胰岛素可通过calpain和proteasome途径促进3T3-L1脂肪细胞三磷酸腺苷结合盒转运体A1的降解  

High insulin level promotes the degradation of high density lipoprotein generation-related functional protein ABCA1 through calpain and proteasome pathway in 3T3-L1 adipocytes

作　　者：苑聪 吴洁[2] 姜志胜[3] 刘厂辉[2] 欧阳泽伟 胡恒境[5] 刘米华 

机构地区：[1]长沙市第一医院心血管二科,410000 [2]南华大学附属第一医院心血管内科 [3]南华大学心血管病研究所动脉硬化学湖南省重点实验室 [4]邵阳市中心医院心血管内科 [5]中南大学湘雅二院心血管内科

出　　处：《中华心血管病杂志》2015年第2期141-145,共5页Chinese Journal of Cardiology

基　　金：湖南省科技厅重大项目（2009SK2010）;南华大学创新科研课题项目（2012XCX19）

摘　　要：目的 探讨高胰岛素环境对高密度脂蛋白（HDL）生成相关功能蛋白三磷酸腺苷结合盒转运体A1（ABCA1）的影响,并探索其具体机制.方法 选取3T3-L1前脂肪细胞,通过经典的“鸡尾酒法”诱导分化为成熟的脂肪细胞,用液体闪烁计数技术检测细胞3H标记胆固醇流出变化,计算外流率;用SYBR Green Ⅰ嵌合荧光法实时荧光定量技术检测胰岛素对3T3-L1成熟脂肪细胞ABCA1mRNA表达的影响;用Western blot技术首先初步观察胰岛素对3T3-L1成熟脂肪细胞ABCA1蛋白水平表达的影响,在加入放线菌酮（cycloheximide,CHX）后进一步观察胰岛素对ABCA1蛋白降解的影响,最后在加入calpain途径抑制剂calpeptin和proteasome途径抑制剂MG-132后,观察胰岛素对脂肪细胞ABCA1影响的可能机制.结果 在不同浓度的胰岛素（0、1、10、102、103 nmol/L）作用下,3T3-L1脂肪细胞胆固醇流出率依次为（7.06±0.27）％、（6.59±0.30）％、（6.34±0.24）％、（5.07±0.40）％和（4.71±0.40）％（P＜0.05）,其中103 nmol/L胰岛素组胆固醇流出率明显低于0 nmol/L组（P＜0.01）;在103 nmol/L浓度的胰岛素环境下,随着孵育时间的延长（0、2、4、6、12 h）,胆固醇流出呈下降趋势,依次为（6.52±0.30）％、（5.59±0.71）％、（5.44±0.37）％、（4.52±0.32）％、（4.38±0.33）％,其中12 h组胆固醇流出率明显低于0h组（P＜0.01）.不同浓度的胰岛素对ABCA1 mRNA的调控无明显差异（P＞0.05）.103 nmol/L胰岛素组ABCA1蛋白水平明显低于0 nmol/L胰岛素组（P＜0.01）.与0h组相比,6h组ABCA1蛋白表达水平开始下降（P＜0.05）,在12h组中ABCA1表达水平则明显降低（P＜0.01）.而calpeptin和MG-132均能减轻胰岛素对ABCA1蛋白的降解作用,与实验阴性对照组（DMSO组）相比,加入calpeptin和MG-132后,ABCA1蛋白水平均明显上调（P均＜0.01）.结论 高浓度胰岛素可通过calpain和proteasome途径促进ABCA1蛋白的降解以及下调Objective To explore effects and potential mechanisms of high insulin environment on high density lipoprotein （HDL） generation-related functional protein ABCA1.Methods [3 H] labeled cholesterol efflux from mature 3T3-L1 adipocytes was detected by liquid scintillation counting.ABCA1 mRNA and protein expression in mature 3T3-L1 adipocytes post stimulation with various concentrations of insulin was detected by real-time fluorescence-based quantitative techniques and Western blot,respectively,in the absence and presence of CHX （cycloheximide,CHX）,calpeptin （calpain pathway inhibitor） or MG-132 （proteasome pathway inhibitor）.Results Cholesterol efflux rates were reduced post insulin stimulation in a dose-dependent manner （（7.06 ± 0.27） %,（6.59 ± 0.30） %,（6.34 ± 0.24） %,（5.07 ± 0.40） %,and （4.71 ±0.40）% at 0,1,10,102,and 103 nmol/L of insulin,P 〈0.05）.Cholesterol efflux rates decreased in a time-dependent manner post 103 nmol/L insulin stimulation （6.52 ± 0.30） %,（5.59 ± 0.71）%,（5.44 ± 0.37）%,（4.52 ± 0.32）%,and （4.38 ± 0.33）% at 0,2,4,6,12 h,respectively）.ABCA1mRNA levels were not affected by insulin（P 〉 0.05）.ABCA1 protein level was significantly downregulated in 103 nmol/L insulin group compared to 0 nmol/L insulin group （P 〈 0.01）.Compared with the 0 h group,ABCA1 protein level was significantly reduced in 6 h group （P 〈 0.05） and further reduced in 12 h group （P 〈 0.01）.Both calpeptin and MG-132 could partly reduce insulin-induced degradation of ABCA1.Compared with the negative control group,ABCA1 protein levels were significantly upregulated by cotreatment with calpeptin and MG-132,respectively （both P 〈 0.01）.Conclusion Our data suggest that high insulin level could promote the ABCA1 protein degradation and reduce cholesterol efflux from mature 3T3-L1 adipocytes through calpain and proteasome pathway,thus,produce a circumference not suitable for nascent HDL formation in 3T3-L1 adipocytes.

关 键 词：动脉粥样硬化 脂细胞 脂蛋白类 HDL 

分 类 号：R543.5[医药卫生—心血管疾病]