Proteomic Analysis of INS-1 Rat Insulinoma Cells: ER Stress Effects and the Protective Role of Exenatide, a GLP-1 Receptor Agonist

Beta cell death caused by endoplasmic reticulum (ER) stress is a key factor aggravating type 2 diabetes. Exenatide, a glucagon-like peptide (GLP)-1 receptor agonist, prevents beta cell death induced by thapsigargin, a selective inhibitor of ER calcium storage. Here, we report on our proteomic studies designed to elucidate the underlying mechanisms. We conducted comparative proteomic analyses of cellular protein profiles during thapsigargin-induced cell death in the absence and presence of exenatide in INS-1 rat insulinoma cells. Thapsigargin altered cellular proteins involved in metabolic processes and protein folding, whose alterations were variably modified by exenatide treatment. We categorized the proteins with thapsigargin initiated alterations into three groups: those whose alterations were 1) reversed by exenatide, 2) exaggerated by exenatide, and 3) unchanged by exenatide. The most significant effect of thapsigargin on INS-1 cells relevant to their apoptosis was the appearance of newly modified spots of heat shock proteins, thimet oligopeptidase and 14-3-3β, ε, and θ, and the prevention of their appearance by exenatide, suggesting that these proteins play major roles. We also found that various modifications in 14-3-3 isoforms, which precede their appearance and promote INS-1 cell death. This study provides insights into the mechanisms in ER stress-caused INS-1 cell death and its prevention by exenatide.

内质网（endoplasmic reticulum, ER）应激介导的β细胞死亡，是加重2型糖尿病的关键诱因。艾塞那肽（exenatide）作为胰高糖素样肽（glucagon-like peptide, GLP）-1受体激动剂，可拮抗毒胡萝卜素（thapsigargin，一种内质网钙储存特异性抑制剂）诱导的β细胞死亡。本研究通过蛋白质组学分析，旨在阐明上述过程的潜在分子机制。本研究以INS-1大鼠胰岛素瘤细胞为模型，分别在艾塞那肽干预与未干预的条件下，对毒胡萝卜素诱导细胞死亡过程中的细胞蛋白质组谱开展了对比分析。毒胡萝卜素可改变参与代谢过程与蛋白质折叠的细胞蛋白表达谱，而艾塞那肽干预可不同程度地调控这些蛋白的表达变化。本研究将受毒胡萝卜素调控的差异蛋白分为三类：其一，表达变化可被艾塞那肽逆转的蛋白；其二，表达变化可被艾塞那肽增强的蛋白；其三，表达变化不受艾塞那肽影响的蛋白。毒胡萝卜素对INS-1细胞最显著的促细胞凋亡（apoptosis）效应，表现为热休克蛋白（heat shock proteins）、嗜甲硫氨酸寡肽酶（thimet oligopeptidase）以及14-3-3β、ε、θ亚型的新型修饰斑点出现，而艾塞那肽可阻断这些修饰斑点的产生，提示这些蛋白在该过程中发挥核心作用。本研究还发现，14-3-3亚型的多种修饰会先于其表达出现，并可促进INS-1细胞死亡。本研究为内质网应激诱导的INS-1细胞死亡机制及其被艾塞那肽拮抗的过程提供了新的科学见解。