文章摘要
王 铮,张少坤,温洪华,韦 晓,刘 超,缪 珩,祝 群.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[J].南京医科大学学报,2020,(6):803~809
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
投稿时间:2019-09-16  
DOI:10.7655/NYDXBNS20200606
中文关键词: 游离脂肪酸  脂多糖  中性神经酰胺酶  β细胞
英文关键词: free fatty acid  lipopolysaccharide  neutral ceramidase  β⁃cells
基金项目:江苏省自然科学基金项目(BK20151577)
作者单位
王 铮 南京医科大学第二附属医院内分泌科江苏 南京 210011 
张少坤 南京医科大学第二附属医院内分泌科江苏 南京 210011 
温洪华 南京医科大学第二附属医院内分泌科江苏 南京 210011 
韦 晓 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 
刘 超 江苏省中西医结合医院内分泌科江苏 南京 210028 
缪 珩 南京医科大学第二附属医院内分泌科江苏 南京 210011 
祝 群 南京医科大学第二附属医院内分泌科江苏 南京 210011 
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中文摘要:
      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pEGFP?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。
英文摘要:
      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 pancreatic β?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 promote β?cell apoptosis and decrease basal insulin secretion through inhibition of the activity of NCDase,a key enzyme of the sphingolipid metabolism.
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