Institute of Medical Biochemistry and Molecular Biology, University Medicine Rostock, Rostock, Germany.
Institute of Medical Biochemistry and Molecular Biology, University Medicine Greifswald, Greifswald, Germany.
Front Endocrinol (Lausanne). 2020 Apr 28;11:232. doi: 10.3389/fendo.2020.00232. eCollection 2020.
Mitochondrial dynamics are important for glucose-stimulated insulin secretion in pancreatic beta cells. The mitochondrial elongation factor MiD51 has been proposed to act as an anchor that recruits Drp1 from the cytosol to the outer mitochondrial membrane. Whether MiD51 promotes mitochondrial fusion by inactivation of Drp1 is a controversial issue. Since both the underlying mechanism and the effects on mitochondrial function remain unknown, this study was conducted to investigate the role of MiD51 in beta cells. Overexpression and downregulation of MiD51 in mouse insulinoma 6 (MIN6) and mouse islet cells was achieved using the pcDNA expression vector and specific siRNA, respectively. Expression of genes regulating mitochondrial dynamics and autophagy was analyzed by quantitative Real-Time PCR, glucose-stimulated insulin secretion by ELISA, and cellular oxygen consumption rate by optode sensor technology. Mitochondrial membrane potential and morphology were visualized after TMRE and MitoTracker Green staining, respectively. Immunofluorescence analyses were examined by confocal microscopy. MiD51 is expressed in insulin-positive mouse and human pancreatic islet and MIN6 cells. Overexpression of MiD51 resulted in mitochondrial fragmentation and cluster formation in MIN6 cells. Mitochondrial membrane potential, glucose-induced oxygen consumption rate and glucose-stimulated insulin secretion were reduced in MIN6 cells with high MiD51 expression. LC3 expression remained unchanged. Downregulation of MiD51 resulted in inhomogeneity of the mitochondrial network in MIN6 cells with hyperelongated and fragmented mitochondria. Mitochondrial membrane potential, maximal and glucose-induced oxygen consumption rate and insulin secretion were diminished in MIN6 cells with low MiD51 expression. Furthermore, reduced Mfn2 and Parkin expression was observed. Based on MiD51 overexpression and downregulation, changes in the mitochondrial network structure similar to those in MIN6 cells were also observed in mouse islet cells. We have demonstrated that MiD51 plays a pivotal role in regulating mitochondrial function and hence insulin secretion in MIN6 cells. We propose that this anchor protein of Drp1 is important to maintain a homogeneous mitochondrial network and to avoid morphologies such as hyperelongation and clustering which are inaccessible for degradation by autophagy. Assuming that insulin granule degradation frequently suppresses autophagy in beta cells, MiD51 could be a key element maintaining mitochondrial health.
线粒体动力学对于胰腺β细胞中的葡萄糖刺激胰岛素分泌很重要。线粒体伸长因子 MiD51 被提议作为一种锚定蛋白，将 Drp1 从细胞质招募到外线粒体膜。MiD51 是否通过失活 Drp1 促进线粒体融合是一个有争议的问题。由于潜在的机制和对线粒体功能的影响仍然未知，因此进行了这项研究以调查 MiD51 在β细胞中的作用。
使用 pcDNA 表达载体和特异性 siRNA 在小鼠胰岛素瘤 6 （MIN6）和小鼠胰岛细胞中分别实现了 MiD51 的过表达和下调。通过定量实时 PCR 分析调节线粒体动力学和自噬的基因表达，通过 ELISA 分析葡萄糖刺激的胰岛素分泌，通过光传感器技术分析细胞耗氧量。用 TMRE 和 MitoTracker Green 染色分别可视化线粒体膜电位和形态。通过共聚焦显微镜检查免疫荧光分析。
MiD51 在胰岛素阳性的小鼠和人胰岛和 MIN6 细胞中表达。MiD51 的过表达导致 MIN6 细胞中线粒体的碎片化和簇形成。MIN6 细胞中高 MiD51 表达导致线粒体膜电位、葡萄糖诱导的耗氧量和葡萄糖刺激的胰岛素分泌降低。LC3 表达保持不变。MiD51 的下调导致 MIN6 细胞中线粒体网络的不均匀性，出现超伸长和碎片化的线粒体。MIN6 细胞中 MiD51 表达降低导致线粒体膜电位、最大和葡萄糖诱导的耗氧量和胰岛素分泌减少。此外，观察到 Mfn2 和 Parkin 表达减少。基于 MiD51 的过表达和下调，MIN6 细胞中的线粒体网络结构变化类似于在 MIN6 细胞中观察到的变化。
我们已经证明 MiD51 在调节 MIN6 细胞中的线粒体功能和胰岛素分泌中起着关键作用。我们提出，这种 Drp1 的锚定蛋白对于维持均匀的线粒体网络很重要，并且可以避免超伸长和簇状等形态，这些形态无法通过自噬进行降解。假设胰岛素颗粒降解经常抑制β细胞中的自噬，那么 MiD51 可能是维持线粒体健康的关键因素。
