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Effects of Vaspin on Pancreatic Beta Cell Function in Type 2 Diabetic Rats by AMPK/mTOR Autophagy Signaling Pathway
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摘要:
目的 探究内脏脂肪特异性丝氨酸蛋白酶抑制剂(visceral adipose tissue-derived serpin, Vaspin)改善2型糖尿病(type 2 diabetes mellitus，T2DM)大鼠胰岛β细胞功能的作用机制。 方法 采用高脂高糖喂养联合腹腔注射链脲佐菌素的方式建立T2DM大鼠模型，并随机分为T2DM组(n=10)、Vaspin组(n=10)，以同周龄正常饲料喂养的SD大鼠为正常对照组(n=10)。记录造模前及Vaspin干预前、干预4周和干预8周时3组大鼠体质量和空腹血糖(fasting blood-glucose, FBG)。Vaspin干预8周时，测定3组大鼠空腹胰岛素(fasting insulin, FINS)、糖耐量与胰岛素敏感性、胰岛β细胞功能及自噬相关蛋白表达水平，观察胰腺组织病理学形态。 结果 与正常对照组比较，T2DM组与Vaspin组干预前、干预4周、干预8周时体质量均下降，FBG均升高(P均＜0.05)；与T2DM组比较，Vaspin组干预8周时大鼠体质量增高，FBG下降(P均＜0.05)。组织病理示，正常对照组大鼠胰腺组织正常，胰岛细胞排列均匀、整齐，形态规则；T2DM组大鼠胰岛结构明显破坏，细胞分布不均匀、形状不规则；Vaspin组大鼠胰岛结构损伤、胰岛细胞形态破坏均较T2DM组减轻。干预8周时，与正常对照组比较，T2DM组及Vaspin组FINS降低，腹腔葡萄糖耐量试验(intraperitoneal glucose tolerance test, IPGTT)及腹腔胰岛素耐量试验(intraperitoneal insulin tolerance test, IPITT)血糖曲线下面积均升高(P均＜0.05)；与T2DM组比较，Vaspin组FINS升高，IPGTT与IPITT血糖曲线下面积均降低(P均＜0.05)。高葡萄糖钳夹试验示，干预8周时，Vaspin组葡萄糖输注速率、第一时相及第二时相胰岛素分泌量虽低于正常对照组，但各指标均较T2DM组升高(P均＜0.05)。免疫组化及Western blot结果示，干预8周时，与正常对照组比较，T2DM组大鼠胰腺组织中胰岛素表达水平及磷酸化哺乳动物雷帕霉素靶蛋白(phosphorylated mammalian target of rapamycin, p-mTOR)/mTOR比值均降低，P62、微管相关蛋白1轻链3(microtubule associated protein1 light chain3, LC3)蛋白水平、磷酸化腺苷酸活化蛋白激酶(phosphorylated adenosine monophosphate activated protein kinase, p-AMPK)/AMPK比值、LC3Ⅱ/LC3Ⅰ比值均升高(P均＜0.05)；与T2DM组比较，Vaspin组大鼠胰腺组织中胰岛素、LC3蛋白水平、p-AMPK/AMPK比值及LC3Ⅱ/LC3Ⅰ比值均升高，p-mTOR/mTOR比值及P62蛋白表达水平均降低(P均＜0.05)。 结论 Vaspin可通过AMPK/mTOR自噬信号通路增强T2DM大鼠胰岛β细胞自噬能力，进而改善胰岛β细胞功能。 -
关键词:
- 内脏脂肪特异性丝氨酸蛋白酶抑制剂 /
- 2型糖尿病 /
- 腺苷酸活化蛋白激酶 /
- 哺乳动物雷帕霉素靶蛋白 /
- 自噬 /
- 胰岛β细胞功能
Abstract:Objective To investigate the mechanism of Vaspin in improving the pancreatic beta cell function in type 2 diabetic (T2DM) rats. Methods The diabetic rat model was established by feeding high-fat and high-sugar diet combined with intraperitoneal injection of streptozotocin. T2DM rats were randomly divided into T2DM group(n=10) and Vaspin group(n=10), with the same age SD rats fed with normal chow as normal control group (n=10). Before modeling, before Vaspin intervention, at 4 weeks and 8 weeks Vaspin intervention, the level of body weight and fasting blood-glucose(FBG) of rats were recorded. At 8 weeks of Vaspin intervention, fasting insulin (FINS), glucose tolerance and insulin sensitivity, pancreatic β-cell function and autophagy-related protein expression levels were measured in three groups of rats, and histopathological morphology of the pancreas was observed. Results Before Vaspin intervention, at the 4 weeks and 8 weeks Vaspin intervention, compared with the normal control group, the level of body weight decreased and FBG increased in T2DM group and Vaspin group(all P < 0.05). At 8 weeks Vaspin intervention, compared with T2DM group, the level of body weight increased and FBG decreased in Vaspin group (all P < 0.05). Histopathology showed that the pancreatic tissue was normal, islet cells were arranged evenly, neatly and regularly in normal control group. In T2DM group, the islet structure was obviously destroyed, and the cells were unevenly distributed and irregularly shaped. Compared with T2DM group, the structural damage and morphological damage of islet cells in Vaspin group were significantly reduced. At the 8 weeks intervention, compared with the normal control group, the level of FINS decreased, while the area under the blood glucose curve of intraperitoneal glucose tolerance test(IPGTT) and intraperitoneal insulin tolerance test(IPITT) increased in T2DM group and Vaspin group(all P < 0.05). Compared with T2DM group, the level of FINS increased, while the area under blood glucose curve of IPGTT and IPITT decreased in Vaspin group(all P < 0.05). At the 8 weeks intervention, the high glucose clamp test showed that the glucose infusion rate, the level of insulin secretion in the first phase and the second phase in Vaspin group were lower than those in the normal control group, but all indexes in Vaspin group were higher than those in T2DM group (all P < 0.05). At the 8 weeks intervention, immunohistochemical and Western blot results showed that compared with the normal control group, the level of insulin expression and the ratio of p-mTOR/mTOR in pancreatic tissue of rats in T2DM group decreased, while the protein levels of P62, microtubule associated protein 1 light chain 3 (LC3), p-AMPK/AMPK ratio and LC3 Ⅱ/LC3 Ⅰ ratio increased. Compared with T2DM group, the levels of insulin, LC3 protein, p-AMPK/AMPK ratio and LC3 Ⅱ/LC3 Ⅰ ratio in pancreatic tissue of rats in Vaspin group increased, while p-mTOR/mTOR ratio and P62 protein expression decreased (all P < 0.05). Conclusion Vaspin can enhance the autophagy of pancreatic beta cells, improve pancreatic beta cells function by AMPK/mTOR signaling pathway in the type 2 diabetic rats. 作者贡献：魏姣姣负责研究实施及论文撰写；刘师伟负责研究设计并指导论文修订；段瑞雪、李楠负责实验设计、数据分析及结果解读；王江娜负责实验过程及文献查询。利益冲突：所有作者均声明不存在利益冲突 -
图 1 3组大鼠糖耐量及胰岛素敏感性比较(n=6)
A.腹腔葡萄糖耐量试验血糖变化；B.腹腔葡萄糖耐量试验的血糖AUC比较；C.腹腔胰岛素耐量试验血糖变化；D.腹腔胰岛素耐量试验的血糖AUC比较
AUC：曲线下面积；T2DM、Vaspin：同表 1
图 2 3组大鼠高葡萄糖钳夹试验结果比较(n=3)
A.试验过程中GIR变化；B.稳态时GIR比较；C.第一时相与第二时相胰岛素分泌量比较
GIR：葡萄糖输注速率；T2DM、Vaspin：同表 1
图 4 3组大鼠胰腺组织胰岛素、P62、LC3蛋白表达免疫组化图(n=3)
A、D、G.胰岛素；B、E、H.LC3蛋白；C、F、I.P62蛋白
LC3：微管相关蛋白1轻链3；T2DM、Vaspin：同表 1
表 1 3组大鼠不同时间点体质量、FBG比较(x±s)
指标 正常对照组
(n=10)T2DM组
(n=10)Vaspin组
(n=10)P值 体质量(g) 造模前 187.6±8.7 187.7±6.0 185.9±8.2 0.844 干预前 515.2±53.9 439.3±35.8† 435.6±46.6† 0.001 干预4周 574.7±64.8 429.1±38.9† 465.7±62.8† 0.000 干预8周 596.1±72.8 400.5±41.7† 466.1±53.6†§ 0.000 FBG(mmol/L) 造模前 5.05±0.31 4.94±0.32 5.00±0.39 0.773 干预前 5.00±0.31 25.99±4.83† 26.10±3.42† 0.000 干预4周 5.26±0.35 26.71±2.04† 24.64±2.67† 0.000 干预8周 5.00±0.60 28.78±2.33† 23.04±3.60†§ 0.000 FBG：空腹血糖；T2DM；2型糖尿病；Vaspin: 内脏脂肪特异性丝氨酸蛋白酶抑制剂；†与正常对照组比较，P＜0.05；§与T2DM组比较，P＜0.05 
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表 2 3组大鼠干预8周时FINS、TC、TG水平比较(x±s)
组别 FINS(μg/L) TC(mmol/L) TG(mmol/L) 正常对照组(n=10) 0.72±0.08 1.62±0.18 0.62±0.17 T2DM组(n=10) 0.37±0.05† 3.19±0.31† 1.78±0.27† Vaspin组(n=10) 0.56±0.05†§ 2.18±0.18†§ 1.07±0.17†§ P值 0.000 0.000 0.000 FINS：空腹胰岛素；TC：总胆固醇；TG：甘油三酯；T2DM、Vaspin：同表 1；†与正常对照组比较，P＜0.05；§与T2DM组比较，P＜0.05
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表 3 3组大鼠干预8周时p-AMPK/AMPK、p-mTOR/mTOR、LC3Ⅱ/LC3Ⅰ、P62水平比较(x±s)
组别 p-AMPK/AMPK p-mTOR/mTOR LC3Ⅱ/LC3Ⅰ P62 正常对照组(n=3) 0.30±0.02 1.69±0.18 1.57±0.10 0.40±0.06 T2DM组(n=3) 0.64±0.04† 1.21±0.07† 2.25±0.12† 1.06±0.04† Vaspin组(n=3) 0.86±0.04†§ 0.37±0.02†§ 2.65±0.05†§ 0.36±0.03†§ P值 0.000 0.000 0.000 0.000 AMPK、p-AMPK、mTOR、p-mTOR：同图 5；LC3：同图 4；T2DM、Vaspin：同表 1；†与正常对照组比较，P＜0.05；§与T2DM组比较，P＜0.05
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