【作者】 齐洁；

【导师】 张钧；

【作者基本信息】 扬州大学 ， 动物与人类运动比较学， 2014， 博士

【摘要】 胰岛素抵抗(insulin resistance, IR)是指胰岛素在其作用靶组织(骨骼肌、肝脏和脂肪组织)摄取和清除葡萄糖的能力低下。人体骨骼肌和脂肪组织是重要的胰岛素效应组织。研究认为,不合理的膳食结构,特别是高脂肪膳食,加上久坐不运动的生活方式,是引起IR的重要原因。运动作为一种有效的非药物防治手段,已广泛应用到防治因IR而引起的慢性代谢性疾病中,但其具体作用机制尚未完全阐明。因此,本研究以高脂饮食喂养诱导大鼠产生IR为模型,探讨不同方案的游泳运动对高脂饮食大鼠IR的影响及其相关机制。研究目的：探讨不同方案的游泳运动对高脂饮食大鼠胰岛素抵抗的影响及其相关机制。研究方法：以SD大鼠为实验对象,随机分为6组：普通饮食对照组(C组)、普通饮食连续90min运动组(CEA组)、普通饮食上下午各45min运动组(CEB组)、高脂饮食R模型组(H组)、高脂饮食IR连续90min运动组(HEA组)和高脂饮食IR上下午各45min运动组(HEB组)。通过8周高脂饲料(热卡比为蛋白质22%,脂肪38%,碳水化合物40%,提供能量约为4243Kcal/1000g)喂养建立IR大鼠动物模型,同时对大鼠实施无负重游泳运动干预。综合利用体重、空腹血糖(FBG)、空腹胰岛素(FINS)、胰岛素抵抗指数(HOMA-IR)、胰岛素敏感指数(ISI)和正糖钳试验评价动物模型的建立和运动干预的效果；通过观察运动对高脂饮食R大鼠血清游离脂肪酸(FFA)、血脂、内脏脂肪重量和骨骼肌脂质异位沉积的影响、骨骼肌PGC-1α、FAT/CD36、MG53、Cav-3和IRSmRNA表达量、骨骼肌炎症因子(CRP、TNF-α、IL-6和INF-y)含量、骨骼肌pIRS-1Ser307和pAktSer473的磷酸化水平以及MG53、IRS-1和Akt的蛋白表达水平的变化,探讨不同运动方案对高脂饮食诱导的IR大鼠骨骼肌的相关机制。通过观察运动对高脂饮食诱导IR大鼠脂肪组织FFA和TNF-a含量、脂肪组织IRS-1mRNA表达量、IKK-β和JNK信号通路的变化、pIRS-1Ser307和pAktSer473的磷酸化水平以及IRS-1和Akt的蛋白表达水平的变化,探讨不同运动方案对高脂饮食诱导的R大鼠脂肪组织的相关机制。研究结果：1.IR动物模型的评价：8周高脂饮食喂养后大鼠血清INS含量显著升高(p<0.01),ISI水平显著下降(p<0.01),HOMA-IR水平显著增加(p<0.01),葡萄糖输注速率(正糖钳实验)显著下降(p<0.01)。说明8周高脂饮食喂养己成功建立了IR动物模型。2.不同运动方案对高脂饮食大鼠IR的干预作用：8周游泳干预后,高脂饮食大鼠血清INS含量和HOMA-IR水平均显著降低(p<0.01),ISI水平和葡萄糖输注速率均显著增加(p<0.05或p<0.01)。但HEA组和HEB组大鼠血清INS、ISI、HOMA-IR和葡萄糖输注速率均无显著性差异(p,0.05),说明连续90min运动和上下午各45min运动均可有效改善高脂饮食大鼠R。3.运动对高脂饮食大鼠IP的影响——骨骼肌组织相关指标变化：①大鼠内脏脂肪重量变化及骨骼肌脂质异位沉积：8周运动干预后,高脂饮食大鼠内脏脂肪重量、脂体比和腹脂指数、骨骼肌FFA、TG、 PGC-1αmRNA和FAT/CD36mRNA表达量均显著下降(p<0.01),骨骼肌脂质异位沉积减少。说明运动可以减少高脂饮食诱导R大鼠脂肪含量和骨骼肌脂质异位沉积,而HEA组和HEB组大鼠无显著性差异(p<0.05)。说明连续90min运动和上下午各45min运动对减少体内脂肪和骨骼肌脂质异位沉积没有差异。②大鼠骨骼肌组织MG53的变化：8周运动干预后,高脂饮食大鼠骨骼肌MG53mRNA和MG53蛋白表达水平均显著下降(p<0.01),但HEA组和HEB组大鼠无显著性差异(p>0.05)。说明连续90min运动和上下午各45min运动可降低高脂饮食大鼠骨骼肌MG53的表达水平,但这两种运动方案对其影响没有差异。③大鼠骨骼肌组织神经酰胺和炎性因子含量的变化：8周运动干预后,高脂饮食大鼠骨骼肌神经酰胺和炎性因子(CRP、TNF-α、IL-6和INF-y)含量均显著降低(p<0.05或p<0.01),但HEA组和HEB组大鼠无显著性差异(p>0.05)。说明连续90min运动和上下午各45min运动可降低高脂饮食大鼠骨骼肌神经酰胺和炎性因子的含量,但这两种运动方案对其影响没有差异。④大鼠骨骼肌组织Cav-3mRNA表达、eNOS和GLUT4含量的变化：8周运动干预后,高脂饮食大鼠骨骼肌Cav-3mRNA表达量、eNOS含量和GLUT4含量显著增加(p<0.05或p<0.01)。但HEA组和HEB组大鼠无显著性差异(p>0.05)。说明连续90min运动和上下午各45min运动可增加高脂饮食大鼠骨骼肌Cav-3mRNA表达、eNOS含量和GLUT4含量,但这两种运动方案对其影响没有差异。⑤大鼠骨骼肌组织IRS-1和Akt表达水平的变化：8周运动干预后,高脂饮食大鼠骨骼肌IRS-1mRNA表达量、IRS-1蛋白表达水平、pAktSer473磷酸化水平和Akt蛋白表达水平均显著增加(p<0.05, P<0.01), pIRS-1Ser307的磷酸化水平显著下降(p<0.01)。但HEA组和HEB组大鼠无显著性差异(p>0.05)。说明连续90min运动和上下午各45min运动可增加高脂饮食大鼠骨骼肌IRS-1和Akt表达水平,但这两种运动方案对其影响没有差异。4.运动对高脂饮食大鼠IR的影响——脂肪组织相关指标变化：①大鼠脂肪组织FFA、TNF-a含量、IKKβ和JNK水平的变化：8周运动干预后,大鼠脂肪组织FFA含量、TNF-a含量、IKKβ蛋白表达水平、pJNKThr183/Tyr185磷酸化水平和JNK蛋白表达水平均显著降低(p<0.01)。但HEA组和HEB组大鼠无显著性差异(p>0.05)。说明连续90min运动和上下午各45min运动可减少高脂饮食大鼠脂肪组织FFA含量、TNF-a含量、IKKβ和JNK表达水平,但这两种运动方案对其影响没有差异。②大鼠脂肪组织IRS-1和Akt表达水平的变化：8周运动干预后,大鼠脂肪组织IRS-1mRNA表达、IRS-1蛋白表达水平、pAktSer473的磷酸化水平和Akt蛋白表达水平均显著增加(p<0.01),pIRS-1Ser307的磷酸化水平显著下降(p<0.01)。但HEA组和HEB组大鼠之间无显著性差异(p>0.05)。说明连续90min运动和上下午各45min运动可增加高脂饮食大鼠脂肪组织IRS-1和Akt表达水平,但这两种运动方案对其影响没有差异。研究结论：1.8周高脂饮食喂养成功建立了大鼠胰岛素抵抗模型。2.8周持续90min运动和上下午各45min运动可有效地改善高脂饮食诱导的胰岛素抵抗。这两种运动方案对改善高脂饮食诱导胰岛素抵抗无差异。3.运动改善胰岛素抵抗的可能机制：骨骼肌的可能机制：运动可通过四条途径,即运动可通过减少高脂饮食大鼠骨骼肌脂质异位沉积、降低大鼠骨骼肌细胞修复因子MG53表达水平、降低骨骼肌炎性因子和神经酰胺水平、增加骨骼肌Cav-3的表达,促进PI3-K/Akt信号转导,改善高脂饮食大鼠IR。脂肪组织的可能机制为：运动可通过抑制脂肪组织IKKβ/NF-κB和JNK/SAPK炎症信号通路,增强IRS-1/PI3-K/Akt信号传导,从而改善高脂饮食大鼠IR。更多还原

【Abstract】 ObjectiveTo investigate the effects of different exercise programs on high-fat-diet induced insulin resistance in rats.MethodsSix-week-old SD rats were randomly assigned to6groups:(1) sedentary rats fed with normal diet group (C group);(2) normal diet rats swimming1time per day(successive90min/time)(CEA group);(3) normal diet rats swimming twice per day(successive45min/time)(CEB group);(4) sedentary rats fed with high-fat diet group (H group);(5) high-fat diet rats swimming1time per day(successive90min/time)(HEA group);(6) high-fat diet rats swimming2time per day(successive45min/time)(HEB group). Rats of H group were fed with high-fat diet for8weeks to induce insulin resistance (IR). The protein provided22%, fat38%and carbohydrate40%of total energy (4243Kcal/1000g) intake. Rats in CEA group and HEA group took swim exercise1time per day(successive90min/time), rats in CEB group and HEB group took swim exercise twice per day(successive45min/time), rats in the four groups were all swam6days for8weeks. Comprehensive using body weight, FBG, FINS, HOMA-IR, ISI and he euglycemic-hyperinsulinemic clamp experiments to evaluate the animal model of IR and effects of exercise intervention. Observing the impacts of high-fat feeding and exercise on FFA, blood lipids, visceral fat, and skeletal muscle lipids deposition. Adopting real-time PCR, ELISA and Western blot to observe the mRNA expression of PGC-la, FAT/CD36, MG53, Cav-3, IRS-1, the contents of CRP, TNF-a, IL-6, INF-y, and the protein expression of MG53, IRS-1, Akt in skeletal muscle respectively, to investigate the effects of different swimming exercise programs on insulin resistance in skeletal muscle of high-fat-diet rats as well as some mechanisms. Adopting real-time PCR, ELISA and Western blot to observe the mRNA expression of IRS-1, the contents of TNF-a, and the protein expression of IKKβ, JNK, IRS-1, Akt in adipose tissue respectively, To investigate the effect of different swimming exercise program on insulin resistance in adipose tissue of high-fat-diet rats as well as some mechanisms.Results1. To evaluate IR animal model:After8weeks, to compared with C group, the level of insulin and HOMA-IR of H group were dramatically increased (p<0.01), the level of ISI and GIR of H group were markedly decreased (p<0.01). It was considered to become IR.2. Effect of different exercise programs on high-fat-diet induced Insulin resistance in rats:After8weeks, to compared with H group, the level of INS and HOMA-IR of HEA group were dramatically decreased (p<0.05), the level of ISI and GIR and HDL-C of HEA group were obviously increased (p<0.01). the level of INS and HOMA-IR of HEB group were dramatically decreased (p<0.01), the level of ISI and GIR and HDL-C of HEB group were obviously increased (p<0.01). But there were no differences between HEA and HEB group. It suggested that both swim exercise1time per day (successive90min/time) and swim exercise twice per day (successive45min/time) for8weeks can ameliorate high-fat-diet induced insulin resistance in3. The effect of different swimming exercise program on insulin resistance in skeletal muscle:①The visceral fat mass and the lipid deposits in skeletal muscle:After8weeks, to compared with H group, the visceral fat mass and the abdominal fat index in both HEA and HEB group were obviously decreased (p<0.01), but there were no differences between HEA and HEB group. both in HEA group and HEB group the level of FFA and TG were dramatically decreased (p<0.01), the mRNA expression of PGC-la and FAT/CD36were also markedly decreased (p<0.01). It suggested that both swim exercise1time per day (successive90min/time) and swim exercise twice per day (successive45min/time) for8weeks can reduce lipid deposits in skeletal muscle in high-fat-diet rat. But the effects of the two exercise programs have no differences.②The mRNA and protein expression of MG53in skeletal muscle:After8weeks, to compared with H group, the mRNA and protein expression of MG53were markedly decreased (p<0.01) both in HEA group and HEB group. But there were no differences between HEA and HEB group. It suggested that both swim exercise1time per day (successive90min/time) and swim exercise twice per day (successive45min/time) for8weeks can reduce the expression of MG53in skeletal muscle in high-fat-diet rat. But the effects of the two exercise programs have no differences.③Ceramide and inflammatory factor in skeletal muscle:After8weeks, to compared with C group, the content of Ceramide and inflammatory factor (CRP、TNF-α、IL-6and INF-y) were significantly increased (p<0.05or p<0.01) of H group; to compared with H group, the content of Ceramide and inflammatory factor (CRP、TNF-α、IL-6and INF-y) were significantly were obviously reduced (p<0.05or p<0.01) both in HEA group and HEB group. But there were no differences between HEA and HEB group. It suggested that both swim exercise1time per day (successive90min/time) and swim exercise twice per day (successive45min/time) for8weeks can decrease the level of Ceramide and inflammatory factor in skeletal muscle in high-fat-diet rat. But the effects of the two exercise programs have no differences.④The mRNA expression of Cav-3, the level of eNOS and GLUT4in skeletal muscle:After8weeks, to compared with H group, the mRNA expression of Cav-3, content of eNOS and GLUT4in skeletal muscle both in HEA group and HEB group were significantly decreased (p<0.05or P<0.01). But there were no differences between HEA and HEB group. It suggested that both swim exercise1time per day (successive90min/time) and swim exercise twice per day (successive45min/time) for8weeks can decrease the level of Cav-3, eNOS and GLUT4in skeletal muscle in high-fat-diet rat. But the effects of the two exercise programs have no differences.⑤The expression of IRS-1and Akt in skeletal muscle:After8weeks, to compared with H group, the mRNA and protein expression of IRS-1, pAktSer473and Akt of skeletal muscle both in HEA group and HEB group were dramatically increased (p<0.05or p<0.01), the protein expression of pIRS-1Ser307was obviously decreased (p<0.01). But there were no differences between HEA and HEB group. It suggested that both swim exercise1time per day (successive90min/time) and swim exercise twice per day (successive45min/time) for8weeks can increase the level of IRS-1and Akt of skeletal muscle in high-fat-diet rat. But the effects of the two exercise programs have no differences.4. The effect of different swimming exercise program on insulin resistance in adipose tissue:①The level of FFA, TNF-α, IKKβ and JNK in adipose tissue:After8weeks, to compared with H group, the content of FFA and TNF-α in adipose tissue of H group were dramaticlly decreased (p<0.01) both in HEA and HEB group, the protein expression of IKK β pJNKThr183/Tyr185and JNK were markedly decreased (p<0.01) both in HEA and HEB group. But there were no differences between HEA and HEB group. It suggested that both swim exercise1time per day (successive90min/time) and swim exercise twice per day (successive45min/time) for8weeks can decrease the level of FFA, TNF-α, IKKβ and JNK in adipose tissue in high-fat-diet rat. But the effects of the two exercise programs have no differences.②The mRNA and protein expression of IRS-1and Akt in adipose tissue:After8weeks, to compared with H group, the mRNA and protein expression of IRS-1, pAktSer473and Akt of adipose tissue both in HEA group and HEB group were dramatically increased (p<0.05or p<0.01), the protein expression of pIRS-1Ser307was obviously decreased (p<0.01). But there were no differences between HEA and HEB group. It suggested that both swim exercise1time per day (successive90min/time) and swim exercise twice per day (successive45min/time) for8weeks can increase the level of IRS-1and Akt of adipose tissue in high-fat-diet rat. But the effects of the two exercise programs have no differences. Conclusion1.8-week high-fat feeding is prone to induce IR model in rat.2. Both swim exercise1time per day (successive90min/time) and swim exercise twice per day (successive45min/time) for8weeks can ameliorate high-fat-diet induced insulin resistance in rats. But the effect of the two exercise program on ameliorate high-fat-diet induced insulin resistance in rats has no difference.3. The action mechanisms of swimming exercise on ameliorating skeletal muscle IR may through four pathways——Swimming exercise can ameliorate high-fat-diet induced skeletal muscle IR in rats through reducing lipid deposits, decreasing the mRNA and protein expression of MG53, making a decline on the level of inflammatory factors, improve the expression of Cav-3, then enhancing the PI3-K/Akt signaling transduction, thus ameliorate high-fat-diet induced skeletal muscle insulin resistance in rats.The action mechanisms of swimming exercise on ameliorating adipose tissue IR may be: swimming exercise can make IRS-1/PI3-K/Akt signaling transduction enhancement through inhibit inflammatory signaling pathways (IKKβ/NF-κB and JNK/SAPK), thus ameliorating high-fat-diet induced adipose tissue insulin resistance in rats.更多还原