Department of Chemistry and Biochemistry, Florida State University , 95 Chieftain Way, Dittmer Building, Tallahassee, Florida 32306, United States.
Department of Mathematics and Program in Neuroscience, Florida State University , Tallahassee, Florida 32306, United States.
Anal Chem. 2016 Nov 1;88(21):10368-10373. doi: 10.1021/acs.analchem.6b02404. Epub 2016 Oct 18.
Glucose-stimulated insulin secretion from pancreatic β-cells within islets of Langerhans plays a critical role in maintaining glucose homeostasis. Although this process is essential for maintaining euglycemia, the underlying intracellular mechanisms that control it are still unclear. To allow simultaneous correlation between intracellular signal transduction events and extracellular secretion, an analytical system was developed that integrates fluorescence imaging of intracellular probes with high-speed automated insulin immunoassays. As a demonstration of the system, intracellular [Ca] ([Ca]) was measured by imaging Fura-2 fluorescence simultaneously with insulin secretion from islets exposed to elevated glucose levels. Both [Ca] and insulin were oscillatory during application of 10 mM glucose with temporal and quantitative profiles similar to what has been observed elsewhere. In previous work, sinusoidal glucose levels have been used to test the entrainment of islets while monitoring either [Ca] or insulin levels; using this newly developed system, we show unambiguously that oscillations of both [Ca] and insulin release are entrained to oscillatory glucose levels and that the temporal correlation of these are maintained throughout the experiment. It is expected that the developed analytical system can be expanded to investigate a number of other intracellular messengers in islets or other stimulus-secretion pathways in different cells.
胰岛β细胞的葡萄糖刺激胰岛素分泌在维持血糖稳态中起着关键作用。尽管这个过程对于维持正常血糖水平是必不可少的，但控制它的细胞内机制仍不清楚。为了允许在细胞内信号转导事件和细胞外分泌之间进行同时相关，开发了一种分析系统，该系统将细胞内探针的荧光成像与高速自动胰岛素免疫测定相结合。作为该系统的演示，通过同时测量暴露于高葡萄糖水平下的胰岛的 Fura-2 荧光来测量细胞内[Ca] ([Ca])。在应用 10 mM 葡萄糖时，[Ca] 和胰岛素都呈振荡性，其时间和定量特征与其他地方观察到的相似。在以前的工作中，已经使用正弦葡萄糖水平来测试胰岛的夹带，同时监测[Ca]或胰岛素水平；使用这个新开发的系统，我们明确地表明，[Ca]和胰岛素释放的振荡都被夹带在振荡的葡萄糖水平中，并且这些的时间相关性在整个实验中都得到了维持。预计开发的分析系统可以扩展到研究胰岛中的其他一些细胞内信使或其他不同细胞中的刺激-分泌途径。
