Fluorescent visualization and evaluation of NPC1L1- mediated vesicular endocytosis during intestinal cholesterol absorption in mice
Received date: 13 Feb 2023
Revised date: 10 Mar 2023
Accepted date: 16 Mar 2023
Published date: 14 Sep 2023
Copyright
Excessive cholesterol absorption from intestinal lumen contributes to the pathogenesis of hypercholesterolemia, which is an independent risk factor for atherosclerotic cardiovascular disease. Niemann-Pick C1-like 1 (NPC1L1) is a major membrane protein responsible for cholesterol absorption, in which the physiological role of vesicular endocytosis is still controversial, and it lacks a feasible tool to visualize and evaluate the endocytosis of NPC1L1 vesicles in vivo. Here, we genetically labeled endogenous NPC1L1 protein with EGFP in a knock-in mouse model, and demonstrated fluorescent visualization and evaluation of the endocytic vesicles of NPC1L1-cago during intestinal cholesterol absorption. The homozygous NPC1L1-EGFP mice have normal NPC1L1 expression pattern as well as cholesterol homeostasis on chow or high-cholesterol diets. The fluorescence of NPC1L1-EGFP fusion protein localizes at the brush border membrane of small intestine, and EGFP-positive vesicles is visualized beneath the membrane as early as 5 min post oral gavage of cholesterol. Of note, the vesicles colocalize with the early endosomal marker early endosome antigen 1 (EEA1) and the filipin-stained free cholesterol. Pretreatment with NPC1L1 inhibitor ezetimibe inhibits the formation of these cholesterol-induced endocytic vesicles. Our data support the notion that NPC1L1-mediated cholesterol absorption is a vesicular endocytic process. NPC1L1-EGFP mice are a useful model for visualizing cellular NPC1L1-cargo vesicle itineraries and for evaluating NPC1L1 activity in vivo in response to diverse pharmacological agents and nutrients.
Xiaojing Wu , Xian-Hua Ma , Jie Lin , Xiaohang Yang , Jian-Hui Shi , Zhifang Xie , Yu-Xia Chen , Weiping J. Zhang . Fluorescent visualization and evaluation of NPC1L1- mediated vesicular endocytosis during intestinal cholesterol absorption in mice[J]. Life Metabolism, 2023 , 2(2) : 81 -89 . DOI: 10.1093/lifemeta/load011
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