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

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.

Fructose and metabolic diseases: too much to be good

Excessive consumption of fructose,the sweetest of all naturally occurring carbohydrates,has been linked to worldwide epidemics of metabolic diseases in humans,and it is considered an independent risk factor for cardiovascular diseases.We provide an overview about the features of fructose metabolism,as well as potential mechanisms by which excessive fructose intake is associated with the pathogenesis of metabolic diseases both in humans and rodents.To accomplish this aim,we focus on illuminating the cellular and molecular mechanisms of fructose metabolism as well as its signaling effects on metabolic and cardiovascular homeostasis in health and disease,highlighting the role of carbohydrate-responsive element–binding protein in regulating fructose metabolism.