Efficient and modified 2-NBDG assay to measure glucose uptake in cultured myotubes
Efficient and modified 2-NBDG assay to measure glucose uptake in cultured myotubes
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DOI：
10.1016/j.vascn.2021.107069
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发表时间：
2021-04-28
影响因子：
1.9
通讯作者：
Mittal, Ashwani
中科院分区：
文献类型：
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作者：
Bala, Manju;Gupta, Prachi;Mittal, Ashwani
在2型糖尿病中，骨骼肌中发生胰岛素抵抗是一个关键缺陷，为了研究这种疾病、其表现和可能的解决方案，葡萄糖摄取的测量是必不可少的。在闪烁计数、流式细胞术、荧光法和分光光度法等多种方法中，2-NBDG溶液是最常用的一种。尽管基于2-NBDG的分析是包括骨骼肌在内的各种细胞中最广泛使用的方法，但即使如此，所有可用的方案也具有巨大的可变性，这会影响整体数据的可重复性。此外，饥饿（使用无葡萄糖/无血清培养基）是葡萄糖摄取试验的先决条件之一，它本身在较长的孵育前期特异性地诱导应激，并改变肌肉细胞的代谢和形态，但这一事实尚未得到适当考虑。因此，在本文中，我们使用特定的骨骼肌细胞，即C2C12肌管，在保持培养肌管形态健康状态的同时，重建了培养前时间、胰岛素、葡萄糖和血清/BSA浓度等条件。我们的实验室标准化方案被观察到在研究肌管中不同应激（氧化和炎症）诱导的胰岛素抵抗状况方面是有效的。与已有方案的比较研究表明，本方法更高效、更有效、更便于研究C2C12的葡萄糖摄取。
Under type-2 diabetes, insulin resistance develops in skeletal muscles as a key defect and to study the disorder, its manifestation, and possible solution, measurement of glucose uptake is a fundamental necessity. Of various approaches (i.e. scintillation counting, flow cytometry, fluorometry and spectrophotometry) fluorescent labelled glucose analogue, 2-NBDG solution is the most popular one. Although 2-NBDG based assay is the most widely used approach in various cells including skeletal muscle, even then all available protocols possess huge variability which impacts the overall data reproducibility. Moreover, starvation (use of glucose/serum free medium), one of the prerequisite condition for glucose uptake assay, itself induces stress specifically during longer preincubation periods and alters muscle cell metabolism and morphology, but the fact has not been duly considered. Therefore in the present article, using specific skeletal muscle cells i.e. C2C12 myotubes, we have reestablished the conditions like pre-incubation time period, concentrations of insulin, glucose and serum/BSA while maintaining the cultured myotubes in morphologically healthy state. Our lab standardized protocols were observed to be effective in studying insulin resistance condition induced by diverse stresses (oxidative & inflammation) in myotubes. Comparative study conducted with already established protocols demonstrates that the present method is more efficient, effective and better improvised for studying glucose uptake in C2C12.