【作者】 李蕾；

【导师】 李之俊；

【作者基本信息】 上海体育学院 ， 运动人体科学， 2009， 博士

【摘要】 研究目的:观察有氧运动对高脂膳食诱导的SD大鼠胰岛素抵抗形成的干预作用及其相关机制探讨。研究方法:本研究以正常SD雄性大鼠为实验对象,统一普通饲料适应性喂养一周后,随机分为4组:普食对照组(C组)、普食运动组(E组)、高脂膳食对照组(H组)、高脂膳食运动组(HE组)。通过10周高脂配方饲料(热卡比蛋白质23.5%,脂肪44.1%,碳水化合物32.4%,提供能量约408Kcal/100g)喂养以诱导胰岛素抵抗,同时对大鼠实施无负重有氧游泳运动干预。综合利用体重、空腹胰岛素(FINS)、空腹血糖(FBG)、胰岛素抵抗指数(HOMA-IR)、胰岛素敏感指数(ISI)、胰岛素耐量试验(ITT)评价动物模型的建立和部分运动干预效果;观察高脂膳食和运动对大鼠血脂指标、内脏脂肪储量、肝重、肝功能谷丙转氨酶、谷草转氨酶(ALT、AST)及对组织(肝脏和肌肉)脂质异位沉积的影响;采用酶联免疫法和实时荧光定量PCR技术系统全面观察血清脂联素、内脏脂肪组织脂联素mRNA表达及骨骼肌和肝脏脂联素受体基因表达在高脂和运动干预条件下所发生的改变,以及SD大鼠肝脏和骨骼肌脂联素受体的分布情况;采用Western blotting方法测定大鼠肝脏和股四头肌腺苷酸蛋白激酶α(AMPKα)蛋白表达及其磷酸化水平在高脂和规律运动后的变化情况。研究结果:1实验前四组大鼠体重无差异。实验末,H组高于C组、E组低于C组、HE组低于H组、HE组高于E组(P<0.01)。平均增重两运动组较两安静组缓慢,以H组增长最快而E组增长最缓慢。FBG、HOMA-IR无显著性差异(P>0.05);FINS指标:H组高于C组、E组低于C组、HE组低于H组(P<0.05);ISI指标:H组低于C组、E组高于C组、HE组高于H组(P<0.05)。2 ITT实验中,各组0时血糖无差异,腹腔注射胰岛素后,H组在15min、30min、60min血糖值均显著高于C组(P<0.05);HE组在15min、30min、60min血糖值均显著低于H组(P<0.05);E组在15min、30min血糖均显著低于C组(P<0.05)。3内脏脂肪重、腹脂指数和肝重,H组均高于C组,E组均低于C组,HE组均低于H组(P<0.01或P<0.05)。4 H组TC、TG、LDL-C、TC/HDL均显著高于C组(P<0.05),HDL-C、FFA无显著性差异(P>0.05);E组和C组比,TC、HDL-C、TC/HDL差异无显著性(P>0.05),TG、LDL-C、FFA显著低于C组(P<0.01或P<0.05);HE组和H组比,HDL-C、LDL-C、FFA差异不具显著性(P>0.05),TC、TG、TC/HDL显著低于H组(P<0.05或P<0.01);HE组和E组比,TC、TG差异无显著性(P>0.05),HDL-C显著低于E组(P<0.05),LDL-C、TC/HDL、FFA显著高于E组(P<0.01或P<0.05)。5 H组和C组肝脏TC、肌TG差异无显著性(P>0.05),肝脏TG非常显著性高于C组(P<0.01),肝脏FFA显著高于C组(P<0.05);E组和C组肝脏TC、肌TG差异无显著性(P>0.05),肝脏TG非常显著性低于C组(P<0.01),肝脏FFA显著低于C组(P<0.05);HE组肝脏TC显著低于H组(P<0.05),肝脏TG和FFA均非常显著性低于H组(P<0.01),肌TG差异不具显著性(P>0.05)。血清ALT和AST未出现显著性差异(P>0.05)。6虽未见显著性差异(P>0.05),但仍可看出血清脂联素水平与FINS和ALT指标均成负相关(R=-0.069,R=-0.179),与HDL呈正相关(R=0.043)。各组大鼠循环脂联素水平未见显著性差异(P>0.05)。H组内脏脂肪组织脂联素mRNA表达显著低于C组(P<0.05);E组略低于C组,HE组略低于H组,但差异不具显著性(P>0.05)。7 AdipoR1mRNA以股四头肌表达较为丰富,AdipoR2mRNA在肝脏高表达,差异有显著性(P<0.05)。H组AdipoR1/R2mRNA表达显著低于C组(P<0.05),而两运动组(E组和HE组)均与同类安静组水平相当(P>0.05)。8各组大鼠股四头肌间AMPKα蛋白表达无显著性差异(P﹥0.05)。HE组AMPKα(Thr172)磷酸化水平显著高于H组(P<0.05),较H组高43.2%;E组AMPKα(Thr172)磷酸化水平非常显著性高于C组(P<0.01),较C组高83.7%。9各组大鼠肝脏组织间AMPKα蛋白表达无显著性差异(P﹥0.05)。HE组AMPKα(Thr172)磷酸化水平显著高于H组(P<0.05),较H组高51.1%;其余各组无显著性差异(P﹥0.05)。主要结论:1高脂膳食饲养10周可诱导正常SD大鼠出现IR。间接评价IR指标ISI较HOMA-IR敏感。运动提高了大鼠组织胰岛素敏感性。2运动干预显著延缓高脂大鼠血脂代谢紊乱、内脏脂肪堆积和肝脏脂质异位沉积。运动用作提高胰岛素敏感性的干预措施时,肌脂和IR之间的关系还不甚清楚。3内脏脂肪组织脂联素mRNA表达降低可能是高脂大鼠IR形成的机制之一。循环脂联素水平变化可能并不是运动延缓高脂大鼠IR形成的主要贡献因素和机制所在。4正常SD大鼠股四头肌以AdipoR1高表达,AdipoR2在肝脏高表达。骨骼肌和肝脏AdipoR1/R2mRNA表达的下调可能是高脂大鼠IR形成的机制之一。未观察到运动干预的显著影响。5运动提高了普食大鼠骨骼肌AMPKα磷酸化水平,提示大鼠骨骼肌对规律耐力运动产生适应可能是由连续激活骨骼肌AMPKα活性引起。运动提高了高脂大鼠肝脏和骨骼肌AMPKα磷酸化水平,提示肝脏和骨骼肌AMPKα活性的连续激活可能是运动延缓高脂大鼠IR形成的关键机制之一。更多还原

【Abstract】 Objective To investigate the effects of exercise intervention during IR formation of high-fat-induced SD rats as well as some mechanisms.Methods Normal male SD rats were divided into four groups: normal dietary control group(C group), normal dietary exercise group(E group), high fat dietary control group(H group)and high fat dietary exercise group(HE group)after one week’s adaptive feeding. H and HE groups were fed 10-weeks’high-fat(23.5% protein,44.1% fat and 32.4% carbohydrate, supply energy about 408Kcal/100g)in order to induce IR, while E and HE groups began to swim without loading. Comprehensive using body weight, FINS, FBG, HOMA-IR, ISI and insulin tolerant test(ITT) to evaluate the building of animal model and effects of exercise intervention. Observing the impacts of high-fat feeding and exercise on blood lipids, visceral fat reserves, liver weight, liver function(ALT and AST), as well as tissue lipids(liver and skeletal muscle)ectopic deposition. Adopting ELISA and real-time PCR to observe adiponectin and its receptors(R1 and R2)mRNA expression systematically and comprehensive, including serum adiponectin, visceral fat tissue adiponectin mRNA expression, skeletal muscle and liver adiponectin receptors mRNA expression and their distributions in SD rats. Using western blotting to determinate AMPKαexpression and its phosphorylation level of liver and red quadriceps(considering the reaction to aerobic exercise is more sensitive on slow muscle fiber)after high-fat-induced and regular exercise.Results (1) There were no difference of weight before experiment while that of H was higher than C group, E was lower than C group, HE was lower than H group, HE was higher than E group after experiment(P<0.01).The average weight gain was the fastest on H group while E group was the slowest. There were no difference on FBG and HOMA-IR(P>0.05); H was higher than C group, E was lower than C group, HE was lower than H group on FINS index(P<0.05); on the contrary, H was lower than C group, E was higher than C group, HE was higher than H group on ISI index(P<0.05). (2) In ITT, there were no difference of FBG at zero point. After intraperitoneal injection of insulin, FBG of H group was higher significantly than C group at 15min, 30min and 60min points(P<0.05); HE group was lower than H group at the same points(P<0.05); E group was lower significantly than C group at 15min and 30min points(P<0.05).(3) H group was higher than C group, E group was lower than C group, and HE group was lower than H group on visceral fat reserves, liver weight and AW/BW(P<0.01orP<0.05).(4) H group was higher than C group on TC, TG, LDL-C, TC/HDL(P<0.05), and there were no difference of HDL-C and FFA(P>0.05); there were no difference between E and C group of TC, HDL-C, TC/HDL(P>0.05), H group were lower than C group on TG, LDL-C, FFA(P<0.01or P<0.05); there were no difference between HE and H group of HDL-C, LDL-C, FFA(P>0.05), HE group were lower than H group on TC, TG, TC/LDL(P<0.05or<0.01); there were no difference between HE and E group of TC,TG(P>0.05), HE group was lower than E group on HDL-C(P<0.05), while higher than E group on LDL-C,TC/LDL and FFA(P<0.01or<0.05).(5) There were no difference between H group and C group of liver TC and skeletal muscle TG(P>0.05), liver TG and FFA were higher than C group significantly(P<0.01or<0.05); There were no difference between E group and C group on liver TC and skeletal muscle TG(P>0.05), liver TG and FFA were lower than C groupsignificantly(P<0.01or<0.05); There were no difference between HE group and H group on skeletal muscle TG(P>0.05), liver TC, TG and FFA were lower than H group significantly(P<0.05or<0.01); There were no difference between four groups on serum ALT and AST(P>0.05).(6) In spite of no significant difference(P>0.05), serum adiponectin was negative correlation with FINS and ALT(R=-0.069,R=-0.179)while positive correlation with HDL(R=0.043).There were no difference between four groups of serum adiponectin(P>0.05). H group was lower than C group on adiponectin mRNA expression of visceral fat tissue(P<0.05), E group was slightly lower than C group and HE group was slightly lower than H group, but the difference were unsignificantly(P>0.05).(7) Adiponectin R1mRNA expression was abundant on quadriceps and R2mRNA expression was abundant on liver tissue(P<0.05). H group were lower than C group significantly on adiponectin R1/R2mRNA expression(P<0.05), while the two exercise groups were as much as the two sedentary groups(P>0.05).(8) There were no significant difference between four groups on AMPKαexpression of quadriceps(P>0.05). HE group was 43.2% higher than H group(P<0.05)and E group was 83.7% higher than C group on AMPKα(Thr172) phosphorylation level(P<0.01).(9) There were no significant difference between four groups of AMPKαexpression of liver tissue(P>0.05). HE group was 51.1% higher than H group on AMPKα(Thr172) phosphorylation level(P<0.05)and there were no difference betweenthe others(P>0.05).conclusions (1) High-fat-feeding for 10 weeks may induce normal SD rats into IR. When evaluating IR indirectly, ISI index was more sensitive than HOMA-IR index. Exercise improved rats insulin sensitivity. (2) Lipid metabolism abnormal may occur before glucose metabolism abnormal during IR formation of high-fat-induced rats. Exercise intervention delayed blood lipids metabolism abnormal, visceral fat accumulation, and liver lipids ectopic deposition significantly. The relationship between skeletal muscle lipids and IR still remained unclear when exercise was for improving insulin sensitivity. (3) Reduction on adiponectin mRNA expression of visceral fat tissue may one of the mechanisms of IR formation of high-fat-induced rats. Cycle adiponectin level may not be the main contribution factor and mechanism of delaying IR formation of high-fat-induced rats by exercise. Energy negative balance caused by exercise may paly the main role.(4) Adiponectin R1/R2 mRNA expressed in both liver and quadriceps of normal SD rats.R1 was high expressed in quadriceps and R2 was in liver. Reduction on adiponectin R1/R2 mRNA expression of skeletal muscle and liver may one of the mechanisms of IR formation of high-fat-induced rats. The study had not observed the significant effects of exercise intervention.(5) Exercise improved skeletal muscle AMPKα(Thr172) phosphorylation level of C group rats, which suggesting that rats skeletal muscle adapted to regular endurance exercise may be caused by continuous activation of skeletal muscle AMPKα. Exercise improved liver and skeletal muscle AMPKα(Thr172) phosphorylation level of H group rats, which suggesting that continuous activation of liver and skeletal muscle AMPKαmay be one of the key mechanisms of delaying IR formation of high-fat-induced rats by exercise.更多还原