Measurement of ATGL activity using adiposomes

Abstract

Adipose triacylglycerol lipase (ATGL) is a dynamic lipid droplet-associated protein involved in cellular lipolysis, which is conserved from bacteria to humans. Recent methods that measure the enzymatic activity of ATGL in vitro are established using lipid emulsions. However, the lipid emulsion platforms contain various membranous structures which reduce the accuracy of enzymatic activity determination. Therefore, a new platform and corresponding method are required for accurate measurement of ATGL enzymatic activity that represents cellular lipid and energy homeostasis. Adiposomes are artificial lipid nanostructures mimicking lipid droplets. Employing adiposome as a platform, we have developed an assay to measure the enzymatic activity of ATGLin vitro. Here, a detailed protocol is described to explain how to measure the activity of ATGL using adiposomes. This method successfully proves the concept of lipid droplet-mimetic lipase activity determining platform and provides a tool to identify the active sites of lipases.

Graphical abstract

Keywords

ATGL / Enzymatic activity / Adiposome / Lipid droplet

Acknowledgements

The authors would like to thank Dr. Yang Wang for her advices on adiposome preparation and TAG hydrolase assay, Dr. Shimeng Xu for his suggestions on site-directed mutation, and Dr. Liujuan Cui for her help of BAT cytosol extraction. The authors also want to thank Dr. Chang Zhou and Dr. Liujuan Cui for their administrative support. The authors thank Hongjie Zhang for his suggestions of radioisotope experiments. This work was supported the National Natural Science Foundation of China (91857201, 91954108, 32170787 and 32100557) and National Key R&D Program of China (2018YFA0800700 and 2018YFA0800900).

Funding

The authors would like to thank Dr. Yang Wang for her advices on adiposome preparation and TAG hydrolase assay, Dr. Shimeng Xu for his suggestions on site-directed mutation, and Dr. Liujuan Cui for her help of BAT cytosol extraction. The authors also want to thank Dr. Chang Zhou and Dr. Liujuan Cui for their administrative support. The authors thank Hongjie Zhang for his suggestions of radioisotope experiments. This work was supported the National Natural Science Foundation of China (91857201, 91954108, 32170787 and 32100557) and National Key R&D Program of China (2018YFA0800700 and 2018YFA0800900).

RIGHTS & PERMISSIONS

2022 The Author(s)