轻度增高的游离脂肪酸与脂多糖协同抑制中性神经酰胺酶诱导INS-1细胞凋亡及胰岛素分泌异常的机制  

Study on the mechanism of mild elevation of free fatty acid and lipopolysaccharide synergistically inhibiting neutral ceramidase to induce apoptosis and abnormal insulin secretion in INS-1 cells

作　　者：王铮 张少坤 温洪华[1] 韦晓 刘超[3] 缪珩[1] 祝群[1] WANG Zheng;ZHANG Shaokun;WEN Honghua;WEI Xiao;LIU Chao;MIAO Heng;ZHU Qun(Department of Endocrinology,the Second Affiliated Hospital of Nanjing Medical University,Nanjing 210011;Research Center of Endocrine and Metabolic Diseases,Jiangsu Province Hospital on Integration of Chinese and Western Medicine,Nanjing 210028,China;Department of Endocrinology,Jiangsu Province Hospital on Integration of Chinese and Western Medicine,Nanjing 210028,China)

机构地区：[1]南京医科大学第二附属医院内分泌科,江苏南京210011 [2]江苏省中西医结合医院内分泌代谢病研究中心,江苏南京210028 [3]江苏省中西医结合医院内分泌科,江苏南京210028

出　　处：《南京医科大学学报（自然科学版）》2020年第6期803-809,共7页Journal of Nanjing Medical University(Natural Sciences)

基　　金：江苏省自然科学基金项目(BK20151577)。

摘　　要：目的:探讨轻度增高的游离脂肪酸与脂多糖(lipopolysaccharide,LPS)协同对大鼠胰岛β细胞株INS-1凋亡及胰岛素分泌的影响及机制。方法:采用0.125 mmol/L棕榈酸和不同浓度的LPS(1、10、50 ng/mL)单独、联合作用INS-1细胞24 h,流式细胞术检测细胞凋亡率,Western blot检测Cleaved caspase-3表达;棕榈酸和LPS(50 ng/mL)单独或联合刺激INS-1细胞24 h后,HPLC法检测中性神经酰胺酶(neutral ceramidase,NCDase)活性,ELISA法检测胰岛素分泌功能;并观察转染p EGFP-C3-NCDase重组质粒后,棕榈酸和LPS刺激的INS-1细胞凋亡率及胰岛素分泌功能的改变。结果:与对照组相比,棕榈酸或LPS(50 ng/mL)单独处理,对INS-1细胞凋亡及胰岛素分泌无明显影响,但两者联合可明显增加凋亡率及Cleaved caspase-3表达(P<0.05)。棕榈酸联合LPS作用24 h后明显抑制基础胰岛素分泌(P<0.01),对细胞内胰岛素含量及葡萄糖刺激的胰岛素分泌无影响。此外,棕榈酸或LPS单独刺激不影响INS-1细胞NCDase,两者联合则显著抑制其活性(P<0.01);而高表达NCDase能明显缓解棕榈酸联合LPS诱导的凋亡和基础胰岛素分泌减少(P<0.05)。结论:轻度增高的游离脂肪酸联合LPS可通过抑制鞘脂信号分子代谢的关键酶——NCDase活性,促进胰岛β细胞凋亡并抑制基础胰岛素分泌。Objective:This study aims to investigate the synergistic effects of mild elevation of free fatty acid and lipopolysaccharide(LPS)on apoptosis and insulin secretion in rat pancreaticβ-cell line INS-1 and its mechanism.Methods:The INS-1 cells were cultured with palmitate(0.125 mmol/L)and different concentrations of LPS(1,10,50 ng/mL)for 24 h alone or together.Apoptosis rate was detected by flow cytometry.Western blot was used to detect the expression of Cleaved caspase-3.Neutral ceramidase(NCDase)activity was investigated by HPLC after INS-1 cells were treated with palmitate and LPS(50 ng/mL)alone or together.Insulin secretion was detected by ELISA.After transfected with recombinant plasmids pEGFP-C3-NCDase,the changes of apoptosis rate and insulin secretion of INS-1 cells stimulated by palmitate and LPS were observed.Results:Compared with the control,palmitate alone or LPS(50 ng/mL)alone had no significant effect on apoptosis or insulin secretion.However,the combination of palmitate and LPS significantly increased the apoptosis rate and up-regulated the expression of Cleaved caspase-3(P<0.05).Intracellular insulin content or glucose-stimulated insulin secretion was not altered while basal insulin secretion was significantly inhibited by palmitate and LPS for 24 h(P<0.01).In addition,NCDase activity was not affected by stimulation with palmitate or LPS alone while its activity was markedly inhibited by combination of palmitate and LPS(P<0.01).NCDase overexpression markedly ameliorated the apoptosis induced by palmitate and LPS in INS-1 cells and increased basal insulin secretion(P<0.05).Conclusion:Mild elevation of free fatty acid and LPS could synergistically promoteβ-cell apoptosis and decrease basal insulin secretion through inhibition of the activity of NCDase,a key enzyme of the sphingolipid metabolism.

关 键 词：游离脂肪酸 脂多糖 中性神经酰胺酶 Β细胞 

分 类 号：R587.1[医药卫生—内分泌]