Abstract
Niemann-Pick C1-like 1 (NPC1L1) is essential for intestinal cholesterol absorption. Together with the cholesterol-rich and Flotillin-positive membrane microdomain, NPC1L1 is internalized via clathrin-mediated endocytosis and transported to endocytic recycling compartment (ERC). When ERC cholesterol level decreases, NPC1L1 interacts with LIMA1 and moves back to plasma membrane. However, how cholesterol leaves ERC is unknown. Here, we find that, in male mice, intracellular bile acids facilitate cholesterol transport to other organelles, such as endoplasmic reticulum, in a non-micellar fashion. When cholesterol level in ERC is decreased by bile acids, the NPC1L1 carboxyl terminus that previously interacts with the cholesterol-rich membranes via the A1272LAL residues dissociates from membrane, exposing the Q1277KR motif for LIMA1 recruitment. Then NPC1L1 moves back to plasma membrane. This study demonstrates an intracellular cholesterol transport function of bile acids and explains how the substantial amount of cholesterol in NPC1L1-positive compartments is unloaded in enterocytes during cholesterol absorption.
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Introduction
Cholesterol is an important lipid that regulates membrane properties and modifies proteins including Hedgehog and Smoothened60. The Npc1l1-Y1306VNxxF→AAAxxA knock-in mouse was generated from C57BL/6JGpt by CRISPR-Cas9 system (GemPharmatech). Neonatal NPC1L1-EGFP knock-in C57BL/6N male mice was used for in vitro intestinal culture assay and 12-week-old male knock-in mice were applied to bile duct cannulation assay. Littermates from the heterozygous of Npc1l1-Y1306VNxxF→AAAxxA crossing were wild-type controls. Mice was housed in plastic cages in a specific pathogen-free animal facility under a 12-hour day/light cycle at 22 °C with a humidity of 50–60%. Mice were fed on chow diet (Bei**g HFK Biosciences, 1026) ad libitum. No gender-based analyses have been performed in this study. We chose male mice in the study because they have been widely used to evaluate cholesterol absorption. In fact, cholesterol absorption efficiency is not significantly different between genders in mice61. Adult mice were sacrificed by cervical dislocation and neonatal mice were sacrificed by decapitation using a sharp pair of scissors.
Ethics
All animal experiments were performed according to the protocols (WDSKY0201408) approved by the Institutional Animal Care and Use Committee of Wuhan University.
Reagents
1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) (850375), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) (840035) and sphingomyelin (LM2312) were from Avanti Polar Lipids. Ham’s F-12 (Ham’s F-12) was from Gibco. Amplex Red Cholesterol Assay Kit (A12216) and HCS LipidTox neutral lipid stain (H34476) were from Invitrogen. Anti-FLAG M2 Affinity Gel (A2220), cholesterol (C8667), deoxycholate (D6750), filipin (F9765), glycocholate (700265 P), glycodeoxycholate (G9910), lithocholic acid (700218 P) and taurodeoxycholate (T0875) were from Sigma-Aldrich. The bile acid (BA) mixture (B822609) was from Macklin. Cholate (C426104), taurocholate (S100834) and taurolithocholate (S171146) were purchased from Alladin. Chenodeoxycholic Acid (HY-76847), gentamicin (HY-A0276A), glycochenodeoxycholate (HY-N2334A), glycolithocholic acid (HY-116374), Linerixibat (HY-16643) and taurochenodeoxycholic acid (HY-N2027) were from Med Chem Express. PTFE Sub-Lite Wall Tube (SUBL-120, 0.012” OD x 0.006” ID), Teflon-coated guide wire (ST003, 0.003”) and RenaSil Silicone Rubber Tubing (SIL-025, 0.025” OD x 0.012” ID) were purchased from Braintree Scientific Inc (MA). Cyclodextrin was purchased from Cyclodextrin Technologies Development. Total cholesterol assay kit and total triglyceride assay kit were purchased from Shanghai Kehua Bio-engineering Co., Ltd. Lipoprotein-deficient serum (LPDS, density >1.215 g/mL) was prepared from newborn calf serum by ultracentrifugation in our laboratory.
Plasmids
The coding sequence of human LIMA1 was inserted into p3×FLAG-CMV14 or pmCherry-N1 to generate pCMV14-LIMA1-3×FLAG (encoding LIMA1-FLAG) or pCMV-LIMA1-mCherry (encoding LIMA1-mCherry), respectively. To generate pCMV-NPC1L1-3×Myc-EGFP plasmid, the coding sequence of human NPC1L1 with an insertion of 3×Myc tag after 966th amino acid was fused with a C-terminal EGFP and then cloned into pcDNA3. The deletion (ΔA1272LAL) or mutation (A1272LAL-to-E1272EEE substitution, A1272LAL → 4E) of pCMV-NPC1L1-3×Myc-EGFP were generated by overlap extension PCR. The coding sequence of NPC1L1-3×Myc-EGFP-ΔA1272LAL or NPC1L1-3×Myc-EGFP-A1272LAL → 4E was also inserted into pLVX-IRES-puro vector by standard PCR, respectively. The sequence of mouse Rab11a was cloned into pEGFP-C1 or pmCherry-C1 to encode EGFP-Rab11a or mCherry-Rab11a, respectively. The sequence of human TFR was cloned into pmCherry-N1 to generate pCMV-TFR-mCherry. The coding region of human NPC1L1-CT67 (1272-1275 a.a.) was amplified from pCMV-NPC1L1-3×Myc-EGFP by standard PCR, and then inserted into the pET28a to obtain pT7-His6-NPC1L1-CT67. The truncations of pT7-His6-NPC1L1-CT67 (Δ1-10, Δ11-20, Δ21-30, Δ31-40, Δ41-50, Δ51-60, Δ21-30, Δ61-67, ΔGPD, ΔVNP and ΔALAL) were prepared by over extension PCR. The D4H, domain 4 of Perfringolysin O carrying D434S mutation, fused with N-terminal His6-mCherry was inserted into pET28a vector62.
Antibodies
The rabbit polyclonal antibody against NPC1L1 (1 μg/mL for immunoblotting and 5 μg/mL for immunohistochemistry) was generated in our laboratory11. The mouse monoclonal antibody against SREBP2 (5 μg/mL for immunoblotting) was produced and purified from hybridoma cell line 1D2 (ATCC, Cat Num #CRL-2545) in our laboratory. The information about commercial antibodies is listed in Supplementary Table 2.
Cell culture
CRL1601 cells were obtained from ATCC (Cat# CRL1601). CRL1601, CRL1601/NPC1L1-3×Myc-EGFP (CRL1601 cells stably expressing NPC1L1-3×Myc-EGFP), CRL1601/NPC1L1-3×Myc-EGFP-ΔA1272LAL (CRL1601 cells stably expressing NPC1L1-3×Myc-EGFP with a deletion of ΔA1272LAL) and CRL1601/NPC1L1-3×Myc-EGFP-A1272LAL → 4E (CRL1601 cells stably expressing NPC1L1-3×Myc-EGFP with A1272LAL-to-E1272EEE substitution) were grown in a monolayer at 37 °C with 5% CO2. Cells were maintained in the Dulbecco’s Modified Eagle Medium (DMEM) containing 100 units/mL penicillin and 100 μg/mL streptomycin sulfate and 10% fetal bovine serum (FBS). To generate CRL1601/NPC1L1-3×Myc-EGFP-ΔA1272LAL or CRL1601/NPC1L1-3×Myc-EGFP-A1272LAL → 4E cell, CRL1601 cells were transfected with lentivirus expressing NPC1L1-3×Myc-EGFP-ΔA1272LAL or CRL1601/NPC1L1-3×Myc-EGFP-A1272LAL → 4E, respectively.
The cholesterol starvation medium was composed of DMEM supplemented with 1 μmol/L lovastatin, 10 μmol/L mevalonate and 5% (w/v) LPDS. For BA treatment assay, cells were washed with 1×PBS once and then incubated with DMEM supplemented with 5% LPDS in the presence or absence of indicated BAs for 10 min or 2 h. For transient overexpression assay, CRL1601 and CRL1601/NPC1L1-3×Myc-EGFP cells were transfected with indicated plasmids using FuGENE HD Transfection Reagent (Promega, E2311) according to the technical manual. Briefly, 1 μg recombinant plasmids and 3 μL FuGENE HD Transfection Reagent were mixed together for 15 min in 100 μL opti-MEM I Reduced Serum Medium (Gibco, 11058021). Six hours later, the media was replaced. After 72 h, cells with indicated treatment were harvested for WB (collected by Tanon Imager 5200 software v2.03) or immunostaining.
RNA interference
On day 0, CRL1601/NPC1L1-3×Myc-EGFP cells were seeded on 12-well plates or 60 mm dishes at a confluency of 30%. Twenty-four hours later, the cells were transfected with negative control siRNA, siRNA against rat Ntcp1 or Lima1 for 48 h using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, 13778150). Briefly, siRNA was dissolved into 10 μmol/L with RNase-free water. 2 μL siRNA and 3 μL transfection reagent were separately added into 75 μL opti-MEM I Reduced Serum Medium for 5 min, then the diluted reagent and siRNA were mixed together and incubated for 5 min. Replace the culture medium with the opti-MEM I Reduced Serum Medium and add siRNA-lipid complex into the medium. Before harvesting, cells were treated with DMEM containing 5% LPDS plus indicated BAs for 10 min or 2 h as described in the Figure legends. The qPCR was performed in Bio-Rad CFX Connect Real-Time System and analyzed by CFX Manager v3.1. The sequence of siRNAs and qPCR primers used in this study are listed in Supplementary Table 1.
Bile acid treatment
The BA mixture from Sus Scrofa was dissolved with 50% ethanol to prepare the 25 mg/mL BA mixture stock solution. The BA mixture stock solution was filtrated with a 0.22 μm filter and diluted with 5% LPDS in DMEM at the ratio of 1:100 to yield a working medium with 0.25 mg/mL BA mixture.
CA, CDCA, DCA and LCA stock solutions were dissolved in DMSO at a concentration of 100 mmol/L. GCA, TCA, GDCA, TDCA, GCDCA, TCDCA, GLCA and TLCA were dissolved in water at a concentration of 100 mmol/L. When in use, the stock solutions of each BA species were diluted 1000 times in DMEM with 5% LPDS and the final working concentration of these bile acids is 0.1 mmol/L. 1.5% or 3% w/v CDX in DMEM harboring 5% LPDS was used to treat cells for 2 h or 10 min as indicated in the Figure legends.
To test the cholesterol mobilizing ability of bile acids, CRL1601/NPC1L1-3×Myc-EGFP cells were treated with DMEM containing 5% LPDS (designed as vehicle), 250 μg/mL BA, or 0.1 mmol/L indicated bile acids in the vehicle medium for 2 h prior to harvest, respectively. If needed, the avasimibe group was added additional 10 μmol/mL avasimibe during the 2-hour incubation. In the short-term treatment of bile acids, CRL1601 or CRL1601/NPC1L1-3×Myc-EGFP cells were treated with control medium, BA, indicated bile acids or 3% w/v CDX for 10 min prior to 4% paraformaldehyde (PFA) fixation, respectively.
LipidTox staining
For LipidTox staining of CRL1601/NPC1L1-3×Myc-EGFP cells, cells grown on coverslip were washed with 1×PBS once, and fixed with 4% paraformaldehyde for 30 min. Cells were washed with 1×PBS for 3 times and incubate with 1×PBS containing 0.1% v/v HCS LipidTox neutral lipid stain for 30 min. Remove the buffer from the cells and wash cells with 1×PBS for 3 times. The coverslips containing cells were mounted with 50% (v/v) glycerol and the edges of coverslips were sealed with nail polish.
For LipidTox staining of intestinal slices, 10-μm-thick frozen sections in the glass slides were incubated at 37 °C for 30 min and washed with 1×PBS for 3 times. The sections were permeabilized with 0.2% v/v triton in 1×PBS for 10 min, washed with 1×PBS for 3 times, and incubated with primary antibody diluted in 1×PBS plus with 1% w/v BSA at 4 °C overnight. After 3 times washing of 1×PBS, the sections were incubated with the secondary antibody diluted in 1% (w/v) BSA at room temperature (RT) for 1 h. The sections were washed with 1×PBS 3 times, stained with 2% v/v HCS LipidTOX neutral lipid stain in 1×PBS at RT for 50 min, and washed with 1×PBS for 5 times. A clean coverslip was mounted onto the sections with 50% (v/v) glycerol and the edges of the coverslip were sealed with nail polish.
Liposome preparation and co-sedimentation assay
100-nm liposomes composed of DOPC, DOPS and sphingomyelin with or w/o cholesterol of different concentrations were prepared as previously described63. Briefly, a total of 1 mg lipid mixture composed of DOPC, DOPS and sphingomyelin with or w/o cholesterol in the indicated molar ratio as described was resuspended in 1 mL chloroform and pipetted into a 50-mL round bottom flask. The organic solvent in the flask was dried at 37 °C in a rotary evaporator with a rotating speed of 50 r/min. Lipids were resuspended in 1 mL 20 mmol/L Hepes (pH 7.2) and then subjected to 5 cycles of fast freeze-thaw test by placing sample vial in liquid nitrogen for 1 min and a 37 °C water bath for 2 min. To generate liposomes, the lipid suspension was forced through a 0.1 μm polycarbonate filter for 21 times by the mini-extruder (Avanti Polar Lipids, 610000-1EA).
To identify the interactions between the liposomes and WT or mutant forms of His6-NPC1L1-CT67 protein, 40 μL liposomes and 10 μL proteins (0.75 μg/μL stock solution) were mixed in 200 μL HEK buffer (20 mmol/L HEPES, pH 7.2, 120 mmol/L potassium acetate and 1 mmol/L MgCl2) and then incubated for 30 min at 37 °C. Liposomes were spun down at 250,000 × g for 30 min. Supernatants (designed as liposome unbound proteins) and pellets (designed as liposome bound proteins) were harvested and applied to western blotting analysis.
To assess the diameter of liposomes, 25 μg liposomes together with indicated bile acids were incubated in 1×PBS with a total volume of 100 μL at RT for 2 h. Then the liposomes were diluted in 2 mL 1×PBS and applied to diameter determination by Zetasizer Nano ZSP (Malvern Instruments, Malvern UK) using a detection angle of 173° at a temperature of 25 °C. The Nano ZSP uses a 10 mW He-Ne laser operating at a wavelength of 633 nm.
In vitro cholesterol mobilizing assay
Liposomes containing DOPC, DOPS and sphingomyelin in the presence or absence of cholesterol in a molar ratio of 2:5:3 or 2:1:3:4 were applied to the mobilizing assay. Liposomes with or w/o cholesterol were separated by a semi-permeable membrane with the pore size allowing the diffusion of molecules below 50 kDa. 0.25 mg/mL bile acids mixture or 0.1 mmol/L indicated bile acid species were added into the cholesterol-liposome side and the permeator was incubated for 3 h at 4 °C on a shaker. Samples from the other side were collected and cholesterol content was measured using Amplex Red Cholesterol Assay Kit following the manufacturer’s instructions. The relative amount of mobilized cholesterol is the specific value that the total cholesterol mobilized to the non-cholesterol liposome divided by the total cholesterol in the cholesterol-liposome.
Measurement of total cholesterol and cholesteryl esters
Cells were homogenized in chloroform/methanol (2:1) to extract lipids and then centrifuged at 20,000 × g for 10 min. The organic phase was harvested and dried using nitrogen flow. The pellets were resuspended and applied to cholesterol and cholesteryl esters with Amplex Red Cholesterol Assay Kit. To measure cholesteryl esters, parallel reactions were made with the presence or absence of cholesterol esterase. The readings were recorded as total cholesterol and free cholesterol respectively. And the difference was calculated as cholesteryl esters.
Bile duct cannulation
12-week-old male C57BL/6J mice or NPC1L1-EGFP knock-in mice were anesthetized with tribromoethanol (0.02 mL/g of 1.2% stock solution) by intraperitoneal injection. Then a small incision was made in the abdominal wall near the duodenal site to visualize the common bile duct. A small hole in the common bile duct was made with the tip of a 29 G insulin syringe. Then PTFE Sub-Lite Wall Tube pre-loaded with a Teflon-coated guide wire was inserted into the common bile duct. After successful catheterization, the cannula was secured with surgical sutures. A guide wire was slowly removed from PTFE tubing and bile would slowly flow into the cannula. Then a softer and larger RenaSil Silicone Rubber Tubing was attached to the PTFE tubing and secured with sutures. The tube was tunneled subcutaneously through the abdominal wall and exteriorized in the scapular region. Sutures were applied to both wound sites to secure the catheter in place. The bile was collected into a tube placed on its back. Mice were placed on a 37 °C heating pad until woke up. Mice were singly housed after surgery.
Intestinal filipin staining
Intestinal samples from Npc1l1-EGFP knock-in mice were washed with ice cold 1×PBS and fixed in 4% PFA at 4 °C for 12 h. The sample was washed with 1×PBS for 3 times and soaked in 30% sucrose for 48 h prior to embedding and freezing in Tissue-Tek OCT (Sakura, 4583). Then the intestine was cut into 10-μm thin sections, and these sections were placed on a hot plate at 37 °C for 1 h. Finally, the sections were stained with 50 μg/mL filipin diluted in 1×PBS for 40 min at RT and mounted in coverslips after 5 times washing in 1×PBS.
In vitro intestinal culture
The in vitro intestinal culture was conducted as described20. The small intestine of the neonatal mouse was longitudinally opened and gently rinsed with ice-cold 1×PBS. The intestine was cut into about 1 cm segments, then intestinal segments were embedded in 1 mL type I collagen gel solution (Wako, 637-00653) and placed into a 30-mm cell culture insert (Millicell, PIHP03050), which had been pre-covered with 1.5 mL type I collagen gel solution. The cell culture insert was incubated with 2 mL Ham’s F12 medium containing 20% (v/v) FCS and 50 μg/mL gentamicin in a 60 mm dish, and the intestinal segments were grown at 37 °C with 5% CO2. On day 4, the 60-mm dishes were washed with 1×PBS and added with 2 mL Ham’s F12 medium containing 10% (v/v) LPDS plus 10 μg/mL cholesterol in the presence or absence of indicated bile acids or BA mixture. If needed, 20 μmol/L avasimibe was added to the LPDS medium. After 2 h incubation, the intestinal segments were carefully collected, fixed with 4% PFA at 4 °C overnight, and then embedded in Tissue-Tek OCT.
D4H staining
CRL1601/NPC1L1-3×Myc-EGFP cells on the coverslips were washed with ice-cold 1×PBS for once and then incubated with 10 μg/mL recombinant mCherry-D4H (dissolved with 1% w/v BSA) for 30 min on ice. The samples were rinsed with ice-cold 1×PBS for 3 times and fixed with 4% PFA at RT. Coverslips were mounted on glass slides using FluorSave (Millipore, 345789) and imaged on a Leica Biosystems SP8 laser scanning microscope.
Immunofluorescence
CRL1601 or CRL1601/NPC1L1-3×Myc-EGFP cells were grown on glass coverslips and fixed with 4% paraformaldehyde (PFA) for 30 min. For impermeable NPC1L1-3×Myc-EGFP staining, cells were serially incubated with primary antibody against Myc tag (1:500 dilution, 1% w/v BSA) and appropriate secondary antibodies (1:500 dilution, 1% w/v BSA) at RT for 1 h, respectively. Then cells were immuno-stained with filipin at RT for another 1 h. The coverslips were washed with 1×PBS for 3 times and mounted by FluorSave.
Quantification and analysis of immunofluorescent images
All confocal images were acquired by a Leica Biosystems SP8 laser scanning microscope with Leica LAS X (v3.5.2.18963). A total of 300 cells from 3 independent trials were quantified, and the average of 100 cells from the same trial represented one statistic on bar chart. For quantification of the relative fluorescent intensity of LipidTox, D4H and surface NPC1L1, the contours of cells were defined manually, and then the background-subtracted fluorescence intensity of LipidTox, D4H or surface NPC1L1 within cell was quantified using Image J (v1.50i). The relative intensity of D4H or surface NPC1L1 was normalized to BA, CDCA or GDCA treated cells as indicated in the relevant legends.
The ERC cholesterol was defined as the cholesterol colocalized with intracellular NPC1L1. To quantify the ERC cholesterol, the cholesterol confocal images were converted to grayscale and subtracted background by the threshold tool. Then, ERC was manually outlined according to their contours in corresponding images. The contour line, namely region of interest (ROI), was copied to the background-subtracted cholesterol image by the ROI manager tool, and the intensity of ROI in the cholesterol images was determined to obtain the intensity of ERC cholesterol. Finally, we measured the cholesterol intensity of the whole cell and calculated the percentage of ERC cholesterol by the ratio.
Similarly, the ERC NPC1L1 referred to intracellular NPC1L1 residing on ERC, and the total NPC1L1 means the whole cell NPC1L1. The contour of ERC or the cell was copied from the image to generate the ROI of ERC NPC1L1 or total NPC1L1, respectively. The intensity of the two ROIs was measured and applied to ERC/total NPC1L1 calculation.
Flow cytometry
CRL1601/NPC1L1-3×Myc-EGFP cells were washed with 1×PBS 2 times, suspended using 2 mM EDTA in 1×PBS and then rinsed with ice-cold 1×PBS once. CRL1601/NPC1L1-3×Myc-EGFP cells were fixed with 4% PFA for 30 min at 4 °C. Next, cells were incubated with anti-Myc antibody and secondary antibody (diluted with 1% w/v BSA) at 4 °C for 1 h in turn. Samples were washed with 1×PBS 3 times, resuspended with 1% BSA in 1×PBS, and then detected by CytoFLEX (Beckman Coulter) with CytExpert (v2.4.0.28). The data from flow cytometry were analyzed by FlowJo (v10.0.7). The gating strategy for analysis of surface NPC1L1 is shown in Figure S11. A total of 6,000 cells from 3 independent trials were counted.
Quantitative real-time PCR
The total RNA was extracted from CRL1601 cells transfected with indicated siRNAs by Trizol as described64. 2 μg mRNA from CRL1601 cells was used for cDNA synthesis and the relative mRNA level of indicated genes was analyzed by the comparative CT method. Rat Gapdh was used as the control.
Co-Immunoprecipitation assay
CRL1601 cells or CRL1601/NPC1L1-3×Myc-EGFP cells were transfected with LIMA1-FLAG and WT or mutant forms of NPC1L1-3×Myc-EGFP as indicated in legends. Cells were treated with BA mixture or indicated bile acid species for 10 min prior to harvest. Then cells were homogenized in NP40 buffer (0.5% NP40 in PBS containing 5 mmol/L EDTA and EGTA) and centrifuged at 3,000 × g for 10 min. The protein concentrations in the lysates were determined by Pierce™ BCA Protein Assay Kits (Thermo Scientific, Cat# 23227) and analyzed by Bio-Rad iMark Microplate Absorbance Reader with Microplate Manager 6 Software (v6.3). The lysates with equivalent protein amounts were then precleared with protein A/G beads at 4 °C for 30 min and centrifuged at 1,000 g for 3 min. The supernatant was incubated with anti-FLAG beads at 4 °C for 2 h. The anti-FLAG beads were spun down at 1000 × g for 3 min and washed with NP40 buffer 5 times. Finally, the beads were mixed with SDS-PAGE Protein Loading Buffer and examined by immunoblotting analysis.
Immunohistochemistry analysis
Intestinal samples from euthanized male mice were fixed with 4% PFA, dehydrated through a series ethanol of different concentration gradients, cleared in xylene, and embedded in paraffin. Samples were cut into 10-μm sections and deparaffinized, then samples were heated at 95 °C for 15 min with Tris-EDTA buffer (20 mmol/L tris base, 1 mmol/L EDTA, pH 9.0) to retrieve antigens. Samples were permeabilized and blocked in PBS with 5% FBS and 0.5% Triton X-100 for 1 h at RT. Sections were then incubated with primary antibodies overnight at 4 °C. After washing with PBS for 5 times, sections were incubated with appropriate Alexa Fluor dye-conjugated secondary antibodies for 1 h at RT. Sections were finally counterstained with Hoechst (Invitrogen) and mounted using FluorSave (Millipore). Images were acquired on a Leica TCS SP8 confocal microscope.
Cholesterol absorption measurement
The cholesterol absorption assays were conducted as described16. The nine-week-old male Npc1l1-Y1306VNxxF→AAAxxA mice were orally gavaged with 100 μL corn oil containing 5 μCi [3H] cholesterol and 0.1 mg unlabeled cholesterol. After 2 h, cholesterol was extracted from the plasma and livers of the mice and counted using the liquid scintillation counter. The percentage cholesterol absorption was calculated as follows: % cholesterol absorption = [3H] in liver (or plasma)/[3H] dosing × 100. Total radioactivity in the plasma was calculated based on the assumption that mice possess approximately 4 mL plasma per 100 g body weight.
For the fecal dual isotope ratio assay, nine-week-old male C57BL/6J mice were pretreated with vehicle or 0.2 mg/mL linerixibat once daily for 3 days prior to cholesterol gavage, and continued to the end of the fecal collection. On the third day, mice were orally gavaged with 100 μL corn oil containing [14C]-cholesterol (0.5 μCi), [3H]-sitosterol (1 μCi) and 0.1 mg unlabeled cholesterol at 2 h after linerixibat treatment. Then the feces from the mice within 2 days were gathered. The [14C]-cholesterol and [3H]-sitosterol in the feces were extracted in chloroform and methanol mixture (with a ratio of 2 to 1) and determined by liquid scintillation counter. The percent cholesterol absorption was calculated as follows: % cholesterol absorption = ([14C]/ [3H] dosing mixture – [14C]/[3H] feces)/(14C/[3H] dosing mixture) × 100.
Statistical analysis
Data were analyzed by GraphPad Prism 9.0 and expressed as mean ± SD. Statistical comparisons between two groups were conducted by Unpaired two-tailed Student’s t test. Comparisons for more than two groups were conducted using a One-way analysis of variance (ANOVA) with Tukey’s post hoc test. Comparisons for two categorical independent variables that each have multiple levels were performed using Two-way ANOVA with Tukey’s post hoc test. Sample sizes, statistical tests and exact P values (calculated to fifteen decimal places) for each experiment are described in the relevant figure or Source Data file. The P values exceeding 0.05 were considered statistically not significant.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
A Reporting Summary for this article describing the experiment details is available as Supplementary Information file. The data supporting the findings of this study are available within the paper and its supplementary information. Source data underlying figures and supplementary figures are provided as a Source Data file. Specific data P values are also included within the Source Data file. Source data are provided with this paper.
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Acknowledgements
We thank Ms. Dan Liang and **g ** for technical assistance. This work was supported by grants from National Natural Science Foundation of China (91954203 and 32021003), 111 Project of the Ministry of Education of China (B16036), the China Postdoctoral Science Foundation Grant (2021M692478) and the Fundamental Research Founds for the Central Universities (2042022kf1045). B.-L. Song acknowledges the support from the Tencent Foundation through the XPLORER PRIZE.
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**ao, J., Dong, LW., Liu, S. et al. Bile acids-mediated intracellular cholesterol transport promotes intestinal cholesterol absorption and NPC1L1 recycling. Nat Commun 14, 6469 (2023). https://doi.org/10.1038/s41467-023-42179-5
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DOI: https://doi.org/10.1038/s41467-023-42179-5
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