Comprehensive multiscale analysis of lactate metabolic dynamics in vitro and in vivo using highly responsive biosensors.
Comprehensive multiscale analysis of lactate metabolic dynamics in vitro and in vivo using highly responsive biosensors.
复制标题
DOI：
10.1038/s41596-023-00948-y
复制
发表时间：
2024-02
期刊：
影响因子：
14.8
通讯作者：
Aoxue Wang;Yejun Zou;Shuning Liu;Xiuze Zhang;Ting Li;Lijuan Zhang;Ruwen Wang;Yale Xia;Xie Li;Zhuo Zhang;Tiemin Liu;Zhenyu Ju;Ru Wang;Joseph Loscalzo;Yi Yang;Yuzheng Zhao
中科院分区：
文献类型：
--
作者：
Aoxue Wang;Yejun Zou;Shuning Liu;Xiuze Zhang;Ting Li;Lijuan Zhang;Ruwen Wang;Yale Xia;Xie Li;Zhuo Zhang;Tiemin Liu;Zhenyu Ju;Ru Wang;Joseph Loscalzo;Yi Yang;Yuzheng Zhao
乳酸作为糖酵解的重要代谢产物，在多种生理和病理过程中发挥着重要作用。然而，由于缺乏高性能的工具，乳酸代谢动力学在体外和体内的全面多尺度分析仍然是一个未解决的问题，直到现在。我们最近开发了一系列遗传编码的乳酸荧光传感器，命名为FiLa，它照亮了细胞，亚细胞器，动物，以及人血清和尿液中的乳酸代谢。在该协议中，我们首先描述了基于FiLa传感器的策略，通过分析乳酸代谢对不同营养和药理学条件的反应来进行实时亚细胞生物能通量分析，这首次在亚细胞尺度上提供了细胞代谢功能的系统水平视图。我们还报告了详细的程序，通过细胞微胶囊系统或重组腺相关病毒在活小鼠中成像乳酸动力学，并快速，简单地测定人体体液中的乳酸。这种全面的多尺度代谢分析策略也可以应用于使用各种分析平台的其他代谢物生物传感器，进一步扩展其可用性。该协议适用于具有生物化学，分子生物学和细胞生物学专业知识的用户。通常，FiLa表达细胞或小鼠的制备需要2天至4周，并且活细胞和体内成像可以在1-2小时内进行。对于基于FiLa的体液测定，在手动测定中，一个样品的整个测量过程通常需要约1分钟，在自动微孔板测定中，96个样品的整个测量过程通常需要约3分钟。
As a key glycolytic metabolite, lactate has a central role in diverse physiological and pathological processes. However, comprehensive multiscale analysis of lactate metabolic dynamics in vitro and in vivo has remained an unsolved problem until now owing to the lack of a high-performance tool. We recently developed a series of genetically encoded fluorescent sensors for lactate, named FiLa, which illuminate lactate metabolism in cells, subcellular organelles, animals, and human serum and urine. In this protocol, we first describe the FiLa sensor-based strategies for real-time subcellular bioenergetic flux analysis by profiling the lactate metabolic response to different nutritional and pharmacological conditions, which provides a systematic-level view of cellular metabolic function at the subcellular scale for the first time. We also report detailed procedures for imaging lactate dynamics in live mice through a cell microcapsule system or recombinant adeno-associated virus and for the rapid and simple assay of lactate in human body fluids. This comprehensive multiscale metabolic analysis strategy may also be applied to other metabolite biosensors using various analytic platforms, further expanding its usability. The protocol is suited for users with expertise in biochemistry, molecular biology and cell biology. Typically, the preparation of FiLa-expressing cells or mice takes 2 days to 4 weeks, and live-cell and in vivo imaging can be performed within 1–2 hours. For the FiLa-based assay of body fluids, the whole measuring procedure generally takes ~1 min for one sample in a manual assay or ~3 min for 96 samples in an automatic microplate assay.