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Inhibition of ASGR1 decreases lipid levels by promoting cholesterol excretion

Abstract

High cholesterol is a major risk factor for cardiovascular disease1. Currently, no drug lowers cholesterol through directly promoting cholesterol excretion. Human genetic studies have identified that the loss-of-function Asialoglycoprotein receptor 1 (ASGR1) variants associate with low cholesterol and a reduced risk of cardiovascular disease2. ASGR1 is exclusively expressed in liver and mediates internalization and lysosomal degradation of blood asialoglycoproteins3. The mechanism by which ASGR1 affects cholesterol metabolism is unknown. Here, we find that Asgr1 deficiency decreases lipid levels in serum and liver by stabilizing LXRα. LXRα upregulates ABCA1 and ABCG5/G8, which promotes cholesterol transport to high-density lipoprotein and excretion to bile and faeces4, respectively. ASGR1 deficiency blocks endocytosis and lysosomal degradation of glycoproteins, reduces amino-acid levels in lysosomes, and thereby inhibits mTORC1 and activates AMPK. On one hand, AMPK increases LXRα by decreasing its ubiquitin ligases BRCA1/BARD1. On the other hand, AMPK suppresses SREBP1 that controls lipogenesis. Anti-ASGR1 neutralizing antibody lowers lipid levels by increasing cholesterol excretion, and shows synergistic beneficial effects with atorvastatin or ezetimibe, two widely used hypocholesterolaemic drugs. In summary, this study demonstrates that targeting ASGR1 upregulates LXRα, ABCA1 and ABCG5/G8, inhibits SREBP1 and lipogenesis, and therefore promotes cholesterol excretion and decreases lipid levels.

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Fig. 1: Asgr1 deficiency increases LXRα, cholesterol excretion and decreases lipid levels.
Fig. 2: Metabolic improvements in Asgr1−/− mice were dependent on LXRα.
Fig. 3: Asialoglycoproteins-ASGR1-AMPK/mTORC1 axis regulates LXRα and SREBP.
Fig. 4: Anti-ASGR1 neutralizing antibody promotes cholesterol excretion.
Fig. 5: Anti-ASGR1 displays synergistic lipid-lowering effects with atorvastatin and ezetimibe.

Data availability

All source data for immunoblotting are shown in Supplementary Fig. 1. All the other source data for Figs. 1, 2, 4, 5 and Extended Data Figs. 1, 2, 412 are provided with this paper. Next-generation sequencing data files that support the findings of this study have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession GSE183013.

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Acknowledgements

We thank J. Jing and D. Liang for technical assistance, K. Liu for proteasome antibodies, G.-H. Shui (Institute of Genetics and Developmental Biology, CAS) for measuring oxysterol, C.-T. Jiang (Peking University) for measuring BAs and W. Qi (ShanghaiTech University) for helpful discussion. This work was supported by grants from the National Natural Science Foundation (China; Nos. 91957103, 91954203 and 32021003) and the Ministry of Science and Technology (China; No. 2018YFA0800700). B.-L.S. acknowledges the support from the Tencent Foundation through the XPLORER PRIZE.

Author information

Authors and Affiliations

Authors

Contributions

B.-L.S. conceived the project and directed the research. J.-Q.W. designed and conducted the overall experiments and analysed the data. A.H. performed the lysosome purification experiments. G.D. and X.-L.Z. measured amino acids. J.W. performed experiments in biochemical assays and Asgr1−/− Lxrα−/−double-knockout mice. L.-L.L. performed the animal experiments with anti-ASGR1 antibody. Y.-F.L., Y.-B.L. and X.-Y.L. performed AAV-shRNA experiments. L.-L.L. and Z.-P.Q. assisted with the characterization of mouse lines, cellular experiments and western blotting assay. B.-L.S., J.-Q.W., X.-J.S. and J.L. wrote the paper with input from others.

Corresponding author

Correspondence to Bao-Liang Song.

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The authors declare no competing interests.

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Nature thanks Daniel Rader, Peter Tontonoz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Characterization of the Asgr1−/− mice and analysis of the effects of ASGR1 on lipid metabolism.

a, Tissue distribution of mouse ASGR1 protein. b, Schematic for the generation of Asgr1-knockout mice. c, DNA genotyping of Asgr1+/+, Asgr1+/− and Asgr1−/− mice. d, Immunoblotting analysis of liver lysates. e–l, n, The mice were as same as those in Fig. 1g–s(n = 6 per group). e, Body weight. f, Food intake (FI). g, Liver-to-body weight ratio. h, Blood glucose. i, Serum levels of aspartate aminotransferase (AST). j, Serum levels of alanine aminotransferase (ALT). k, Biliary phospholipid. l, Biliary cholesterol: phospholipid ratio (C: P). m, Eight-week-old male Asgr1−/− and their WT male littermates were randomly grouped (n = 8 per group) and fed a high fat/high cholesterol/bile salts (HF/HC/BS) diet for 4 weeks. The ratio of cholic acid (CA) to muricholic acid (MCA). n, Immunoblotting analysis of liver tissue lysates (n = 6 per group). o, Huh7 cells and the Huh7 cells deficient in ASGR1 (ASGR1 KO) were refreshed with DMEM containing 10% FBS or cholesterol depletion medium (DMEM containing 5% LPDS, 1 μM lovastatin and 10 μM Mevalonate) for 16 h. Then the cells were treated with the indicated concentrations of 25-hydroxycholesterol (25-HC) in cholesterol depletion medium for 5 h. p, Huh7 cells and the Huh7 cells stably expressed ASGR1 (ASGR1-OE) were treated as described in (o). q, Huh7 cells were transfected with scrambled siRNA (siControl) or siRNAs targeting ASGR1 or ASGR2. Fifty-six hours after transfection, cells were switched to DMEM containing 10% FBS or cholesterol depletion medium. After 16 h, cells were harvested, lysed and subjected to immunoblotting. r, Huh7 cells were transfected with indicated plasmids. Thirty-two hours after transfection, cells were switched to DMEM containing 10% FBS or cholesterol depletion medium. After 16 h, cells were harvested, lysed, and subjected to immunoblotting. s, Silencing ASGR1 did not influence Dil-LDL uptake. Huh7 cells were transfected with siRNA. After 72 h, cells were incubated in cholesterol depletion medium at 4 °C for 30 min and then subjected to the fresh cholesterol depletion medium containing 10 μg/ml Dil-LDL at 4 °C for 1 h. After PBS wash twice, cells were switched to 37 °C for the indicated time points. Scale bar 20 μm. t, Quantification of the internalized Dil-LDL in (s). The internalized Dil-LDL in WT cells at 0 h is defined as 0 (n = 45 cells). u, Silencing ASGR1 did not change the PCSK9-induced LDLR degradation. Huh7 cells were transfected with indicated siRNA. The cells were incubated in DMEM containing 10% FBS or cholesterol depletion medium for 12 h. The cells were treated with indicated amounts of PCSK9 protein for additional 4 h. v, The LDL clearance assay. The 10-week-old male WT and Asgr1−/− mice (n = 4 per group) were fed an HF/HC/BS diet for 2 days. Before measuring the uptake rate, the mice were fasted for 17 h and refed for 6 h. Then the mice were intravenously injected with the 3H-cholesteryl oleate labelled human LDL (5× 105 dpm/mouse, 200 μl). The blood samples were collected at indicated time points. The radioactivity at 30 s post injection as the starting point. w–x, VLDL secretion assay. The 10-week-old male WT and Asgr1−/− mice (n = 4 per group) were fed an HF/HC/BS diet for 2 days. The mice were fasted for 4 h and intraperitoneal (i. p.) injected Poloxamer 407 (1 mg/kg in PBS). Blood samples were collected at indicated time points. Total cholesterol (w) and triglyceride (x) were measured. All values are presented as mean ± SEM. P values were calculated by One-way ANOVA followed by Tukey’s test (el), or unpaired two-tailed Student’s t-test (m), or two-way ANOVA followed by Šídák's multiple comparisons test (t, vx). Experiments in a, c, d, os, u, were performed as indicated twice with similar results.

Source data

Extended Data Fig. 2 Characterization of female Asgr1−/− mice.

ao, Eight-week-old female Asgr1−/− mice and their WT (Asgr1+/+) female littermates were randomly grouped (n = 6 per group) and allowed ad libitum access to water and an HF/HC/BS diet for 5 weeks. a, Immunoblotting analysis of liver tissue lysates (n = 6 mice per group). b, The mRNA levels of the genes in the liver were determined by RT-qPCR. c, Body weight. d, Liver-to-body-weight ratio. e, Serum TC. f, Serum TG. g, TC in liver. h, TG in liver. i, serum ALT. j, Serum AST. k, Volume of bile in gallbladder. l, Biliary cholesterol concentration. m, Total biliary cholesterol. n, Biliary BAs concentration. o, Total BAs. pz, The same mice as shown in Fig. 2 (n = 6 per group) p, Relative amounts of mRNAs in livers analysed by RT-qPCR. q, Liver-to-body weight ratio. r, H&E and Oil Red O stains of the liver sections. Scale bars represent 50 μm. s, Body weight. t, Food intake. u, Blood glucose. v, Serum ALT. w, Serum AST. x, Biliary cholesterol concentration. y, Biliary BAs concentration. z, Volume of bile in gallbladder. All values are presented as mean ± SEM. P values were calculated by unpaired two-tailed Student’s t-test (bq, sz).

Source data

Extended Data Fig. 3 Mechanisms of ASGR1 regulating LXRα degradation.

a, Huh7 cells were transfected with indicated plasmids. Forty-three hours after transfection, cells were switched to medium containing 10 μM MG132. b, Huh7 cells and the Huh7 cells stably expressing ASGR1 (ASGR1 OE-A and -B) were incubated at 37 °C for 43 h. Then cells were incubated with 10 μM MG132 for 5 h. The lysates were subjected to immunoprecipitation with anti-LXRα coupled agarose to pull down LXRα. Input and pellet fractions were immunoblotted with anti-ubiquitin, anti-LXRα and anti-ASGR1 antibodies. c, Huh7 cells and the Huh7 cells deficient in ASGR1 (ASGR1 KO-A and -B) were incubated at 37 °C for 43 h. Then cells were treated as described in b. d, Huh7 cells were transfected with scrambled (control) or siRNA targeting BRCA1 and BARD1 (siBA1+BD1). After 12 h, cells were transfected with the indicated plasmids. The cells were harvested for immunoblotting after 48 h. e, Immunoblotting analysis of WT and ASGR1 knockout Huh7 cells. f, Huh7 cells were transfected with siRNA. After 72 h, cells were subjected to immunoblotting. g, Immunoblotting analysis of Huh7 cells transfected with the indicated plasmids. h, Huh7 cells and the Huh7 cells deficient in ASGR1 (ASGR1 KO-A and -B) were incubated at 37 °C for 48 h. Then cells were harvested for immunoblotting. i, Huh7 cells and the Huh7 cells stably expressing ASGR1 (ASGR1 OE-A and -B) were incubated at 37 °C for 48 h. j, The Ki-67 and proliferating cell nuclear antigen (PCNA) stain of liver sections from male WT and Asgr1−/− mice by immunohistochemistry. The male p53−/−&Ptch1+/− medulloblastoma samples were used as positive control for staining. Representative images are shown. Scale bars, 50 μm. k, Huh7 cells and the ASGR1 KO-A and -B cells were incubated at 37 °C for 48 h. Then cells were harvested for immunoblotting. l, Huh7 cells and the ASGR1 OE-A and -B cells were incubated at 37 °C for 48 h. Then cells were harvested for immunoblotting. m, The schematic illustration of the mouse ASGR1-ligand complex. ASGR1 has a single transmembrane domain. The 1–39 a.a. is located in cytosol. The 40–60 a.a. is the transmembrane domain. The stalk domain is 61–140 a.a. The 141–284 a.a. acts as the carbohydrates recognition domain (CRD). The D239, D241, W243, E252, N264 and D265 residues in mouse ASGR1 are essential for ligand binding. n, Huh7 cells were transfected with indicated plasmids. After 8 h, cells were switched to DMEM containing 10% FBS or not. After 40 h, cells were harvested and analysed with immunoblotting. o, Huh7 cells were transfected with the indicated plasmids. After 35 h, cells were incubated in MEM for 6 h. Then the cells were incubated in MEM containing different concentrations of asialofetuin A for 8 h. p, Huh7 cells were transfected and incubated in MEM containing asialoorosomucoid. q, Huh7 cells were transfected with indicated plasmids. The 6A mutation means all of the 6 residues (Q239, D241, W243, E252, N264 and D265) were changed to alanines. After transfection for 48 h, cells were harvested and analysed with immunoblotting. r, The ASGR1(WT) and ASGR1(6A) plasmids were transfected into Huh7 cells. After 36 h, cells were treated as described in o. s, Huh7 cells were transfected with scrambled (siControl) or CHC-targeting siRNA (siCHC-1 and siCHC-2). After 12 h, cells were transfected with the indicated plasmids. After 48 h, cells were subjected to immunoblotting. t, Huh7 cells were transfected with plasmids as indicated. After 46 h, cells were switched to the medium with or without 20 nM Bafilomycin A1. After 2 h, cells were lysed and subjected to immunoblotting. u, Huh7 cells were treated as described in Fig. 3g with different concentration of dorsomorphin (0, 3 and 10 μM) for 5 h. v, Huh7 cells were transfected with plasmids as indicated. After 43 h, cells were treated with 100 μM A769662 or not. After 5 h, cells were lysed and subjected to immunoblotting. Experiments in ai, kl, nv were performed as indicated twice with similar results.

Extended Data Fig. 4 ASGR1-mediated glycoprotein uptake increases lysosomal a.a. and actives mTORC1.

a, Assessment of immunopurified lysosomes. Upper panel illustrates TMEM192-3×FLAG as the bait protein for rapid isolation of lysosomes. 0.5% of cell lysate and 10% of immunoprecipitated samples were analysed in lane 1-2 or 3-4, respectively. The mock-transduced Huh7 cells were used as a control. b, Fold changes of lysosomal a.a.s in WT or ASGR1-KO Huh7 cells with or without asialofetuin A treatment (30 μg/mL, 2 h). Data were normalized to control group (WT cell without asialofetuin A treatment). n = 3 biological repeats were shown and plotted. c, The lysosomal a.a. transporter SLC38A9 is required for asialofetuin A-mediated mTORC1 activation. WT or SLC38A9-KO Huh7 cells were starved in EBSS with 5 mM glucose for 3 h. Followed by treatment with saline (−), 30 μg/mL (+) or 100 μg/mL (++) asialofetuin A for 3 h. Cells were harvested for immunoblotting. d, Rescue experiments. WT and the mutant SLC38A9 (with an N-terminal HA tag) were expressed in SLC38A9-KO Huh7 cells by lentiviral transduction. Cells were then treated as in c with 30 μg/mL asialofetuin A. e, Asialofetuin A promotes lysosomal translocation of mTOR. HepG2 cells grown on coverslips were starved in EBSS with 5 mM glucose for 3 h and supplied with 100 μg/mL asialofetuin A for 3 h. Cells were then fixed, stained with indicated antibodies and examined under a laser confocal microscope. f, Colocalization rate was quantified by the built-in function in Leica LAS X software. n = 45 cells per group. Box denotes median, upper and lower quartiles. g, Comparison of asialofetuin A and albumin as the a.a. source to stimulate S6K phosphorylation. The experiments were performed as in c. Experiments in c, d, g were performed as indicated twice with similar results. All values are presented as mean ± SEM. P values were calculated by unpaired two-tailed Student’s t-test (b, f).

Source data

Extended Data Fig. 5 Injection of asialofetuin A activates hepatic mTORC1, inhibits AMPK and increases lipid levels.

Eight-week-old Asgr1 knockout male mice (Asgr1−/−) and WT male littermates (Asgr1+/+) were randomly grouped (n = 8 for WT and 6 for Asgr1−/−) and allowed ad libitum access to water and an HF/HC/BS diet for 2 days. Then the mice were i.p. injected with asialofetuin A (0.75 mg/g) for 9 h. a, Immunoblotting analysis of liver samples. b, The mRNA levels of the genes in the liver were determined by RT-qPCR. Cyclophilin was used as an internal control. c, Body weight. d, Liver-to-body-weight ratio. e, Blood glucose. f, Serum ALT. g, Serum AST. h, Serum TC. i, Serum TG. j, TC in liver. k, TG in liver. l, Biliary cholesterol concentration. m, Biliary phospholipid. n, Ratio of biliary cholesterol to phospholipid. o, Biliary BAs concentration. p, Volume of bile in gallbladder. q, Total biliary BAs. r, Total biliary cholesterol. s, The representative image of the bile. t, The H&E stain (upper) and Oil Red O stain (down) of the liver sections of mice. Representative images are shown. Scale bars represent 50 μm. All values are presented as mean ± SEM. P values were calculated by unpaired two-tailed Student’s t-test (b–r).

Source data

Extended Data Fig. 6 Rapamycin inhibits mTORC1, activates AMPK, increases cholesterol excretion and lowers lipid levels.

The 10-week-old male Asgr1 knockout mice (Asgr1−/−) and WT male littermates (Asgr1+/+) were randomly grouped (n = 6 per group) and allowed ad libitum access to water and an HF/HC/BS diet. The mice were intragastrically administered with vehicle or 5 mg/kg/d rapamycin daily for 2 weeks. a, Immunoblotting analysis of liver samples (n = 3 randomly chosen mice per group). b, The mRNA levels of the genes in the liver were determined by RT-qPCR. Cyclophilin was used as an internal control. c, Body weight. d, Liver-to-body-weight ratio. e, Blood glucose. f, Food intake. g, Serum TC. h, Serum TG. i, TC in liver. j, TG in liver. k, Serum ALT. l, Serum AST. m, Biliary cholesterol concentration. n, Biliary phospholipid. o, Ratio of biliary cholesterol to phospholipid. p, Biliary BAs concentration. q, Volume of bile in gallbladder. r, Total biliary cholesterol. s, Total biliary BAs. t, Faecal cholesterol. u, The representative image of the bile. v, The H&E staining (upper) and Oil Red O (ORO) staining (down) of the liver sections of mice. Representative images are shown. Scale bars represent 50 μm. All values are presented as mean ± SEM. P values were calculated by unpaired two-tailed Student’s t-test (b–t).

Source data

Extended Data Fig. 7 Knock-down of hepatic AMPK decreases cholesterol excretion and increases lipid levels.

The 8-week-old male Asgr1 knockout mice (Asgr1−/−) and male WT littermates (Asgr1+/+) were randomly grouped and injected with 1×1010 viral genome (v.g.) of AAV2/8-shAMPKα1, AAV2/8-shAMPKα2 or AAV2/8-shControl (Ctrl) (from left to right, n = 8, 8, 8, 6, 5 per group). After fed an HF/HC/BS diet or normal chow diet for 4 weeks, the mice were analysed. a, Immunoblotting analysis of liver samples. Two mice per group were randomly chosen for analysis. b, Gene expression in liver measured by RT-qPCR. c, Body weight. d, Liver/body weight. e, Blood glucose. f, Food intake. g, Serum TC. h, Serum TG. i, TC in liver. j, TG in liver. k, Serum ALT. l, Serum AST. m, Biliary cholesterol concentration. n, Biliary phospholipid. o, Ratio of biliary cholesterol concentration to biliary phospholipid. p, Biliary BAs concentration. q, Volume of bile in gallbladder. r, Total biliary cholesterol. s, Total biliary BAs. t, Faecal cholesterol. u, Liver sections were stained with H&E (upper) or Oil Red O (ORO) (down). Representative images are shown. Scale bars represent 50 μm. All values are presented as mean ± SEM. P values were calculated by unpaired two-tailed Student’s t-test (bt).

Source data

Extended Data Fig. 8 Coadministration of A769662 and GW3965 recapitulates the phenotypes of Asgr1−/− mice.

The 8-week-old male Asgr1 knockout mice (Asgr1−/−) and WT male littermates (Asgr1+/+) were randomly grouped (n = 6 per group) and allowed ad libitum access to water and an HF/HC/BS diet. The mice were intragastrically administered with vehicle or 20 mg/kg/d GW3965 or intraperitoneally injected with A769662 30 mg/kg twice daily for 2 weeks. a, Immunoblotting analysis of liver samples. Three mice per group were randomly chosen for analysis. b, Gene expression in liver measured by RT-qPCR. c, Serum TC. d, Serum TG. e, TC in liver. f, TG in liver. g, Serum AST. h, Serum ALT. i, Biliary cholesterol concentration. j, Biliary phospholipid. k, Ratio of biliary cholesterol to biliary phospholipid. l, Biliary BAs concentration. m, Volume of bile in gallbladder. n, Total biliary cholesterol. o, Total biliary BAs. p, Faecal cholesterol. q, Liver sections were stained with H&E (upper) or Oil Red O (ORO) (down). Representative images are shown. Scale bars represent 50 μm. All values are presented as mean ± SEM. P values were calculated by unpaired two-tailed Student’s t-test (bp).

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Extended Data Fig. 9 Silencing of Asgr1 increases cholesterol excretion and prevents atherosclerosis.

Eight-week-old Balb/c male mice were injected with 1×1011 viral genome (v.g.) of AAV2/8-shASGR1 or AAV2/8-shControl (Ctrl) (n = 8 per group) and fed an HF/HC/BS diet for 4 weeks. a, Immunoblotting analysis of liver samples. Six mice per group were randomly chosen for analysis. b, Gene expression in liver measured by RT-qPCR. c, Serum TC. d, Serum TG. e, Liver TC. f, Liver TG. g, Biliary cholesterol concentration. h, Biliary BAs concentration. i, Volume of bile in gallbladder. j, Total biliary cholesterol. k, Total biliary BAs. l, Body weight. m, Ratio of liver to body weight. n, Serum ALT. o, Serum AST. p, q, Liver sections were stained with H&E (p) or Oil Red O (q). Representative images are shown. Scale bars represent 50 μm. r–z, Nine-week-old male Ldlr−/− mice were injected with 1×1011 viral genome (v.g.) of AAV2/8-shAsgr1 or AAV2/8-shControl (Ctrl) (n = 8, 8, 8, 10, 16). The age matched WT male C57BL/6J mice were used as control. The mice were fed chow diet or an HF/HC/BS diet for 8 weeks. r, Blood TC. s, Blood TG. t, Serum ALT. u, Serum AST. v, Body weight. w, Liver-to-body-weight ratio. x, Representative images of aortas tree staining by Sudan IV. y, Quantifications of atherosclerotic lesions shown in (x). z, Liver sections were stained with H&E (upper) or Oil Red O (down). Representative images are shown. Scale bars represent 50 μm. All values are presented as mean ± SEM. P values were calculated by unpaired two-tailed Student’s t-test (bo, rw, y).

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Extended Data Fig. 10 Preparing anti-ASGR1 neutralizing antibody.

a, Schematic representation of procedure to develop monoclonal anti-ASGR1 neutralizing antibody. b, The representative coomassie blue staining of the ASGR1 protein purified from HEK293T cells. c, Primary hepatocytes were isolated from 8-week-old male C57B/L6 mice. The cells were incubated with different clones of anti-ASGR1 antibodies for 72 h. d, Primary hepatocytes were isolated from 8-week-old male C57B/L6 mice. Cells were incubated with the 4B9 IgG for 72 h. Then cells were harvested and subjected to RNA extraction and RT-qPCR analysis (n = 3 biological repeats). e, Huh7 cells were transfected with indicated plasmids. Forty-eight hours after transfection, cells lysates were analysed by immunoblotting with 4B9-IgG, anti-Flag, anti-ASGR1 or anti-Actin antibodies. fl, The mice were as same as those in Fig. 4a (n = 6 per group) f, Relative amounts of mRNAs in livers. g, Body weight. h, Food intake. i, Liver-to-body-weight ratio. j. Blood glucose. k. Serum ALT. l, Serum AST. All values are presented as mean ± SEM. P values were calculated by unpaired two-tailed Student’s t-test (d, fl). Experiments in b, c, e were performed as indicated twice with similar results.

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Extended Data Fig. 11 Characterization of the mice receiving anti-ASGR1 (4B9-IgG) and atorvastatin.

The mice were as same as those in Fig. 5a–l (n = 6 per group). a, b, Gene expression in liver analysed by RT-qPCR. c, Body weight. d, Food intake. e, Liver-to-body-weight ratio. f, Blood glucose. g, Serum ALT. h, Serum AST. i, Biliary cholesterol concentration. j, Biliary BAs concentration. k, l, H&E (k) or Oil Red O staining (l) of the liver sections. Representative images are shown. Scale bars represent 50 μm. All values are presented as mean ± SEM. P values were calculated by unpaired two-tailed Student’s t-test (aj).

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Extended Data Fig. 12 Characterization of the mice receiving anti-ASGR1(4B9-IgG) and ezetimibe.

The mice were as same as those in Fig. 5i–p (n = 6 per group). EZ: ezetimibe. a, b, Gene expression in liver analysed by RT-qPCR. c, Body weight. d, Food intake. e, Liver-to-body-weight ratio. f, Blood glucose. g, Serum ALT. h, Serum AST. i, Biliary cholesterol concentration. j, Biliary BAs concentration. k, H&E staining of the liver sections of mice. Representative images are shown. Scale bars represent 50 μm. All values are presented as mean ± SEM. P values were calculated by unpaired two-tailed Student’s t-test (aj).

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Supplementary information

Supplementary Information

Supplementary Fig. 1, the raw images of the immunoblotting experiments, Supplementary Table 1, which lists total endogenous LXR ligands in Asgr1+/+ and Asgr1−/− mice livers, and Supplementary Table 2, containing the nucleotide sequences of the qPCR primers.

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Wang, JQ., Li, LL., Hu, A. et al. Inhibition of ASGR1 decreases lipid levels by promoting cholesterol excretion. Nature 608, 413–420 (2022). https://doi.org/10.1038/s41586-022-05006-3

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