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  3. 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

  • Nat Protoc. 2024 Feb 2. doi: 10.1038/s41596-023-00948-y.
Aoxue Wang # 1 2,  Yejun Zou # 1 2,  Shuning Liu # 1,  Xiuze Zhang # 1,  Ting Li 3 4,  Lijuan Zhang 1,  Ruwen Wang 5,  Yale Xia 1,  Xie Li 1 2,  Zhuo Zhang 1 2,  Tiemin Liu 6,  Zhenyu Ju 7,  Ru Wang 8,  Joseph Loscalzo 9,  Yi Yang 10,  Yuzheng Zhao 11 12
Affiliations

Affiliations

  • 1 Optogenetics & Synthetic Biology Interdisciplinary Research Center, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai, China.
  • 2 Research Unit of New Techniques for Live-cell Metabolic Imaging, Chinese Academy of Medical Sciences, Beijing, China.
  • 3 Optogenetics & Synthetic Biology Interdisciplinary Research Center, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai, China. tingli@ecust.edu.cn.
  • 4 Research Unit of New Techniques for Live-cell Metabolic Imaging, Chinese Academy of Medical Sciences, Beijing, China. tingli@ecust.edu.cn.
  • 5 School of Exercise and Health, Shanghai University of Sport, Shanghai, China.
  • 6 State Key Laboratory of Genetic Engineering, Department of Endocrinology and Metabolism, Institute of Metabolism and Integrative Biology, Human Phenome Institute, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai, China.
  • 7 Key Laboratory of Regenerative Medicine of Ministry of Education, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou, China.
  • 8 School of Exercise and Health, Shanghai University of Sport, Shanghai, China. wangru@sus.edu.cn.
  • 9 Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
  • 10 Optogenetics & Synthetic Biology Interdisciplinary Research Center, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai, China. yiyang@ecust.edu.cn.
  • 11 Optogenetics & Synthetic Biology Interdisciplinary Research Center, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai, China. yuzhengzhao@ecust.edu.cn.
  • 12 Research Unit of New Techniques for Live-cell Metabolic Imaging, Chinese Academy of Medical Sciences, Beijing, China. yuzhengzhao@ecust.edu.cn.
  • # Contributed equally.
PMID: 38307980 DOI: 10.1038/s41596-023-00948-y
Abstract

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.

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