Shahrestanaki Mohammad Keyvanloo, Arasi Fatemeh Panahi, Aghaei Mahmoud
Department of Clinical Biochemistry, School of Pharmacy & Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
J Cell Biochem. 2019 May;120(5):7759-7770. doi: 10.1002/jcb.28050. Epub 2018 Nov 11.
Chronic exposure to high glucose induces endoplasmic reticulum (ER) stress in pancreatic beta cells (PBCs). The previous evidence showed that adenosine modulate PBCs viability and insulin secretion. The aim of this study was to evaluate possible involvement of adenosine in protection of MIN6 β-cells from Tunicamycin (Tu)-induced ER stress. MIN6 cells were cotreated with Tu and different concentrations of adenosine. Cell viability, proliferation, and apoptosis were evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT), 5-bromo-2'-deoxyuridine (Brdu), and colony formation assays. Caspase-12 activity was assayed using the fluorometric method. Thioflavin T (ThT) staining was used for the evaluation of protein aggregation. Insulin secretion was evaluated using specific an ELISA kit. Ca mobilization assayed using Fura2/AM probe. BIP, CHOP, XBP-1, and XBP-1s expression in both messenger RNA (mRNA) and protein levels were evaluated using the reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Bcl-2, p-eIF2α/eIF2α, and GADD34 levels also determined with Western blot analysis. Adenosine protected MIN6 cells against Tu-induced ER stress in a dose-dependent manner and increased their proliferation. Decreased caspase-12 activity and upregulated Bcl-2 protein may explain antiapoptotic effects of adenosine. ThT staining indicated an attenuated aggregation of misfolded proteins. Adenosine effectively increased insulin secretion in Tu-treated cells. BIP, CHOP, XBP1, and sXBP1 expression were decreased significantly in cotreated cells, indicating alleviation of ER stress. However, adenosine potentiated the expression of GADD34 and decreased p-eIF2α/eIF2α ratio. Adenosine increased cytosolic Ca levels, which may promote adenosine triphosphate (ATP) synthesis in mitochondria, helping ER to preserve protein hemostasis. Taken together, adenosine upregulated Bcl-2 and GADD34 to protect PBCs against Tu-induced apoptosis and increase Insulin secretion.
长期暴露于高糖环境会诱导胰岛β细胞（PBCs）发生内质网（ER）应激。先前的证据表明，腺苷可调节PBCs的活力和胰岛素分泌。本研究的目的是评估腺苷在保护MIN6β细胞免受衣霉素（Tu）诱导的内质网应激中可能发挥的作用。将MIN6细胞与Tu和不同浓度的腺苷共同处理。使用3-[4,5-二甲基噻唑-2-基]-2,5-二苯基溴化四氮唑（MTT）、5-溴-2'-脱氧尿苷（Brdu）和集落形成试验评估细胞活力、增殖和凋亡。使用荧光法检测半胱天冬酶-12的活性。使用硫黄素T（ThT）染色评估蛋白质聚集情况。使用特定的ELISA试剂盒评估胰岛素分泌。使用Fura2/AM探针检测钙动员情况。分别使用逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹分析评估信使核糖核酸（mRNA）和蛋白质水平上的结合免疫球蛋白蛋白（BIP）、C/EBP同源蛋白（CHOP）、X盒结合蛋白1（XBP-1）和剪接型XBP-1（XBP-1s）的表达。Bcl-2、磷酸化真核翻译起始因子2α/真核翻译起始因子2α（p-eIF2α/eIF2α）和生长停滞和DNA损伤诱导蛋白34（GADD34）的水平也通过蛋白质印迹分析来测定。腺苷以剂量依赖的方式保护MIN6细胞免受Tu诱导的内质网应激，并增加其增殖。半胱天冬酶-12活性降低和Bcl-2蛋白上调可能解释了腺苷的抗凋亡作用。ThT染色表明错误折叠蛋白的聚集减弱。腺苷有效地增加了Tu处理细胞中的胰岛素分泌。在共同处理的细胞中，BIP、CHOP、XBP1和sXBP1的表达显著降低，表明内质网应激得到缓解。然而，腺苷增强了GADD34的表达并降低了p-eIF2α/eIF2α的比值。腺苷增加了细胞质钙水平，这可能促进线粒体中三磷酸腺苷（ATP）的合成，帮助内质网维持蛋白质稳态。综上所述，腺苷上调Bcl-2和GADD34以保护PBCs免受Tu诱导的凋亡并增加胰岛素分泌。
