Wu Xiaojing, Ma Xian-Hua, Lin Jie, Yang Xiaohang, Shi Jian-Hui, Xie Zhifang, Chen Yu-Xia, Zhang Weiping J
Department of Pathophysiology, Naval Medical University, Shanghai 200433, China.
NHC Key Laboratory of Hormones and Development, Chu Hsien-I Memorial Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin 300134, China.
Life Metab. 2023 Mar 16;2(2):load011. doi: 10.1093/lifemeta/load011. eCollection 2023 Apr.
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 . 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 response to diverse pharmacological agents and nutrients.
肠道腔内胆固醇吸收过多会导致高胆固醇血症的发病机制，而高胆固醇血症是动脉粥样硬化性心血管疾病的独立危险因素。尼曼-皮克C1样蛋白1（NPC1L1）是负责胆固醇吸收的主要膜蛋白，其囊泡内吞作用的生理作用仍存在争议，并且缺乏一种可行的工具来可视化和评估NPC1L1囊泡的内吞作用。在此，我们在基因敲入小鼠模型中用EGFP对内源性NPC1L1蛋白进行基因标记，并在肠道胆固醇吸收过程中对NPC1L1-货物囊泡的内吞作用进行了荧光可视化和评估。纯合子NPC1L1-EGFP小鼠在正常饮食或高胆固醇饮食下具有正常的NPC1L1表达模式以及胆固醇稳态。NPC1L1-EGFP融合蛋白的荧光定位于小肠的刷状缘膜，在口服胆固醇后5分钟，膜下即可观察到EGFP阳性囊泡。值得注意的是，这些囊泡与早期内体标记物早期内体抗原1（EEA1）和 Filipin 染色的游离胆固醇共定位。用NPC1L1抑制剂依泽替米贝预处理可抑制这些胆固醇诱导的内吞囊泡的形成。我们的数据支持NPC1L1介导的胆固醇吸收是一个囊泡内吞过程的观点。NPC1L1-EGFP小鼠是一种有用的模型，可用于可视化细胞内NPC1L1-货物囊泡的行程，并评估NPC1L1对各种药物和营养物质的反应活性。
