运动与生酮饮食对STZ诱导的Ⅱ型糖尿病小鼠葡萄糖稳态及肝脏脂代谢的作用及机制研究

发布时间：2018-04-21 04:33

本文选题：生酮饮食 + 运动　；参考：《华东师范大学》2017年硕士论文

【摘要】：糖尿病是威胁全球人类身体健康的非传染性疾病之一,不但给患者个人造成了极大的健康负担,而且也给国家和社会造成了沉重的经济负担。国际糖尿病联盟(IDF)最新统计数据显示,2016全球约约有4.15亿成年糖尿病患者,据估计,2040年全球成年糖尿病患者将达到6.42亿。糖尿病发病机制极为复杂,至今为止,尚无任何方法可完全治愈糖尿病。目前二型糖尿病主要治疗手段有药物治疗、饮食治疗以及合理的体育活动。由于药物的安全性以及除二甲双胍外大多数糖尿病药物疗效有限或价格昂贵,因此,非药疗法越来越受到人们的重视。临床研究发现,生酮饮食(Ketogenic Diet,KD)作为一种糖尿病的干预手段可对患者的糖代谢稳态产生良性影响,并开始用于二型糖尿病患者的临床治疗。然而,近几年有研究发现糖尿病小鼠长期生酮饮食可能导致肝脏组织脂肪代谢紊乱,甚至诱发脂肪肝。临床及动物研究显示不同方式运动对脂肪肝的预防、治疗均有一定的积极作用,但是运动能否改善生酮饮食治疗所导致的肝脏组织脂肪代谢紊乱仍待研究。目的:(1)探究生酮饮食对糖尿病小鼠糖、脂代谢的影响及其机制。(2)探究生酮饮食联合不同方式运动对糖尿小鼠肝脏脂代谢影响及其机制。方法:雄性C57BL/6J小鼠(3-5周龄),共85只,适应性饲养3天,随机挑选出10只作为正常对照组(NC),采用标准普通饲料喂养。另外75只小鼠首先采用45%高脂饲料喂养4周,之后100 mg/kg BW腹腔注射STZ,连续三次空腹血糖水平高于11.1 mmol/l,判断为Ⅱ型糖尿病造模成功。此次实验中共有58只小鼠造模成功,根据体重和血糖水平选取32只,将其分为糖尿病对照组(HFD,n=8)、生酮饮食组(KD,n=8)、生酮饮食高强度间歇运动组(KH,n=8)、生酮饮食有氧运动组(KA,n=8)。每次运动前、后进行5min,8m/min的准备活动和整理活动,KH组采取8周无坡度跑台训练,高强度运动4 min,积极性休息2 min,速度由17m/min增至24m/min,每次运动60min,3次/周;KA组采取8周无坡度跑台训练,12m/min速度运动60min,每次运动60min,5次/周。结果:(1)与 NC 组相比较,HFD 组小鼠 FBG(p0.01)、HbAlc(p0.05),血清 Glu(p0.05)、Insulin(p0.05),HOMA-IR(p0.05)及日饮水量(p0.05)显著上升,HOMA-ISI(p0.05)显著降低;KD组HOMA-ISI(p0.05)显著上升,血清 Insulin(p0.05)、HOMA-IR(p0.05)体重(p0.05)显著下降,FBG、HbAlc、血清Glu以及日饮水量无显著差异;KH组HOMA-ISI(p0.05)显著上升,HOMA-IR(p0.05)、血清 Insulin(p0.05)和体重(p0.05)显著下降,FBG、HbAlc、血清Glu及日饮水量无显著差异;KA组小鼠血清Insulin(p0.05)、体重(p0.05)显著下降,FBG、HbAlc、血清 Glu、HOMA-IR、HOMA-ISI 以及日饮水量无显著差异;与HFD组相比较,KD组、KH组以及KA组HOMA-ISI(p0.05)、均显著升高;KD 组、KH 组以及 KA 组 FBG(p0.01)、HbAlc(p0.05),血清 Insulin(p0.05)、Glu(p0.05),HOMA-IR(p0.05)、体重(p0.05)及日饮水量(p0.05)显著下降。与NC组相比较,KD组、KH组以及KA组小鼠G6PC以及PCK1 mRNA表达均显著降低(p0.01);与HFD组相比较,KD组、KH组、KA组小鼠G6PC以及PCK1 mRNA表达均显著降低(p0.01)。(2)与 NC 组相比较,HFD 组血清 ALT(p0.01)、TC(p0.05)、LDL-C(p0.05)、肝指数(p0.01)、肝脏TG(p0.01)、NEFA(p0.01)水平显著升高,附睾脂肪%显著降低(p0.05),血清TG、HDL-C、AST水平无显著变化;KD组血清 TC(p0.05)、TG(p0.05)、HDL-C(p0.05)、ALT(p0.01)、AST(p0.05),附睾脂肪%(p0.01)、肝指数(p0.01)、肝脏 TG(p0.01)、NEFA(p0.05)显著升高;与HFD组相比较,KD组血清HDL-C(p0.05)、附睾脂肪%(p0.01)显著升高,血清LDL-C水平、肝指数显著降低(p0.05),血清AST、ALT、TG、TC及肝脏组织TG、NEFA水平无显著差异。肝脏HE以及油红染色显示,NC组小鼠肝脏的肝小叶结构完整,HFD组小鼠肝脏脂肪空泡较多,细胞膜结构不清晰,KD组小鼠肝脏也出现较多明显的脂肪空泡;肝脏MASSON染色显示,与NC组相比较,HFD组及KD组小鼠肝脏组织均出现了不同程度的纤维化。(3)与NC组相比,HFD组小鼠肝脏组织脂代谢相关基因CIDEA(p0.01)、SCD1(p0.05)、ACC1(p0.01)、ACC2(p0.01)、SREBP1(p0.05)、CPT1A(p0.001)的mRNA水平出现显著上升,FAS、AMPKa1的mRNA表达水平未有显著变化,AMPKa2的mRNA水平显著降低(p0.05);KD组CIDEA、FAS、SCD1、SREBP1、ACC1、ACC2、CPT1A 的 mRNA 水平均显著上升(p0.01),AMPKa1(p0.05)、AMPKa2(p0.01)的 mRNA 表达水平显著下降;与 HFD组相比较可以发现,生KD组小鼠肝脏组织AMPKa2的mRNA表达水平显著下降,SCD1(p0.05)、AMPKa2(p0.05)以及 CPT1A(p0.01)的 mRNA 相对表达均显著上升,CIDEA、FAS、SREBP1、ACC1、ACC2、AMPKa1 的 mRNA表达水平未有显著差异。(4)与NC组相比,HFD组小鼠肝脏的FAS蛋白表达水平显著上升(p0.01),ATGL、P-ACC/ACC 蛋白表达水平出现显著下降(p0.05),HSL、MGL、PPARα、PPARγ、CPT1A蛋白表达水平以及P-AMPK/AMPK蛋白比值未发生显著变化,生酮饮食组小鼠肝脏P-AMPK/AMPK蛋白比值出现显著下降(p0.05),FAS(p0.01)、PPARγ(p0.05)、CPT1A(p0.05)蛋白表达出现显著上升,HSL、ATGL、MPGL、PPARα蛋白表达以及P-ACC/ACC蛋白比值无显著变化;与糖尿病对照组相比,生酮饮食组小鼠肝脏FAS蛋白及P-AMPK/AMPK蛋白表达比值显著下降(p0.05),ATGL、CPT1A、PPARα蛋白表达显著提高(p0.05),而HLS、MGL、PPARy蛋白表达以及P-ACC/ACC蛋白比值无显著变化。(5)与KD组相比较,KA组糖尿病小鼠血清TG(p0.05)、ALT(p0.01)、AST(p0.05)、HDL-C(p0.01),肝脏 TG(p0.01)、NEFA(p0.01)水平显著降低,血清LDL-C(p0.01)的水平显著升高;肝指数及附睾脂肪%未发生明显差异,KH 组血清 LDL-C(p0.01)水平显著提高,血清 ALT(p0.05)、TC(p0.05)、肝脏TG(p0.05)水平显著降低,血清TG、AST水平、肝脏组织NEFA含量及肝指数、附睾脂肪%未发生明显变化。肝脏HE、油红染色显示,与KD组相比,KA组小鼠肝细胞形态趋向于正常,脂肪脂滴含量明显减少,KH组肝细胞结构较为清晰,肝细胞内脂滴含量减少;肝脏MASSON染色显示,与KD组相比,KH纤维化程度稍有降低,KA组肝脏纤维化显著降低。(6)与KD组相比,KH组及KA组小鼠肝脏组织G6PC的mRNA相对表达量均显著降低(p0.05),KA组小鼠肝脏组织G6PC活性显著下降(p0.05),而KH组无显著差异。与KD组相比,生酮饮食联合高强度间歇运动或有氧运动干预均可使糖尿病小鼠肝脏PCK1的mRNA相对表达量显著降低(p0.01),且均可显著降低小鼠肝脏组织PCK1的活性(p0.05)。与KD组相比,KH组小鼠肝脏组织 CIDEA(p0.01)、FAS(p0.01)、SCD1(p0.05)、SREBP1(p0.01)、ACC1(p0.01)的 mRNA 相对表达显著降低,ACC2(p0.05)、AMPKa2(p0.01)、CPT1A(p0.05)的mRNA相对表达含量显著提高,AMPKa1的mRNA影响则不明显。KA组小鼠肝脏组织CIDEA、FAS、SCD1、SREBP1、ACC1、ACC2的mRNA相对表达量(p0.01)显著降低,肝脏AMPKa1、AMPKa2、CPT1A(p0.01)的mRNA相对表达量显著提高。(7)与KD组相比较,KA组肝脏FAS、HSL、ATGL蛋白表达无显著变化,MGL、CPT1A的蛋白表达、P-AMPK/AMPK、P-ACC/ACC蛋白表达的比值均显著升高(p0.05),KH 组 HSL、ATGL、MGL、CPT1A 的蛋白表达、P-ACC/ACC蛋白表达的比值均无显著变化,FAS蛋白表达显著降低(p0.05)P-AMPK/AMPK蛋白表达的比值均显著上升(p0.05)。结论:(1)生酮饮食及生酮饮食联合运动干预均可显著改善STZ诱导的Ⅱ型糖尿病小鼠血糖控制。(2)长期生酮饮食,有可能会导致STZ诱导的T2DM小鼠肝脏组织脂肪积累,产生脂肪肝。(3)生酮饮食结合有氧运动干预或生酮饮食结合高强度间歇运动干预,均可有效缓解单独使用生酮饮食所导致糖尿病小鼠肝脏组织脂肪积累,且有氧运动效果更明显。(4)有氧运动可能通过激活AMPK/ACC/CPT1A信号通路加速糖尿病小鼠肝脏组织脂肪氧化,减缓脂肪肝发生进程,而高强度间歇运动则不依赖此调节途径。
[Abstract]:Diabetes is one of the non communicable diseases that threaten the health of the world. It not only creates a huge health burden on individuals, but also creates a heavy economic burden on the country and society. The latest statistics of the International Diabetes Association (IDF) show that about 415 million adult diabetic patients around the world are about 2016, estimated to be in 2040. The incidence of diabetes in adults will reach 642 million. The pathogenesis of diabetes is very complicated. So far, there is no way to cure diabetes completely. At present, the main treatment of type two diabetes is medications, diet and physical exercise. Nondrug therapy is becoming more and more important. Therefore, non drug therapy is becoming more and more important. Clinical studies have found that Ketogenic Diet (KD), as an intervention for diabetes, can have a benign effect on the homeostasis of glucose metabolism in patients and began to be used in the clinical treatment of patients with type two glycometabolism. However, in recent years, there have been studies on the discovery of sugar. The long-term ketone diet may lead to lipid metabolism disorder in the liver tissue and even induce fatty liver. Clinical and animal studies have shown that different ways of exercise have positive effects on the prevention and treatment of fatty liver. But whether the exercise can improve the lipid metabolism disorder in the liver tissue caused by the treatment of ketogenic diet still needs to be studied. (1) to explore the effect of ketogenic diet on glucose and lipid metabolism in diabetic mice and its mechanism. (2) to explore the effect and mechanism of ketogenic diet combined with different exercise on liver lipid metabolism in diabetic mice. Methods: male C57BL/6J mice (3-5 weeks of age), 85, adaptive feeding for 3 days, and randomly selected 10 as normal control group (NC), using standard general popularization The other 75 mice were fed with 45% high fat diet for 4 weeks, and then 100 mg/kg BW were intraperitoneally injected with STZ, and three consecutive fasting blood glucose levels were higher than 11.1 mmol/l. It was judged to be a successful model of type II diabetes. In this experiment, 58 mice were successfully built, and 32 were selected according to the weight and blood glucose level, and they were divided into diabetes pairs. The group (HFD, n=8), the ketogenic diet group (KD, n=8), the high intensity intermittent exercise group (KH, n=8) and the aerobic exercise group (KA, n=8) in the ketogenic diet (KA, n=8). Before each exercise, the preparation activities and the activities of 8m/min were carried out. The KH group took 8 weeks without slope running platform training, the high intensity exercise was 4 min, and the active rest was 2. The speed was increased from 2 Each exercise was 60min, 3 times per week, group KA took 8 weeks without slope running, and 12m/min speed exercise was 60min, each exercise was 60min, 5 times per week. (1) compared with group NC, HFD group mice FBG (P0.01), HbAlc (P0.05), serum Glu and daily drinking water increased significantly. HOMA-ISI (P0.05) increased significantly, serum Insulin (P0.05), HOMA-IR (P0.05) weight (P0.05) decreased significantly, FBG, HbAlc, serum Glu and daily drinking water had no significant difference. Serum Insulin (P0.05), body weight (P0.05) decreased significantly, FBG, HbAlc, serum Glu, HOMA-IR, HOMA-ISI and daily drinking water amount had no significant difference. Compared with the HFD group, KD, KH and KA groups were all significantly higher. Weight (P0.05) and daily drinking water (P0.05) decreased significantly. Compared with the NC group, the G6PC and PCK1 mRNA expressions in KD, KH and KA groups were significantly decreased (P0.01). The liver index (P0.01), liver TG (P0.01), NEFA (P0.01) level significantly increased, the epididymal fat% decreased significantly (P0.05), the serum TG, HDL-C, AST level had no significant changes; KD group serum TC (P0.05), fat% of epididymis, liver index, liver index, significantly higher; The serum HDL-C (P0.05) and epididymal fat% (P0.01) in the KD group were significantly higher, the serum LDL-C level, the liver index decreased significantly (P0.05), the serum AST, ALT, TG, TC and liver tissue TG were not significant differences. The liver MASSON staining showed that the liver tissues of group HFD and KD group had different degrees of fibrosis in KD group. (3) compared with the NC group, the lipid metabolism related gene CIDEA (P0.01), SCD1 (P0.05), ACC1 (P0.05) in the HFD group were compared with the NC group. 5, the level of mRNA in CPT1A (p0.001) increased significantly, and the level of mRNA expression in FAS and AMPKa1 was not significantly changed, and the mRNA level of AMPKa2 decreased significantly (P0.05). The expression level of mRNA in the liver tissue of KD mice decreased significantly, and the relative expressions of SCD1 (P0.05), AMPKa2 (P0.05) and CPT1A (P0.01) were significantly increased. (4) there was no significant difference in the expression level of the mRNA. The expression level of P0.01, ATGL and P-ACC/ACC decreased significantly (P0.05), HSL, MGL, PPAR a, PPAR gamma, CPT1A protein expression level and P-AMPK/AMPK protein ratio did not change significantly, the ratio of P-AMPK/AMPK protein in the liver of the ketone diet mice decreased significantly (P0.05). There was no significant increase in the expression of HSL, ATGL, MPGL, PPAR alpha and P-ACC/ACC protein. Compared with the control group, the ratio of FAS protein and P-AMPK/AMPK protein in the liver of the ketone diet group decreased significantly (P0.05), ATGL, CPT1A, and PPAR alpha protein expression significantly increased (P0.05). There was no significant change in white ratio. (5) compared with group KD, serum TG (P0.05), ALT (P0.01), AST (P0.05), HDL-C (P0.01), liver TG (P0.01), liver TG (P0.01), liver index and epididymal fat% were significantly higher in KA group. ALT (P0.05), TC (P0.05), liver TG (P0.05) level decreased significantly, serum TG, AST level, liver tissue NEFA content and liver index, epididymal fat% did not change obviously. Liver HE, oil red staining showed that the hepatocyte morphology of KA group mice tended to be normal, fat lipid droplet content decreased obviously, liver cell structure was clearer, The lipid droplet content in liver cells decreased, and the liver MASSON staining showed that the degree of KH fibrosis decreased slightly and the liver fibrosis in the group KA decreased significantly compared with the KD group. (6) the mRNA relative expression of G6PC in the KH and KA groups decreased significantly (P0.05), and the G6PC activity of the liver tissue in the KA group was significantly lower than that in the KD group. Compared with the KD group, the relative expression of PCK1 mRNA in the liver of diabetic mice could be reduced significantly (P0.01) by combination of ketogenic diet combined with high intensity intermittent exercise or aerobic exercise (P0.01), and the activity of PCK1 in liver tissue of mice was significantly reduced (P0.05). The liver tissue of KH mice was compared with that of the KD group. The relative expression of mRNA in BP1 (P0.01) and ACC1 (P0.01) decreased significantly, while ACC2 (P0.05), AMPKa2 (P0.01), CPT1A (P0.05) increased significantly. (7) there was no significant change in the expression of FAS, HSL, ATGL protein in the liver of KA group compared with the KD group. The protein expression of MGL, CPT1A, P-AMPK/AMPK, and P-ACC/ACC protein expression increased significantly (P0.05). The ratio of the expression of (P0.05) P-AMPK/AMPK protein increased significantly (P0.05). Conclusion: (1) the combination of ketogenic diet and ketogenic diet combined exercise could significantly improve the control of blood glucose in type II diabetic mice induced by STZ. (2) the long-term ketone diet may lead to the accumulation of fat in the liver tissue of STZ induced T2DM mice and the production of fatty liver. (3) Ketone diet combined with aerobic exercise intervention or ketogenic diet combined with high intensity intermittent exercise intervention can effectively alleviate the lipid accumulation in liver tissue of diabetic mice induced by single use of ketone diet, and the effect of aerobic exercise is more obvious. (4) aerobic exercise may accelerate the liver tissue lipid in diabetic mice by activating the AMPK/ACC/ CPT1A signaling pathway Fat oxidation slowed down the development of fatty liver, while high intensity intermittent exercise did not depend on this regulation.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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