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
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江苏省自然科学基金项目(BK20151577)


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
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    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。

    Abstract:

    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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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(自然科学版),2020,(6):803-809

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  • 收稿日期:2019-09-16
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  • 在线发布日期: 2020-07-07
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