Abstract

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.

中文翻译：

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新型的secretagogin / ATF4途径参与氧化的LDL诱导的内质网应激和胰岛β细胞凋亡

摘要

β细胞中胆固醇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细胞过量摄取胆固醇会降低促分泌素（SCGN）的表达，然后减弱胰岛素的分泌。在这里，我们旨在确定胆固醇诱导的SCGN降低是否与胰腺β细胞内质网应激和细胞凋亡的调节有关。在这项研究中，用氧化的低密度脂蛋白（ox-LDL）处理MIN6细胞24小时，然后量化细胞内脂质滴和细胞凋亡，并通过Western blot分析鉴定SCGN和ER应激标记。此外，进行了小干扰RNA（siRNA）介导的SCGN敲低和重组质粒介导的SCGN修复实验，以确认SCGN在ER应激和相关细胞凋亡中的作用。最后，通过计算机预测了SCGN与ATF4的相互作用，然后通过免疫共沉淀法进行了验证。我们发现ox-LDL处理可增加ER应激标志物的水平，例如磷酸化蛋白激酶样内质网激酶，磷酸化真核生物起始因子2 alpha，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，促进MIN6细胞凋亡；此外，SCGN的表达下调。siRNA介导的SCGN敲低会通过增加ATF4表达而加剧β细胞内质网应激。用重组SCGN预处理MIN6细胞可部分拮抗ox-LDL诱导的ER应激和凋亡。此外，共同免疫沉淀试验揭示了MIN6细胞中SCGN和ATF4之间的相互作用。综上所述，这些结果表明，ox-LDL处理诱导的胰腺β细胞凋亡可部分归因于依赖SCGN / ATF4的ER应激反应。