Unit for Diabetes Research, Department of Clinical Science and Education, Södersjukhuset, Sjukhusbacken 10, Research Center 3rd Floor, Karolinska Institutet, 118 83 Stockholm, Sweden.
J Cell Biochem. 2012 May;113(5):1635-44. doi: 10.1002/jcb.24032.
Pancreatic β-cells have a well-developed endoplasmic reticulum (ER) and express large amounts of chaperones and protein disulfide isomerases (PDI) to meet the high demand for synthesis of proteins. We have observed an unexpected decrease in chaperone protein level in the β-cell model INS-1E after exposure to the ER stress inducing agent thapsigargin. As these cells are a commonly used model for primary β-cells and has been shown to be vulnerable to ER stress, we hypothesize these cells are incapable of mounting a chaperone defense upon activation of ER stress. To investigate the chaperone expression during an ER stress response, induced by thapsigargin in INS-1E cells, we used quantitative mass spectrometry based proteomics. The results displayed a decrease of GRP78/BiP, PDIA3 and PDIA6. Decrease of GRP78/BiP was verified by Western blot and occurred in parallel with enhanced levels of p-eIF2α and CHOP. In contrast to INS-1E cells, GRP78/BiP was not decreased in MIN6 cell or rat and mouse islets after thapsigargin exposure. Investigation of the decreased protein levels of GRP78/BiP indicates that this is not a consequence of reduced mRNA expression. Rather the reduction results from the combined effect of reduced protein synthesis and enhanced proteosomal degradation and possibly also degradation via autophagy. Induction of ER stress with thapsigargin leads to lower protein levels of GRP78/BiP, PDIA3 and PDIA6 in INS-1E cells which may contribute to the susceptibility of ER stress in this β-cell model.
胰岛β细胞具有发达的内质网（ER），并表达大量的伴侣蛋白和蛋白质二硫键异构酶（PDI），以满足蛋白质合成的高需求。我们观察到，在暴露于内质网应激诱导剂他普西龙后，β细胞模型 INS-1E 中的伴侣蛋白水平出人意料地下降。由于这些细胞是常用的原代β细胞模型，并且已经显示易受内质网应激的影响，我们假设这些细胞在激活内质网应激时无法建立伴侣蛋白防御。为了研究内质网应激反应期间的伴侣蛋白表达，我们使用基于定量质谱的蛋白质组学方法，在 INS-1E 细胞中用他普西龙诱导内质网应激。结果显示 GRP78/BiP、PDIA3 和 PDIA6 的表达减少。GRP78/BiP 的减少通过 Western blot 得到验证，并与 p-eIF2α 和 CHOP 水平的升高平行发生。与 INS-1E 细胞不同，MIN6 细胞或大鼠和小鼠胰岛在他普西龙暴露后 GRP78/BiP 没有减少。对 GRP78/BiP 减少的蛋白质水平的研究表明，这不是 mRNA 表达减少的结果。相反，这种减少是由于蛋白质合成减少、蛋白酶体降解增强以及可能通过自噬降解的综合作用所致。用他普西龙诱导内质网应激导致 INS-1E 细胞中 GRP78/BiP、PDIA3 和 PDIA6 的蛋白质水平降低，这可能导致该β细胞模型对内质网应激的敏感性增加。
