Poldip2在胰岛素信号通路中的作用及对2型糖尿病脂代谢的调节

发布时间：2018-10-12 09:01

【摘要】：目的:本研究旨在研究细胞水平中高糖对细胞的脂代谢作用及poldip2的表达水平;并研究2型糖尿病小鼠注入Poldip2腺病毒后对胰岛素信号通路中的作用及对KKAy小鼠脂代谢的调节作用。方法:(1)将Mc Ardle 7777细胞分别培养于高糖培养基和正常葡萄糖培养基中,检测在高糖和正常葡萄糖培养下TG,TCH水平及Poldip2的表达情况。(2)利用不同胰岛素剂量建立不同水平的胰岛素抵抗,检测Poldip2在胰岛素作用下的剂量反应关系、时间效应关系;以及在胰岛素最优剂量和时间下,Poldip2对Mc Ardle7777细胞的脂代谢作用。(3)体内实验中,KKAy小鼠经过4周高脂饮食后,将KKAy小鼠随机分为Poldip2病毒组;GFP组;Saline组。此外,将C57BL/6J小鼠作为空白对照组测定小鼠中血脂四项,餐后脂耐受水平,血清游离脂肪酸,胰岛素,瘦素,肝脏脂水平,肝脏中PTP1B酶活性、H_2O_2水平,并检测胰岛素信号通路中相关蛋白的表达水平。结果:细胞实验中,高糖情况下Poldip2的表达水平显著下降,同时高糖刺激能够降低脂代谢水平。在胰岛素刺激实验中,正常葡萄糖下,胰岛素剂量为20μmol,24h内Poldip2蛋白的表达最为丰富;高糖下,胰岛素剂量5μmol,培养6h后Poldip2的表达最为丰富,其细胞TCH水平显著高于未加胰岛素刺激下的TCH水平。同时发现在高糖培养下,较低剂量的胰岛素,就会使Poldip2的表达抑制。体内动物试验中,Poldip2腺病毒能够显著改善KKAy小鼠的血糖水平,并能降低血清中TG,TCH,LDL-C及肝脏中TCH的水平;降低血清中游离脂肪酸及胰岛素水平;对小鼠新鲜肝脏中PTP1B酶活性和H_2O_2的检测发现,Poldip2能提高肝脏中H_2O_2水平,并显著降低PTP1B酶的活性;通过餐后脂耐受水平检测发现Poldip2可加速小鼠外源性脂肪代谢。通过对胰岛素信号通路中相关蛋白的检测,Poldip2能够降低KKAy小鼠中PTEN,PTP1B蛋白的表达,激活Akt的活性进而促进胰岛素信号通路的传递;另外Poldip2可能通过降低PEPCK蛋白的表达,进而抑制糖异生的合成,使肝糖葡萄糖输出减少。结论:细胞在高糖培养下发现高糖对细胞具有高毒作用,产生了脂代谢紊乱,这可能与细胞在高糖培养下Poldip2的蛋白水平下降相关。Poldip2能够扭转外源性脂代谢的异常,改善小鼠的脂代谢情况。Poldip2改善糖尿病小鼠糖脂代谢的机制可能是通过Poldip2的表达升高,激活Nox4产生H_2O_2,过度氧化抑制了PTP1B,PTEN的活性,进而激活Akt蛋白的活性,改善了胰岛素信号通路及糖脂代谢紊乱。
[Abstract]:Aim: to study the effect of high glucose on lipid metabolism and the expression of poldip2 in cells, and to study the role of Poldip2 adenovirus in insulin signaling pathway and the regulation of lipid metabolism in KKAy mice in type 2 diabetic mice. Methods: (1) Mc Ardle 7777 cells were cultured in high glucose medium and normal glucose medium respectively. The levels of TG,TCH and the expression of Poldip2 were detected under high glucose and normal glucose culture. (2) different levels of insulin resistance were established with different insulin doses. The dose-response relationship, time-effect relationship and lipid metabolism of Poldip2 on Mc Ardle7777 cells under the optimal insulin dose and time were measured. (3) in vivo, the KKAy mice were fed a high-fat diet for 4 weeks. KKAy mice were randomly divided into Poldip2 virus group, GFP group and Saline group. In addition, C57BL/6J mice were used as blank control group to determine four items of blood lipids, postprandial lipid tolerance, serum free fatty acids, insulin, leptin, liver lipid, liver PTP1B enzyme activity and H_2O_2 level. The expression of related proteins in insulin signaling pathway was detected. Results: in the cell experiment, the expression of Poldip2 was significantly decreased under high glucose condition, and the lipid metabolism was decreased by high glucose stimulation. In insulin stimulation experiment, the expression of Poldip2 protein was the most abundant in normal glucose at 20 渭 mol,24h, and in high glucose at 5 渭 mol, for 6 h. The level of TCH was significantly higher than that of TCH without insulin stimulation. At the same time, it was found that the low dose of insulin inhibited the expression of Poldip2 in high glucose culture. In animal experiment in vivo, Poldip2 adenovirus could significantly improve the blood glucose level of KKAy mice, and decrease the levels of TG,TCH,LDL-C in serum and TCH in liver, free fatty acid and insulin in serum. The detection of PTP1B enzyme activity and H_2O_2 in fresh liver of mice showed that Poldip2 could increase the level of H_2O_2 in liver and decrease the activity of PTP1B enzyme significantly, and Poldip2 could accelerate the exogenous fat metabolism of mice by detecting the level of postprandial lipid tolerance. By detecting the related proteins in insulin signaling pathway, Poldip2 can reduce the expression of PTEN,PTP1B protein in KKAy mice, activate the activity of Akt and promote the transmission of insulin signal pathway. In addition, Poldip2 may decrease the expression of PEPCK protein. Furthermore, the synthesis of glucose allogeneic was inhibited, and the glucose output of liver was decreased. Conclusion: high glucose has high toxicity to cells in high glucose culture and leads to lipid metabolism disorder, which may be related to the decrease of protein level of Poldip2 in high glucose culture. Poldip2 can reverse the abnormality of exogenous lipid metabolism. Improving lipid metabolism in mice. The mechanism of Poldip2 in improving glucose and lipid metabolism in diabetic mice may be through the increase of Poldip2 expression and activation of Nox4 to produce H _ 2O _ 2. Excessive oxidation inhibits the activity of PTP1B,PTEN and then activates the activity of Akt protein. Improved insulin signaling pathway and glycolipid metabolism disorder.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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