Fluorescent visualization and evaluation of NPC1L1- mediated vesicular endocytosis during intestinal cholesterol absorption in mice

Abstract

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.

Keywords

cholesterol homeostasis / NPC1L1-EGFP / cholesterol transportation / vesicle endocytosis / CRISPR/Cas9 / knock-in mice

References

[1] Luo J , Yang H , Song BL . Mechanisms and regulation of cholesterol homeostasis. Nat Rev Mol Cell Biol 2020; 21: 225- 45.

[2] Benjamin EJ , Muntner P , Alonso A et al. American Heart Association Council on Epidemiology and Prevention Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics-2019 update: a report from the American Heart Association. Circulation 2019; 139: e56- e528.

[3] Soppert J , Lehrke M , Marx N et al. Lipoproteins and lipids in cardiovascular disease: from mechanistic insights to therapeutic targeting. Adv Drug Deliv Rev 2020; 159: 4- 33.

[4] Ference BA , Ginsberg HN , Graham I et al. Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel. Eur Heart J 2017; 38: 2459- 72.

[5] Altmann SW , Davis HR Jr , Zhu LJ et al. Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption. Science 2004; 303: 1201- 4.

[6] Davies JP , Scott C , Oishi K et al. Inactivation of NPC1L1 causes multiple lipid transport defects and protects against diet-induced hypercholesterolemia. J Biol Chem 2005; 280: 12710- 20.

[7] Davies JP , Levy B , Ioannou YA . Evidence for a Niemann-pick C (NPC) gene family: identification and characterization of NPC1L1. Genomics 2000; 65: 137- 45.

[8] Wang J , Chu BB , Ge L et al. Membrane topology of human NPC1L1, a key protein in enterohepatic cholesterol absorption. J Lipid Res 2009; 50: 1653- 62.

[9] Zhang JH , Ge L , Qi W et al. The N-terminal domain of NPC1L1 protein binds cholesterol and plays essential roles in cholesterol uptake. J Biol Chem 2011; 286: 25088- 97.

[10] Li PS , Fu ZY , Zhang YY et al. The clathrin adaptor Numb regulates intestinal cholesterol absorption through dynamic interaction with NPC1L1. Nat Med 2014; 20: 80- 6.

[11] Xie 谢畅 C , Zhou 周章森 ZS , Li 李钠 N et al. Ezetimibe blocks the internalization of NPC1L1 and cholesterol in mouse small intestine. J Lipid Res 2012; 53: 2092- 101.

[12] Temel RE , Tang W , Ma Y et al. Hepatic Niemann-Pick C1-like 1 regulates biliary cholesterol concentration and is a target of ezetimibe. J Clin Invest 2007; 117: 1968- 78.

[13] Alrefai WA , Annaba F , Sarwar Z et al. Modulation of human Niemann-Pick C1-like 1 gene expression by sterol: role of sterol regulatory element binding protein 2. Am J Physiol Gastrointest Liver Physiol 2007; 292: G369- 76.

[14] Pramfalk C , Jiang ZY , Cai Q et al. HNF1alpha and SREBP2 are important regulators of NPC1L1 in human liver. J Lipid Res 2010; 51: 1354- 62.

[15] Yu L , Bharadwaj S , Brown JM et al. Cholesterol-regulated translocation of NPC1L1 to the cell surface facilitates free cholesterol uptake. J Biol Chem 2006; 281: 6616- 24.

[16] Jia L , Betters JL , Yu L . Niemann-pick C1-like 1 (NPC1L1) protein in intestinal and hepatic cholesterol transport. Annu Rev Physiol 2011; 73: 239- 59.

[17] Ge L , Wang J , Qi W et al. The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1. Cell Metab 2008; 7: 508- 19.

[18] Zhang YY , Fu ZY , Wei J et al. A LIMA1 variant promotes low plasma LDL cholesterol and decreases intestinal cholesterol absorption. Science 2018; 360: 1087- 92.

[19] Xie C , Li N , Chen ZJ et al. The small GTPase Cdc42 interacts with Niemann-Pick C1-like 1 (NPC1L1) and controls its movement from endocytic recycling compartment to plasma membrane in a cholesterol-dependent manner. J Biol Chem 2011; 286: 35933- 42.

[20] Ge L , Qi W , Wang LJ et al. Flotillins play an essential role in Niemann-Pick C1-like 1-mediated cholesterol uptake. Proc Natl Acad Sci U S A 2011; 108: 551- 6.

[21] Garcia-Calvo M , Lisnock J , Bull HG et al. The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1). Proc Natl Acad Sci U S A 2005; 102: 8132- 7.

[22] Weinglass AB , Kohler M , Schulte U et al. Extracellular loop C of NPC1L1 is important for binding to ezetimibe. Proc Natl Acad Sci U S A 2008; 105: 11140- 5.

[23] Johnson TA , Pfeffer SR . Ezetimibe-sensitive cholesterol uptake by NPC1L1 protein does not require endocytosis. Mol Biol Cell 2016; 27: 1845- 52.

[24] Liu G , Zhou L , Zhang H et al. Regulation of hepatic lipogenesis by the zinc finger protein Zbtb20. Nat Commun 2017; 8: 14824.

[25] Petersen NH , Færgeman NJ , Yu L et al. Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells. J Lipid Res 2008; 49: 2023- 37.

[26] Xie P , Zhu H , Jia L et al. Genetic demonstration of intestinal NPC1L1 as a major determinant of hepatic cholesterol and blood atherogenic lipoprotein levels. Atherosclerosis 2014; 237: 609- 17.

RIGHTS & PERMISSIONS

2023 The Author(s) 2023. Published by Oxford University Press on behalf of Higher Education Press.