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Hepatic nonvesicular cholesterol transport is critical for systemic lipid homeostasis

Nature Metabolism volume 5, pages 165–181 (2023)Cite this article

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Abstract

In cell models, changes in the ‘accessible’ pool of plasma membrane (PM) cholesterol are linked with the regulation of endoplasmic reticulum sterol synthesis and metabolism by the Aster family of nonvesicular transporters; however, the relevance of such nonvesicular transport mechanisms for lipid homeostasis in vivo has not been defined. Here we reveal two physiological contexts that generate accessible PM cholesterol and engage the Aster pathway in the liver: fasting and reverse cholesterol transport. During fasting, adipose-tissue-derived fatty acids activate hepatocyte sphingomyelinase to liberate sequestered PM cholesterol. Aster-dependent cholesterol transport during fasting facilitates cholesteryl ester formation, cholesterol movement into bile and very low-density lipoprotein production. During reverse cholesterol transport, high-density lipoprotein delivers excess cholesterol to the hepatocyte PM through scavenger receptor class B member 1. Loss of hepatic Asters impairs cholesterol movement into feces, raises plasma cholesterol levels and causes cholesterol accumulation in peripheral tissues. These results reveal fundamental mechanisms by which Aster cholesterol flux contributes to hepatic and systemic lipid homeostasis.

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Fig. 1: Asters transport accessible PM cholesterol in hepatocytes.
Fig. 2: Asters facilitate hepatocyte PM to ER cholesterol transport during fasting.
Fig. 3: Adipose-derived fatty acids liberate sequestered PM cholesterol during fasting.
Fig. 4: Fasting induces Smpd3 expression to regulate cholesterol metabolism and VLDL production.
Fig. 5: Aster-C is a direct target gene of FXR in mouse liver.
Fig. 6: Hepatic Aster deficiency impairs transport of lipoprotein-derived cholesterol and RCT.
Fig. 7: Asters are required for storage of dietary cholesterol as CEs in hepatocytes.
Fig. 8: Hepatic Aster deficiency disrupts systemic cholesterol homeostasis.

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Data availability

The RNA-seq dataset generated for this paper has been deposited to the National Center for Biotechnology Information and is available at accession no. GSE206278. For RNA-seq analysis, trimmed FASTQ files were aligned to GRCm38/mm10 using STAR40. Source data are provided with this paper.

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Acknowledgements

We thank all members of the Tontonoz, Tarling-Vallim, Edwards, Villanueva, Young and Bensinger laboratories at UCLA for useful advice and discussions and for sharing reagents and resources. We thank K. Williams, G. Su and staff at UCLA Lipidomics core for the lipidomics analysis. Confocal microscopy was performed at the California NanoSystems Institute of Advanced Light Microscopy/Spectroscopy Facility. We thank the Vector Core of the University of Michigan for AAV packaging. We thank J. Smothers and A. Radhakrishnan for the helpful suggestions about the ALOD4 protein purification. This work was supported by NIH grant R01 DK126779 and Fondation Leducq Transatlantic Network of Excellence (19CVD04). X.X. was supported by AHA Postdoctoral Fellowship (18POST34030388). J.P.K. is supported by AHA Postdoctoral Fellowship (903306). A.F. was funded by Ermenegildo Zegna Founder’s Scholarship (2017) and by American Diabetes Association postdoctoral fellowship (1-19-PDF-043-RA). Y.G. is supported by Damon Runyon Cancer Research Foundation and Mark Foundation postdoctoral fellowship (DRG2424-21). R.T.N. was supported by a T32GM008042 grant to the UCLA-Caltech Medical Scientist Training Program. A.N. was supported by the NIDDK of the National Institutes of Health under Award Number T32DK007180.

Author information

Author notes

  1. These authors contributed equally: Xu Xiao, John Paul Kennelly.

Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    Xu Xiao, John Paul Kennelly, Alessandra Ferrari, Yajing Gao, Kevin Qian, Jaspreet Sandhu, Alexander Nguyen, Rohith T. Nagari & Peter Tontonoz

  2. Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA, USA

    Xu Xiao, John Paul Kennelly, Alessandra Ferrari, Yajing Gao, Kevin Qian, Rohith T. Nagari, Thomas Q. de Aguiar Vallim & Peter Tontonoz

  3. Department of Medicine, Division of Cardiology, University of California, Los Angeles, Los Angeles, CA, USA

    Bethan L. Clifford, Kelsey E. Jarrett, Madelaine C. Brearley-Sholto & Thomas Q. de Aguiar Vallim

  4. Pediatric Gastroenterology, Hepatology and Nutrition, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    Emily Whang

  5. Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Jaspreet Sandhu & Thomas Q. de Aguiar Vallim

  6. Vatche and Tamar Manoukian Division of Digestive Diseases, Department of Medicine David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    Alexander Nguyen

  7. Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA, USA

    Min Sub Lee & Steven J. Bensinger

  8. Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA, USA

    Sicheng Zhang & Claudio J. Villanueva

  9. Department of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    Thomas A. Weston & Stephen G. Young

  10. Department of Human Genetics, University of California, Los Angeles, Los Angeles, CA, USA

    Stephen G. Young

  11. Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA

    Steven J. Bensinger

  12. Jonsson Comprehensive Cancer Center (JCCC), University of California, Los Angeles, Los Angeles, CA, USA

    Thomas Q. de Aguiar Vallim

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Contributions

Conceptualization was carried out by X.X., J.P.K. and P.T. Methodology was the responsibility of X.X., J.P.K., B.L.C., Y.G., K.Q., J.S., A.N., R.T.N. and M.S.L. Investigation was conducted by X.X., J.P.K., A.F., B.L.C., E.W., Y.G., K.Q., J.S., K.E.J, M.C.B., A.N., R.T.N., M.S.L., S.Z. and T.W. Writing was carried out by X.X., J.P.K. and P.T. Funding was acquired by P.T. Resources were the responsibility of S.G.Y., S.J.B., C.J.V., T.Q.d.A.V. and P.T. Supervision was carried out by P.T.

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Correspondence to Peter Tontonoz.

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Nature Metabolism thanks David Cohen Andrew Brown and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Isabella Samuelson, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Generation of Aster-C 3xHA KI and Asters hepatocyte specific KO mice.

a, Quantitative PCR of Asters expression in mouse liver (n = 6). b, Evaluation of 3xHA-Aster-C KI mice. Genotyping results (upper) and anti-HA tag immunoblot (bottom) for 3xHA-Aster-C KI mice. The genotyping result is representative of at least 50 similar results. The anti-HA-Aster-C western blot was repeated independently in Fig. 5e. c, Strategy for generating Gramd1a (Aster-A) and Gramd1c (Aster-C) Flox/Flox (F/F) mice. Coding exons are depicted in black. Exons that correspond to the GRAM domain, ASTER domain, and transmembrane (TM) domain are depicted in green, blue, and red respectively. Scale bar represents 1 kb. d, Genotyping results for L-A KO, L-C KO and L-A/C KO mice. The genotyping results are representative of at least 100 similar results per condition. (e, f and g) Expression levels of the indicated genes in liver from F/F control and L-A KO mice (e, n = 8/11); L-C KO mice (f, n = 5/6); L-A/C KO mice (g, n = 12/6). h, Gramd1a, Gramd1b and Gramd1c expression level in the testis of F/F control and L-A/C KO mice (n = 12/6). All data are presented as mean ± SEM. P values were determined by two-sided Student’s t-test with Benjamini, Krieger and Yekutieli correction for multiple comparisons (e, f, g and h).

Source data

Extended Data Fig. 2 Fasting stimulates hepatic PM–ER cholesterol transport.

a, Gross appearance of livers from mice fasted for 4- or 16-h. b, Hepatic triglycerides in mice fasted for 4- or 16-h (n = 3/3). c, Hepatic CE in mice fasted for 4- or 16-h (n = 3/3). d, Significantly upregulated pathways in the livers of mice fasted for 16-h compared to 4-h according to pathway analysis of RNA Sequencing data. e, Significantly downregulated pathways in the livers of mice fasted for 16-h compared to 4-h according to pathway analysis of RNA Sequencing data. f, Hepatic mRNA expression of SREBP-2 pathway targets from the livers of mice fasted for 4- or 16-h (n = 3/3). g, Quality control of plasma membrane isolation from the mouse liver. Cadherin: PM maker; Calnexin: ER maker; ATGL: lipid droplet (LD) maker; Actin: cytoskeleton maker. This analysis was completed once as the organelle isolation method has been previously validated (further method details are in the Methods section). h, Free cholesterol analysis from purified PMs of wild-type mice (n = 3/3). i, TLC analysis of free cholesterol (FC), sphingomyelin (SM) and phosphatidylcholine (PC) from the livers of mice fasted for either 4 or 16 h. j, PM total lipids as measured by mass-spec from livers of F/F control and L-A/C KO after 4- and 16-h fasting (n = 3/3). All data are presented as mean ± SEM. P values were determined by two-sided Student’s t-test (b, c and h), or two-sided Student’s t-test with Benjamini, Krieger and Yekutieli correction (f).

Source data

Extended Data Fig. 3 Aster-mediated cholesterol transport determines the size of the accessible PM cholesterol pool.

a, PM total lipids from mouse liver after 4- and 16-h fasting as determined by mass spec (n = 3/3). b, Immunoblot analysis of ALOD4 binding in F/F control and L-A/C KO primary hepatocytes after treatment with vehicle, nSmase (100 mU/ml) and GW4869 (10 μM) for 1-h. Actin was used as a loading control. c, Expression levels of the indicated genes in primary hepatocyte that had been cultured in Maintenance medium and Ro 48-8071 (1μM) overnight before being treated with or without oleic acid (30μM) for 6-h (n = 3). d, Immunoblot analysis of ALOD4 binding in primary hepatocytes after treatment with vehicle or indicated concentration of glucagon, acetoacetate (Ac-Ac), beta-hydroxybutyrate (BHB) or MβCD-cholesterol (35 µM) for 1-h. Calnexin was used as a loading control. e, Immunoblot analysis of ALOD4 binding in primary hepatocytes after treatment with vehicle or insulin (300 nM), glucose (50 mM) and both for 1-h. Calnexin was used as a loading control. All data are presented as mean ± SEM.

Source data

Extended Data Fig. 4 Asters transport lipoprotein-derived cholesterol from the PM to ER in hepatocytes.

a. Immunoblot analysis of ALOD4 binding in control or Sr-b1 knockdown HAECs after treatment with vehicle or HDL (400 ug/ml) for 1-h. Calnexin was used as a sample processing control. b, [14C] counts in liver unesterified cholesterol of mice from Fig. 6c (n = 10/6). c, mRNA expression levels of the indicated genes in the livers of mice from Fig. 6c (n = 10/6). d, [14C] counts in unesterified cholesterol in the livers of mice from Fig. 6g (n = 7/9). e, mRNA expression levels of the indicated genes in the livers of mice from Fig. 6g (n = 7/9). f, The rate of [14C] lipoprotein clearance from the circulation (n = 6/5), related to Figs. 6j–6m. Data are represented as mean ± SEM with individual animals noted as dots. *p < 0.05. g. Fecal cholesterol analysis of F/F and L-A/C KO mice fed a cholesterol-free diet for 48 hours (n = 7/7). h, Growth curves for F/F and L-A/C KO mice fed a Western diet from 8 weeks of age (n = 8/12). Masses are shown as mean ± SEM. i, Whole-body cholesterol content from F/F control and L-A/C KO mice on chow diet at 15 weeks old (n = 5/7). All data are presented as mean ± SEM. P values were determined by two-sided Student’s t-test (b, d, g and i), two-sided Student’s t-test with Benjamini, Krieger and Yekutieli correction (c and e), or two-way ANOVA with Sidak’s correction for multiple comparisons (h).

Source data

Extended Data Fig. 5 Model for the role of hepatic Asters in liver and systemic cholesterol homeostasis.

In normal physiology (left side of schematic), fasting stimulates fatty acid release from adipose tissue to promote hepatic sphingomyelinase activity, which liberates sequestered cholesterol in the hepatocyte PM. Aster proteins recognize this newly accessible cholesterol and transport it to the ER for CE formation, suppression of SREBP-2 pathway targets, bile acid synthesis and VLDL production. Hepatic Asters are also induced by FXR and function in the RCT pathway by moving HDL-derived cholesterol (and LDL-derived cholesterol) within hepatocytes. Loss of hepatic Aster function (right side of the schematic) impairs CE formation and VLDL output during fasting. Loss of Asters in the liver also decreases the appearance of HDL-derived cholesterol in bile and feces, raises plasma cholesterol levels (due to enhanced liver cholesterol efflux), and causes peripheral cholesterol accumulation (for example, adrenal gland, brown adipose tissue).

Extended Data Fig. 6 Western blot quantifications.

a, Western blot quantifications of ALOD4 binding from Fig. 1c. Normalized to loading control Actin (n = 1). b, Western blot quantifications of indicated proteins from Fig. 1c. Normalized to sample processing control calnexin (n = 5). c, Western blot quantifications of indicated proteins from Fig. 2g. Normalized to sample processing controls calnexin or lamin a/c (n = 4). d, Western blot quantifications of ALOD4 binding from Fig. 3c. Normalized to loading control actin (n = 1). e and f, Western blot quantifications of SMPD3 from Fig. 4b and Fig. 4c. Normalized to loading control actin (n = 3). g, Western blot quantifications of indicated proteins from Fig. 4i. Equal amounts of protein was loaded for each line (n = 4). h, Western blot quantifications of indicated proteins from Fig. 5b. Normalized to sample processing control calnexin (n = 4). i, Western blot quantifications of indicated proteins from Fig. 5e. Normalized to actin which served as a loading control for SRB1 and a sample processing control for Aster-C (n = 1). j, Western blot quantifications of ABCA1 from Fig. 8c. Normalized to sample processing control calnexin (n = 5). k, l and m, Western blot quantifications of ALOD4 binding from extended data Figs. 3b, 3d and 3e. Normalized to loading control actin or calnexin (n = 1). n, Western blot quantifications of ALOD4 binding and Sr-B1 from Fig. 4a. Normalized to calnexin which served as a loading control for ALOD4 and a sample processing control for SRB1 (n = 1). Data are presented as mean ± SEM. P values were determined by two-sided Student’s t-test (b, c, e, f and h).

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Xiao, X., Kennelly, J.P., Ferrari, A. et al. Hepatic nonvesicular cholesterol transport is critical for systemic lipid homeostasis. Nat Metab 5, 165–181 (2023). https://doi.org/10.1038/s42255-022-00722-6

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