文献类型：期刊文献

英文题名：Comprehensive multiscale analysis of lactate metabolic dynamics in vitro and in vivo using highly responsive biosensors

作者：Wang, Aoxue[1,2];Zou, Yejun[1,2];Liu, Shuning[1];Zhang, Xiuze[1];Li, Ting[1,2];Zhang, Lijuan[1];Wang, Ruwen[3];Xia, Yale[1];Li, Xie[1,2];Zhang, Zhuo[1,2];Liu, Tiemin[4];Ju, Zhenyu[5];Wang, Ru[3];Loscalzo, Joseph[6];Yang, Yi[1];Zhao, Yuzheng[1,2]

机构：[1]East China Univ Sci & Technol, Optogenet & Synthet Biol Interdisciplinary Res Ctr, Shanghai Frontiers Sci Ctr Optogenet Tech Cell Met, Sch Pharm,State Key Lab Bioreactor Engn, Shanghai, Peoples R China;[2]Chinese Acad Med Sci, Res Unit New Tech L Cell Metab Imaging, Beijing, Peoples R China;[3]Shanghai Univ Sport, Sch Exercise & Hlth, Shanghai, Peoples R China;[4]Fudan Univ, Zhongshan Hosp,State Key Lab Genet Engn,Dept Endoc, Inst Metab & Integrat Biol, Human Phenome Inst, Shanghai, Peoples R China;[5]Jinan Univ, Inst Aging & Regenerat Med, Key Lab Regenerat Med,Minist Educ, Guangzhou Regenerat Med & Hlth Guangdong Lab, Guangzhou, Peoples R China;[6]Harvard Med Sch, Brigham & Womens Hosp, Dept Med, Boston, MA USA

年份：2024

卷号：19

期号：5

起止页码：1311

外文期刊名：NATURE PROTOCOLS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:001156450300001)】；

基金：This research is supported by National Key Research and Development Program of China (2019YFA0904800 to Y. Zhao and 2021YFA0804900 to A.W.), National Natural Science Foundation of China (32150030, 32030065, 32121005 and 92049304 to Y. Zhao; 91857202, 21937004 and 32150028 to Y.Y.; 82030039 to Z.J; 32000920 to A.W.; 32201230 to Y. Zou), the Shanghai Science and Technology Commission (20JC1412000), the Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism (Y. Zhao), the Research Unit of New Techniques for Live-cell Metabolic Imaging (Chinese Academy of Medical Sciences, 2019-I2M-5-013 to Y. Zhao), the innovative research team of high-level local universities in Shanghai, the State Key Laboratory of Bioreactor Engineering, the Fundamental Research Funds for the Central Universities, US National Institutes of Health (HL155107, HL155096 and HL166137 to J.L.) and the American Heart Association (AHA2020CV-19 and AHA957729 to J.L.).

语种：英文

摘要：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 similar to 1 min for one sample in a manual assay or similar to 3 min for 96 samples in an automatic microplate assay.