0.0003), and there is a negative correlation between the FGF21 and the feed intake in the blood (R=-0.502, P=0.0047).6.Val on the amino acid patterns in the blood: no matter the effect on the pattern of amino acids in the blood: In the wild mice or in the FGF21 heterozygous mice, Val deficiency significantly reduced the free Val and Met concentration in the blood of (P0.05) mice and significantly increased the concentration of (P0.01) His. There was a good linear (R2=0.79, P0.0001) and two regression relationship between the concentration of Val and the feed intake in the blood (R2= 0.794, P0.0001). The expression level also has a certain linear (R2=0.56, P0.0001) and two regression relations (R2=0.615, P=0.0002). The concentration of Val in blood also has a very significant negative correlation with the concentration of FGF21 in the blood (R=-0.47, P=0.008). In addition, the concentration of His in the blood and the intake of the blood have a certain linear relationship (R2=0.58, P0.0001).7.Val lacks the central appetite peptide. The effect of signal pathway activity: Val deficiency had no significant effect on the rate of NPY positive cells in the hypothalamus (P0.05). Similar was that Val deficiency had no significant influence on the number of p-mTOR positive cells in the hypothalamic arcuate nucleus (P0.05), but significantly increased the number of p-eIF2 alpha positive cells in the anterior pyriform cortex (P0.05). In summary, the mice could quickly induce Val in the diet. The lack of food intake and change of feeding behavior, mainly by reducing the feeding time and food intake per meal to reduce the short-term intake of.Val in mice, promote the expression of CCK in the small intestine, increase the concentration of His in the blood, reduce the concentration of Val in the blood and stimulate the expression of FGF21 in the liver, and increase the number of p-eIF2 alpha positive cells in the central pyriform cortex. The long-term intake of the mice was inhibited and weight loss was reduced.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S816

【参考文献】