【作者】 刘闯；

【导师】 伍国耀； 武振龙；

【作者基本信息】 中国农业大学 ， 动物营养与饲料科学， 2014， 博士

【摘要】 本研究通过两个试验探讨了日粮蛋白质水平对SD大鼠体脂含量的影响,并从能量代谢的角度研究了其内在机制。(1)第一个试验旨在研究日粮蛋白质水平对生长期SD大鼠体脂含量的影响。试验选用30只30日龄的生长期SD大鼠,日粮酪蛋白水平分别10%、20%和40%。试验期为80d,以日采食量最低组为标准进行配对饲养。在试验期第1天、第30天和第80天分别进行大鼠呼吸测热试验。试验期满后进行断颈处死,腹主动脉采血后,取组织器官称量并取适量冻存备用,分析组织器官游离氨基酸谱,分析肝脏和褐色脂肪中与能量代谢相关蛋白的表达情况。结果表明,20%日粮蛋白质组和40%日粮蛋白质组体重没有显著差异,而10%日粮蛋白质组体重显著低于其它两组(P<0.01)。相对于20%日粮蛋白质组而言,10%日粮蛋白质组和40%日粮蛋白质组的白色脂肪组织(P<0.01)指数都显著降低。处理第30天时,高日粮蛋白质水平显著降低了生长大鼠呼吸交换率(P<0.01),这种改变延续到处理的第80天。低日粮蛋白质水平在处理第80天时,显著增加了生长大鼠二氧化碳产生量(P<0.01)、氧气消耗量(P<0.01)、产热量(P<0.01)和静息代谢率(P<0.01)。在试验大鼠肝脏中随着日粮蛋白质水平的升高,磷酸化的AMP激活的蛋白激酶(p-AMPKa,P<0.01)、激素敏感酯酶(HSL, P<0.01)、p-HSL(P<0.01)、固醇调节元件结合蛋白(SREBP-1, P<0.01)、p-SREBP-1(P<0.01)和细胞色素C(P<0.01)的表达水平显著地降低,p-AMPKa占AMPKa的比率(P<0.01)也显著地下降,而日粮蛋白质水平的降低和升高都能下调肝脏中AMPKa (P<0.01)的表达。日粮蛋白质水平影响了生长期大鼠组织器官游离氨基酸谱,这些浓度变化的氨基酸可能参与了高蛋白质日粮减少体脂的作用,并可作为生物标记物为以后的研究提供参考。(2)第二个试验旨在研究日粮蛋白质水平对高脂诱导肥胖SD大鼠体脂含量的影响。选取48只10周龄SD大鼠,分别饲喂正常日粮或高脂日粮,诱导肥胖12周后,从各组选取12只大鼠分别进行正常蛋白质水平日粮(酪蛋白水平20%)和高蛋白质水平日粮(酪蛋白水平40%)的饲喂,以高脂-40%日粮蛋白质组大鼠采食量为基础进行配对饲养,在不同日粮蛋白质水平处理期第70天进行大鼠呼吸测热试验；饲喂10周后CO2窒息致死,腹主动脉采血后,取组织器官称量并取适量冻存备用；通过高效液相色谱检测血浆、肝脏、肌肉、空肠和腹部白色脂肪中游离氨基酸谱,分析肝脏、肌肉和脂肪中与能量代谢相关蛋白的表达情况,分析肝脏、肌肉和脂肪中与蛋白质代谢相关蛋白的表达情况。结果表明,高日粮蛋白质水平显著降低了大鼠白色脂肪含量(P<0.05)；高蛋白质日粮显著地降低了肥胖大鼠呼吸交换率(P<0.01)；在肥胖大鼠腹部白色脂肪中,高蛋白质日粮显著降低了p-AMPKa的表达水平,并降低了p-AMPKa占AMPKa的比率；在肥胖大鼠肝脏中,高蛋白质日粮显著降低了p-HSL占HSL的比率；日粮蛋白质水平影响了试验大鼠组织器官游离氨基酸谱,这些浓度变化的氨基酸可能参与了高蛋白质日粮减少体脂的作用,并可作为生物标记物为以后的研究提供参考。综上所诉,在能量摄入量相似的情况下,高日粮蛋白质水平仍然有减少大鼠白色脂肪组织含量的作用。这种作用可能是通过改变机体能量代谢而实现的,同时组织器官中变化的游离氨基酸也可能参与了这一调控过程。更多还原

【Abstract】 Two experiments were conducted to investigate effects of dietary protein levels on white adipose tissue content in Sprague-Dawley (SD) rats, and to understand the mechanisms responsible for the changes in energy metabolism.(1) The objective of the first experiment was to determine effects of dietary protein levels on adipose tissue content in growing rats. Thirty male SD (30d of age) were randomly allotted into one of3groups (n=10) fed purified isocaloric diets containing10%casein (low-level protein, LP),20%casein (medium-level protein, MP), or40%casein (high-level protein, HP) for80days. Pair-feeding per kg body weight was performed by offering the amount of food taken by HP rats. On d1, d30and d80day of different dietary protein treatment, the energy expenditure measurements were conducted. At the end of the study period, the animals were sacrificed by cervical dislocation. Blood and tissue samples were collected rapidly, snap-frozeing in liquid nitrogen, and stored at-80℃for future analysis. The tissues and organs were used to analyze the free amino acids profiles and the expressions of proteins related to the energy metabolism. Results indicated that there was no significant difference in body weight between MP and HP group, but the body weight of LP rats was lower (P<0.01) than that of the other two groups. Rats in LP and HP groups had less white adipose tissue (P<0.01) than MP rats. On d30and d80of treatment, HP diet resulted in a higher respiratory exchange ratio (P<0.01) in young rats; on d80of treatment, LP rats produced more CO2(P<0.01), consumed more O2per kg body weight (P<0.01), had a higher resting metabolic rate(P<0.01), and generated more heat (P<0.01) than the rats of other two groups. In the liver of rats, higher dietary protein level lower the expressions of phospho-AMP-activated protein kinase a (p-AMPKa. P<0.01), hormone-sensitive lipase (HSL, P<0.01), p-HSL(P<0.01), sterol regulatory element binding protein (SREBP-1, P<0.01), p-SREBP-1(P<0.01) and cytochrome c (P<0.01), as well as the ratio of p-AMPKa to AMPKa (P<0.01). Compared with the MP group, LP and HP diets decreased the expression of AMPKa (P<0.01) in the liver. Dietary protein level changed the profiles of free amino acids in tissues and organs of rats. Those changed amino acids may play roles in the fat-reducing effect of high-protein diet, and can be selected as biomarkers for the further studies.(2) The objective of the second experiment was to study effects of HP diet on adipose tissue content in diet-induced obese (DIO) rats. Forty eight male SD rats (10weeks old) were randomly allotted into two groups (n=20/group), fed normal (NF) or high fat (HF) diet for12weeks to induce obesity. Basing on the body weight,12rats from each group were randomly allotted into two subgroups (n=6), fed either the MP or HP diet for10weeks. Pair-feeding was performed by offering the amount of food taken by the rats in the HF-HP group. On d70of different dietary protein treatment, the energy expenditure measurements were conducted. At the end of the study period, rats were sacrificed by CO2asphyxiation. Blood and tissue samples were collected rapidly, snap-frozen in liquid nitrogen and stored at-80℃for future analysis. The tissues and organs were used to analyze the free amino acids profiles and the expressions of proteins related to energy and protein metabolism. Results showed that HP diet reduced major white adipose tissue mass (P<0.05) in rats; DIO rats fed a HP diet had a lower RER (P<0.01), lower protein levels for p-AMPKa and a lower ratio of p-AMPKa to AMPKa in the white adipose tissue, and a lower ratio of p-HSL to HSL in the liver; Dietary protein level changed the profiles of free amino acids in tissues and organs of rats. Those changed amino acids may play roles in the fat-reducing effect of high-protein diet, and can be selected as biomarkers for the further studies. In conclusion, under the similar dietary energy intake, high dietary protein also can reduce fat in rats. This effect may due to changed energy metabolism induced by high protein diet, and the changed free amino acids may participate in this regulation.更多还原