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(NPC1...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.展开更多
基金This work was supported by grants from the National Key R&D Program and the National Natural Science Foundation of China(2019YFA0802503,91857203,2018YFA0800602)Collaborative Innovation Program of Shanghai Municipal Health Commission(2020CXJQ01).
文摘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.
