Dynamic Changes in the Protein Localization in the Nuclear Environment in Pancreatic β‑Cell after Brief Glucose Stimulation

Characterization of molecular mechanisms underlying pancreatic β-cell function in relation to glucose-stimulated insulin secretion is incomplete, especially with respect to global response in the nuclear environment. We focus on the characterization of proteins in the nuclear environment of β-cells after brief, high glucose stimulation. We compared purified nuclei derived from β-cells stimulated with 17 mM glucose for 0, 2, and 5 min using quantitative proteomics, a time frame that most likely does not result in translation of new protein in the cell. Among the differentially regulated proteins, we identified 20 components of the nuclear organization processes, including nuclear pore organization, ribonucleoprotein complex, and pre-mRNA transcription. We found alteration of the nuclear pore complex, together with calcium/calmodulin-binding chaperones that facilitate protein and RNA import or export to/from the nucleus to the cytoplasm. Putative insulin mRNA transcription-associated factors were identified among the regulated proteins, and they were cross-validated by Western blotting and confocal immunofluorescence imaging. Collectively, our data suggest that protein translocation between the nucleus and the cytoplasm is an important process, highly involved in the initial molecular mechanism underlying glucose-stimulated insulin secretion in pancreatic β-cells.目前关于胰腺β细胞（pancreatic β-cell）功能与葡萄糖刺激胰岛素分泌（glucose-stimulated insulin secretion）相关分子机制的阐释仍不完整，尤其在核环境的全局应答维度。本研究聚焦于短时高糖刺激后，β细胞核环境内蛋白质组的特征解析。我们采用定量蛋白质组学（quantitative proteomics）技术，对比了经17 mM葡萄糖分别刺激0、2、5分钟的β细胞纯化细胞核——该时间窗口大概率不会引发细胞内新蛋白质的翻译。在差异调控蛋白中，我们鉴定出20种参与核组织过程的组分，涵盖核孔组织、核糖核蛋白复合物以及前mRNA转录相关过程。我们发现核孔复合物发生改变，同时还检测到钙/钙调蛋白结合分子伴侣的表达变化，这类分子伴侣可介导蛋白质与RNA在细胞核与细胞质之间的导入与输出。在差异蛋白中还鉴定出潜在的胰岛素mRNA转录相关因子，并通过蛋白质印迹（Western blotting）与共聚焦免疫荧光成像（confocal immunofluorescence imaging）技术完成了交叉验证。综上，我们的研究数据表明，细胞核与细胞质间的蛋白质转运是一项关键过程，深度参与胰腺β细胞葡萄糖刺激胰岛素分泌的初始分子机制。