Tews D, Lehr S, Hartwig S, Osmers A, Paslack W, Eckel J
Institute of Clinical Biochemistry and Pathobiochemistry, German Diabetes Center, Düsseldorf, Germany.
Horm Metab Res. 2009 Apr;41(4):294-301. doi: 10.1055/s-0028-1105911. Epub 2008 Dec 15.
Glucagon like peptide-1 (Glp-1) exhibits beneficial effects on beta cell mass by both enhancing proliferation and inhibiting apoptosis. The precise mechanism of the anti-apoptotic effect of Glp-1 and Glp-1 mimetics like exendin-4 has remained elusive. Here, we studied cytokine-induced apoptosis in the pancreatic beta cell line INS-1 and performed a comparative mitochondrial protein pattern analysis using two-dimensional difference gel electrophoresis (2D-DIGE). Cytokine incubation of INS-1 cells increased caspase-3 activity about 3-fold, which was reduced by 60% in the presence of exendin-4. Production of reactive oxygen species in response to cytokines was completely prevented after preincubation with exendin-4. Highly purified mitochondria were obtained and mitochondrial proteins were labeled with Cy-dyes and separated on overlapping zoom 2D gels spanning a pH-range of 4-9. Protein spots with significant changes after cytokine and exendin-4 treatment were identified by MALDI mass spectrometry. Comparing all treatment conditions, comparative mitochondrial proteome analysis allowed to identify 33 different proteins, which were significantly altered between comparison groups. Changes in protein patterns revealed involvement of cytokine-induced electron transport chain damage. Thus, cytochrome bc1 complex subunit I and ATP synthase subunit beta were downregulated by 30-40%. This was abrogated by the presence of exendin-4. In conclusion, this study provides further insights into the role of mitochondria in cytokine-induced apoptosis. We show here that exendin-4 significantly counter-regulates the reduced abundance of electron transport chain proteins, leading to a reduction of oxidative stress and most likely contributing to the anti-apoptotic action of this drug.
胰高血糖素样肽-1（Glp-1）通过增强增殖和抑制凋亡对β细胞量产生有益作用。Glp-1和诸如艾塞那肽-4之类的Glp-1模拟物的抗凋亡作用的确切机制仍不清楚。在此，我们研究了细胞因子诱导的胰腺β细胞系INS-1中的凋亡，并使用二维差异凝胶电泳（2D-DIGE）进行了比较线粒体蛋白质模式分析。INS-1细胞经细胞因子孵育后，半胱天冬酶-3活性增加了约3倍，在存在艾塞那肽-4的情况下降低了60%。在用艾塞那肽-4预孵育后，对细胞因子产生的活性氧的产生被完全阻止。获得了高度纯化的线粒体，并用Cy染料标记线粒体蛋白质，并在pH范围为4-9的重叠变焦2D凝胶上进行分离。通过基质辅助激光解吸电离质谱法鉴定细胞因子和艾塞那肽-4处理后有显著变化的蛋白质斑点。比较所有处理条件，比较线粒体蛋白质组分析能够鉴定出33种不同的蛋白质，这些蛋白质在比较组之间有显著改变。蛋白质模式的变化揭示了细胞因子诱导的电子传递链损伤的参与。因此，细胞色素bc1复合体亚基I和ATP合酶亚基β下调了30%-40%。这在艾塞那肽-4存在的情况下被消除。总之，本研究进一步深入了解了线粒体在细胞因子诱导的凋亡中的作用。我们在此表明，艾塞那肽-4显著地反向调节电子传递链蛋白丰度的降低，导致氧化应激的降低，并且很可能有助于这种药物的抗凋亡作用。
