Department of Metabolism & Endocrinology, The First Affiliated Hospital of the University of South China, Hengyang 421001, China.
Guangxi Key Laboratory of Diabetic Systems Medicine, Guilin Medical University, Guilin 541199, China.
Acta Biochim Biophys Sin (Shanghai). 2021 Jan 12;53(1):54-62. doi: 10.1093/abbs/gmaa142.
Excessive accumulation of cholesterol in β cells initiates endoplasmic reticulum (ER) stress and associated apoptosis. We have reported that excessive uptake of cholesterol by MIN6 cells decreases the expression of secretagogin (SCGN) and then attenuates insulin secretion. Here, we aimed to determine whether cholesterol-induced SCGN decrease is involved in the modulation of ER stress and apoptosis in pancreatic β cells. In this study, MIN6 cells were treated with oxidized low-density lipoprotein (ox-LDL) for 24 h, and then intracellular lipid droplets and cell apoptosis were quantified, and SCGN and ER stress markers were identified by western blot analysis. Furthermore, small interfer RNA (siRNA)-mediated SCGN knockdown and recombinant plasmid-mediated SCGN restoration experiments were performed to confirm the role of SCGN in ER stress and associated cell apoptosis. Finally, the interaction of SCGN with ATF4 was computationally predicted and then validated by a co-immunoprecipitation assay. We found that ox-LDL treatment increased the levels of ER stress markers, such as phosphorylated protein kinase-like endoplasmic reticulum kinase, phosphorylated eukaryotic initiation factor 2 alpha, activating transcription factor 4 (ATF4), and transcription factor CCAAT-enhancer-binding protein homologous protein, and promoted MIN6 cell apoptosis; in addition, the expression of SCGN was downregulated. siRNA-mediated SCGN knockdown exacerbated β-cell ER stress by increasing ATF4 expression. Pretreatment of MIN6 cells with the recombinant SCGN partly antagonized ox-LDL-induced ER stress and apoptosis. Furthermore, a co-immunoprecipitation assay revealed an interaction between SCGN and ATF4 in MIN6 cells. Taken together, these results demonstrated that pancreatic β-cell apoptosis induced by ox-LDL treatment can be attributed, in part, to an SCGN/ATF4-dependent ER stress response.
胆固醇在β细胞中的过度积累会引发内质网（ER）应激和相关的细胞凋亡。我们已经报道，MIN6 细胞摄取过多的胆固醇会降低分泌素（SCGN）的表达，从而减弱胰岛素的分泌。在这里，我们旨在确定胆固醇诱导的 SCGN 减少是否参与调节胰岛β细胞的 ER 应激和细胞凋亡。在这项研究中，MIN6 细胞用氧化型低密度脂蛋白（ox-LDL）处理 24 小时，然后定量细胞内脂滴和细胞凋亡，并用 Western blot 分析鉴定 SCGN 和 ER 应激标志物。此外，通过小干扰 RNA（siRNA）介导的 SCGN 敲低和重组质粒介导的 SCGN 恢复实验来确认 SCGN 在 ER 应激和相关细胞凋亡中的作用。最后，通过计算预测和免疫共沉淀实验验证了 SCGN 与 ATF4 的相互作用。我们发现 ox-LDL 处理增加了 ER 应激标志物的水平，如磷酸化蛋白激酶样内质网激酶、磷酸化真核起始因子 2α、激活转录因子 4（ATF4）和转录因子 CCAAT 增强子结合蛋白同源蛋白，并促进 MIN6 细胞凋亡；此外，SCGN 的表达下调。siRNA 介导的 SCGN 敲低通过增加 ATF4 表达加剧了β细胞 ER 应激。MIN6 细胞用重组 SCGN 预处理部分拮抗 ox-LDL 诱导的 ER 应激和细胞凋亡。此外，免疫共沉淀实验显示 MIN6 细胞中 SCGN 和 ATF4 之间存在相互作用。综上所述，这些结果表明 ox-LDL 处理诱导的胰岛β细胞凋亡部分归因于 SCGN/ATF4 依赖性 ER 应激反应。
