【作者】 蔡元丽；

【导师】 林海；

【作者基本信息】 山东农业大学 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 本研究利用外源糖皮质激素(地赛米松,DEX)导入的方法建立肉仔鸡的应激模型,从肉仔鸡的生长前期和后期两个阶段探讨应激对肉仔鸡脂肪代谢的影响。通过研究应激对肉仔鸡脂肪沉积规律影响的基础上,重点考察了应激对肝脏脂肪从头合成的影响机制。应激对肉仔鸡生长前期脂肪代谢的影响选取7日龄体重相近的Arbor Acres雄性肉鸡108只,随机分为三个处理:应激组(DEX)、对照组(Control)和采食量配对组(Pair-fed)。应激组每天早8:00腹部皮下注射地塞米松(1mg/ml),剂量为2.0mg/Kg体重,自由采食和饮水;对照组注射与应激组相同体积的生理盐水,自由采食和饮水;采食量配对组注射与应激组相同体积的生理盐水,同时饲喂应激组前一天的采食量。结果发现,应激显著降低了14日龄肉仔鸡的平均日增重和饲料转化率,增加了颈脂、腹脂、腿脂以及肝脏的脂肪沉积;应激显著增加了血浆中胰岛素水平,VLDL(极低级密度脂蛋白)也有增加的趋势,这表明血液中脂类的流量增加;应激还提高肝脏FAS(脂肪酸合成酶)以及ME(苹果酸酶)的活性,促进肝脏ACC(乙酰辅酶A羧化酶)、FAS以及ME mRNA的表达。但颈脂和腹脂中LPL(脂蛋白脂酶)、PPARγ(过氧化物酶体增殖物激活受体)以及ATGL(脂肪细胞甘油三酯酶) mRNA的水平没有受到应激的影响。这表明,应激导致脂肪沉积增加的主要原因是肝脏脂肪酸从头合成的增强以及血浆中脂类流量的增加。应激对肉仔鸡生长后期脂肪代谢的影响选取35日龄体重相近的Arbor Acres雄性肉仔鸡196只,随机分为三个处理:应激组(DEX,剂量为2.0mg/Kg体重)、对照组(Control)和采食量配对组(Pair-fed),自由采食、饮水。注射3d后,于38日龄分别随机选取8只鸡于饲喂和空腹状态下采集血液、腹脂、皮下脂肪(腿部、颈部皮下)和肝脏样品。结果发现,DEX处理极显著增加肝脏和颈脂的相对重量,DEX处理有增加腹脂和腿脂相对重量的趋势;在禁食状态下,DEX处理显著增加血浆中TG(甘油三酯)、VLDL、尿酸和胰岛素的含量,使肝素后血浆中LPL活性升高;在饲喂状态下,DEX处理显著增加血糖、TG、VLDL、尿酸和胰岛素含量;DEX处理显著增加38日龄肉仔鸡在禁食状态下ME的活性,肝脏FAS的活性也有增加的趋势,但DEX对饲喂状态下肝脏FAS和ME的活性没有影响。DEX处理极显著地增加38日龄肝脏禁食状态下ACC和FAS的mRNA水平,与配对组相比,DEX处理也有增加ME的mRNA表达量的趋势,在饲喂状态下,与配对组相比,DEX处理显著增加肝脏ACC和ME mRNA的水平;与配对组相比,DEX处理促进禁食状态下腹脂FAS的mRNA水平,但对禁食状态下腹脂ME的mRNA水平以及饲喂状态下FAS、ME的mRNA水平都没有显著影响;DEX处理显著增加饲喂状态下腹脂LPL mRNA的水平,在禁食状态下,LPL mRNA也有增加的趋势,而腹脂ATGL和PPARγ不论是禁食还是饲喂状态都没有受到DEX处理的影响。这表明,肝脏脂肪酸从头合成能力增加以及血浆中脂肪流量增多是38日龄应激肉仔鸡脂肪沉积增加的主要原因,这与应激对肉仔鸡生长前期的影响也是一致的;血浆LPL活性增加以及腹脂LPL mRNA表达量的上调是应激肉仔鸡脂肪沉积增加的另一个原因,这一点与应激对肉仔鸡生长前期的影响不同,这可能与生长后期腹脂明显增多有关系。DEX和胰岛素对离体培养的肉仔鸡肝细胞脂肪酸合成的影响分离并培养6日龄肉仔鸡肝细胞,培养15h后开始正式试验,分为4个处理,每个处理6个重复。对照组:基础培养液,为含5%新生牛血清的Williams’E培养基;DEX组:基础培养液中含DEX 200nmol/L;INS组:基础培养液中含胰岛素100nmol/L;DEX+INS组:在基础培养液中含DEX 200nmol/L、胰岛素100nmol/L。在CO2培养箱中37℃静置培养24h。结果发现,DEX和胰岛素共同作用于肝细胞时,能显著增加其FAS、ME以及ACC mRNA的表达量,而DEX或胰岛素单独处理没有改变FAS、ME以及ACC mRNA的表达量。DEX或胰岛素单独处理没有改变肉仔鸡肝细胞LXRα和SREBP-1c mRNA的水平,但DEX和胰岛素共同作用于肝细胞时,LXR(P=0.0036)的mRNA的水平显著增加了,对于SREBP-1c mRNA的水平,两种激素共同作用与对照组或DEX组相比有增加的趋势(P=0.0719),而与胰岛素处理相比没有显著差别。这表明,是DEX和胰岛素的协同作用促进了肝脏脂肪酸的从头合成,这与活体试验即DEX处理以及由此导致的高胰岛素血症促进了肝脏脂肪酸合成的结果是一致的。而生脂基因mRNA表达量的上调可能受到核转录因子LXRα和/或SREBP-1的调控。更多还原

【Abstract】 The effects of dexamethasone on lipid metabolism were studied in vivo and in primary culture of hepatocytes in broiler chickens.Effect of dexamethasone (DEX, a synthetic glucocorticoid) on lipid metabolism in broiler chickens (Gallus gallus domesticus) was investigated in the early growing stage. Male Arbor Acres chickens (1 wk old, n=30) were injected with DEX or saline for 1 wk, and a pair-fed group was included. DEX administration resulted in enhanced lipid deposition in adipose tissues. Plasma insulin increased about 3.3 fold in DEX injected chickens as against the control and hepatic triglyceride was higher as compared with the pair-fed chickens. In DEX injected chickens, the hepatic activities of malic enzyme (ME) and fatty acid synthetase (FAS) was significantly increased, while the mRNA levels of acetyl CoA carboxylase (ACC), ME, and FAS was significantly up-regulated, compared with the control. Although the mRNA levels of lipoprotein lipase (LPL), peroxisome proliferator-activated11 receptor-γ(PPARγ) and adipose triglyceride lipase (ATGL) genes in adipose tissue were not affected by DEX injection, ME activity and mRNA levels in abdominal fat pad of chickens treated with DEX is higher than those of control chickens. The results indicated that the increased hepatic de novo lipogenesis and in turn, the increased circulating lipid flux contributes to the augmented fat deposition in adipose tissues and liver in DEX-challenged chickens. The results suggest that glucocorticoids together with the induced hyperinsulinemia should be responsible for the up-regulated hepatic lipogenesis.Effects of DEX on lipid metabolism in broiler chickens (Gallus gallus domesticus) were investigated in the late growing stage. Male Arbor Acres chickens (35 d of age, n=30) were injected with DEX or saline for 3d, and a pair-fed group was included. When the samples were collected at 38 d of age, one half of chickens were in the fed status, another half were fasted for 12h. DEX administration resulted in enhanced lipid deposition in cervical fat, abdomnal fat and thigh fat also had increased trend. DEX injection increased plasma TG, VLDL and insulin concentration not only in fasted status but also in fed status. DEX administration led to higher post-heparin LPL activity and plasma glucose level in fasted status. In fasted status, DEX administration resulted in increased ME activity in liver, liver FAS activity tended to increase in DEX injected chicken. But the FAS and ME activity in DEX chickens had no significant change in fed status. DEX injection resulted in enhanced ACC and FAS mRNA levels in liver in fasted status. Compared to pair-fed, ME mRNA level in liver tended to increase in DEX chickens in fasted status. In fed status, DEX administration led to enhanced liver ACC and ME mRNA expressions compared to pair-fed chickens. DEX administration up-regulated FAS mRNA expression in abdominal fat in fasted status, but the ME and FAS mRNA levels of abdominal fat in fed status were not altered by DEX injection. DEX injection resulted in LPL mRNA expression of abdominal fat in fed status, and in fasted status LPL mRNA level trended to increase by DEX injection. We also measured the mRNA levels of PPARγand ATGL in abdominal fat in fasted and fed status. Neither of these two genes’mRNA expression was altered by DEX injection. The results suggested that the increased hepatic de novo lipogenesis and in turn, the increased circulating lipid flux contributes to the augmented fat deposition in adipose tissues in DEX-challenged chickens which agreed to the former experiments. Up-regulation of LPL mRNA level in abdominal fat and increased plasma LPL activity also contribute to the enhanced fat deposits which were not accordant with results in the early growing stage of chickens perhaps because of more abdominal fat deposit. The results indicated that glucocorticoids together with the induced hyperinsulinemia should be responsible for the up-regulated hepatic lipogenesis.The effects of DEX and insulin on the fatty acid synthesis of hepatocytes cultured in vitro were investigated in the third experiment. Hepatocytes were isolated from livers of 6 day old chick. Cells were incubated in Williams’Medium E containing 10% serum. After 15 h incubation, the medium was changed to contain 5% serum. There were four treatments, one of which had the same composition supplemented with DEX (200nmol/L), insulin (100nmol/L) or both. The control group contained neither of the hormones. Afer 24h of treatment, total RNA was isolated and lipogenic genes were measured by real-time PCR. The results found that in combination with DEX, insulin increased significantly FAS, ACC and ME mRNA level, but when added alone, DEX or insulin had no effent on these lipogenic genes. The effects of the two hormones on nucleus transcriptional factors were also evaluated. DEX and insulin synergically promoted LXRαmRNA level. When DEX plus insulin were added to the medium, SREBP-1c tended to increase compared to DEX or control group. The results suggested that it was insulin plus DEX that enhanced hepatic de novo lipogenesis. The transcription of lipogenic genes perhaps were modulated by LXRαand/or SREBP-1.更多还原