School of Engineering, University of Applied Sciences Upper Austria, Stelzhamerstraße 23, 4600 Wels, Austria.
FFoQSI GmbH-Austrian Competence Centre for Feed and Food Quality, Safety and Innovation, Technopark 1C, 3430 Tulln, Austria.
Int J Mol Sci. 2020 Oct 27;21(21):7964. doi: 10.3390/ijms21217964.
Due to the global rise of type 2 diabetes mellitus (T2DM) in combination with insulin resistance, novel compounds to efficiently treat this pandemic disease are needed. Screening for compounds that induce the translocation of glucose transporter 4 (GLUT4) from the intracellular compartments to the plasma membrane in insulin-sensitive tissues is an innovative strategy. Here, we compared the applicability of three fluorescence microscopy-based assays optimized for the quantitation of GLUT4 translocation in simple cell systems. An objective-type scanning total internal reflection fluorescence (TIRF) microscopy approach was shown to have high sensitivity but only moderate throughput. Therefore, we implemented a prism-type TIR reader for the simultaneous analysis of large cell populations grown in adapted microtiter plates. This approach was found to be high throughput and have sufficient sensitivity for the characterization of insulin mimetic compounds in live cells. Finally, we applied confocal microscopy to giant plasma membrane vesicles (GPMVs) formed from GLUT4-expressing cells. While this assay has only limited throughput, it offers the advantage of being less sensitive to insulin mimetic compounds with high autofluorescence. In summary, the combined implementation of different fluorescence microscopy-based approaches enables the quantitation of GLUT4 translocation with high throughput and high content.
由于 2 型糖尿病（T2DM）在全球范围内的发病率上升，同时伴随着胰岛素抵抗，因此需要新型化合物来有效治疗这种流行疾病。筛选能够诱导胰岛素敏感组织中葡萄糖转运蛋白 4（GLUT4）从细胞内区室向质膜易位的化合物是一种创新策略。在这里，我们比较了三种基于荧光显微镜的测定法在简单细胞系统中定量 GLUT4 易位的适用性，这些测定法经过优化，可用于检测 GLUT4 易位。基于扫描全内反射荧光（TIRF）显微镜的客观型方法显示出高灵敏度，但通量适中。因此，我们为适应微板培养的大细胞群体的同时分析，实施了棱镜型 TIR 读取器。该方法具有高通量和足够的灵敏度，可用于鉴定活细胞中的胰岛素模拟化合物。最后，我们将共聚焦显微镜应用于从表达 GLUT4 的细胞形成的巨大质膜囊泡（GPMV）。虽然该测定法通量有限，但它具有对具有高自发荧光的胰岛素模拟化合物的灵敏度较低的优点。总之，不同荧光显微镜测定法的综合实施能够实现高通量和高内涵的 GLUT4 易位定量。
