摘要
目的:研究脂肪细胞因子Vaspin对棕榈酸诱导的INS-1细胞自噬水平的影响。方法:培养INS-1细胞,分为正常组:用普通培养基培养24 h;棕榈酸(palmitic acid,PA)组:以0.5 mmol/L PA培养24 h;PA+Vaspin组:先加入320 ng/mL Vaspin预培养2 h,再以0.5 mmol/L PA+320 ng/mL Vaspin共培养24 h;PA+Vaspin+雷帕霉素(rapamycin)组:先以50 nmol/L Rapamycin预培养2 h,再以0.5 mmol/L PA+320 ng/mL Vaspin共培养24 h;PA+Vaspin+3-甲基腺嘌呤(3-methyladenine,3-MA)组:先以1 mmol/L 3-MA预培养2 h,再以0.5 mmol/L PA+320 ng/mL Vaspin共培养24 h。葡萄糖(3.3 mmol/L和16.7 mmol/L)刺激胰岛素分泌实验检测各组胰岛素分泌水平,Western blot检测哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、pmTOR、Beclin-1、LC3-Ⅰ、LC3-Ⅱ及P62蛋白水平,mRFP-GFP-LC3检测自噬流水平,流式细胞仪检测细胞凋亡水平。结果:与正常组相比,PA组INS-1细胞上清液中胰岛素分泌水平在3.3 mmol/L葡萄糖和16.7 mmol/L葡萄糖刺激下均降低[(1.02±0.08)ng/mL vs.(0.54±0.06)ng/mL,P=0.000;(1.15±0.13)ng/mL vs.(0.71±0.05)ng/mL,P=0.000],PA+Vaspin组较PA组升高[(0.76±0.03)ng/mL vs.(0.54±0.06)ng/mL,P=0.001;(0.91±0.04)ng/mL vs.(0.71±0.05)ng/mL,P=0.011];与正常组相比,PA组INS-1细胞p-mTOR与mTOR蛋白比值低(2.04±0.14 vs.1.19±0.09,P=0.000),PA+Vaspin组p-mTOR与mTOR蛋白比值较PA组高(1.51±0.05 vs.1.19±0.09,P=0.005);同时,PA组自噬相关蛋白Beclin-1、LC3Ⅱ/LC3Ⅰ比值与正常组相比明显增加(0.81±0.07 vs.0.65±0.04,P=0.005;2.35±0.25 vs.1.16±0.11,P=0.000),P62蛋白水平降低(1.04±0.19 vs.0.60±0.08,P=0.000);PA+Vaspin组Beclin-1、LC3Ⅱ/LC3Ⅰ蛋白水平较PA组低(0.58±0.04 vs.0.81±0.07,P=0.001;1.95±0.08 vs.2.35±0.25,P=0.007),P62蛋白增加(0.82±0.03 vs.0.60±0.08,P=0.032);PA组自噬溶酶体数量较正常组多(8.33±1.53 vs.0.33±0.58,P=0.000),Vaspin干预后降低(3.67±1.53 vs.8.33±1.53,P=0.001);同时,PA干预INS-1细胞24 h后细胞凋亡明显增加(7.91±0.50 vs.22.15±2.05,P=0.000),PA+Vaspin组较PA组细胞凋亡减少(13.23±1.97 vs.22.15±2.05,P=0.000)。结论:Vaspin能够改善棕榈酸诱导的INS-1细胞过度自噬和细胞凋亡,改善胰岛β细胞分泌功能。
Objective:To investigate the effect of the adipocytokine vaspin on the level of palmitic acid(PA)-induced autophagy in INS-1 cells.Methods:INS-1 cells were divided into groups and cultured as follows:normal group:cultured in an ordinary medium for 24 hours;PA group:cultured with 0.5 mmol/L PA for 24 hours;PA+vaspin group:pre-cultured with 320 ng/ml vaspin for 2 hours,and then co-cultured with 0.5 mmol/L PA and 320 ng/mL vaspin for 24 hours;PA+vaspin+rapamycin group:pre-cultured with 50 nmol/L rapamycin for 2 hours,and then co-cultured with 0.5 mmol/L PA and 320 ng/mL vaspin for 24 hours;PA+vaspin+3-methyladenine(3-MA)group:pre-cultured with 1 mmol/L 3-MA for 2 hours,and then co-cultured with 0.5 mmol/L PA and 320 ng/mL vaspin for 24 hours.The glucose-stimulated insulin secretion(GSIS)test(with glucose levels of 3.3 and 16.7 mmol/L)was performed to determine the insulin level in each group;Western blot was used to determine the levels of mammalian target of rapamycin(mTOR),p-mTOR,Beclin-1,LC3-Ⅰ,LC3-Ⅱ,and P62;mRFP-GFP-LC3 and flow cytometry were used to determine the autophagic flow and cell apoptosis,respectively.Results:Compared with the normal group,the PA group had significantly reduced GSIS levels in the INS-1 cell supernatant[(1.02±0.08)ng/mL vs.(0.54±0.06)ng/mL,P=0.000;(1.15±0.13)ng/mL vs.(0.71±0.05)ng/mL,P=0.000],while the vaspin-treated group had significantly increased GSIS levels than the PA group[(0.76±0.03)ng/mL vs.(0.54±0.06)ng/mL,P=0.001;(0.91±0.04)ng/mL vs.(0.71±0.05)ng/mL,P=0.011].Compared with the normal group,the PA group had a significantly reduced p-mTOR-to-mTOR ratio(2.04±0.14 vs.1.19±0.09,P=0.000),while the vaspin-treated group had a significantly increased ratio compared with the PA group(1.51±0.05 vs.1.19±0.09,P=0.005).Compared with the normal group,the PA group had significant increases in the level of the autophagy-associated protein Beclin-1 and the ratio of LC3-Ⅱ to LC3-Ⅰ(0.65±0.04 vs.0.81±0.07,P=0.005;1.16±0.11 vs.2.35±0.25,P=0.000)and a significant reduction in P62 level(1.04±0.19 vs.0.60±0.08,P=0.000);the vaspin-treated group had significant decreases in the Beclin-1 level and the LC3-Ⅱ-to-LC3-Ⅰratio(0.58±0.04 vs.0.81±0.07,P=0.001;1.95±0.08 vs.2.35±0.25,P=0.007)and a significant increase in P62 level(0.82±0.03 vs.0.60±0.08,P=0.032)compared with the PA group.The PA group had a significantly higher number of autolysosomes than the normal group(8.33±1.53 vs.0.33±0.58,P=0.000),while the vaspin-treated group had a significantly reduced number of autolysosomes compared with the PA group(3.67±1.53 vs.8.33±1.53,P=0.001).After treatment for 24 hours,the PA group had a significantly increased apoptosis rate of INS-1 cells compared with the normal group(22.15±2.05 vs.7.91±0.50,P=0.000),while the vaspin-treated group had a significantly reduced apoptosis rate compared with the PA group(13.23±1.97 vs.22.15±2.05,P=0.000).Conclusion:Vaspin can inhibit PA-induced excessive autophagy and apoptosis of INS-1 cells,thus improving the secretory function of isletβcells.
作者
段瑞雪
刘师伟
常冰梅
吴亚茹
汪湄湄
张佳新
郭胜慧
李欣
张琦
Duan Ruixue;Liu Shiwei;Chang Bingmei;Wu Yaru;Wang Meimei;Zhang Jiaxin;Guo Shenghui;Li Xin;Zhang Qi(Department of Graduate School,Shanxi Medical University;Department of Endocrinology,Shanxi Bethune Hospital;Department of Endocrinology,Taiyuan Central Hospital)
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2020年第3期331-337,共7页
Journal of Chongqing Medical University
基金
国家自然科学基金资助项目(编号:81471025)
山西省回国留学人员科研资助项目(编号:2015-109)
山西省科技成果转化引导专项资助项目(编号:201604D131003)
山西省留学回国人员科技活动择优资助项目(编号:2017)
白求恩·默克糖尿病研究基金资助项目(编号:G2017044)
山西省自然科学基金资助项目(编号:201701D121150)。
作者简介
段瑞雪,Email:duanruixuenfm@163.com,研究方向:胰岛素抵抗与代谢综合征;通信作者:刘师伟,Email:lswspring6@aliyun.com。
