Abstract
Alcohol-associated liver disease (ALD) in its earliest form is evidenced as hepatic steatosis which may progress to liver cirrhosis. The mechanisms behind this are poorly understood and therapeutics limited. Liver is a specialized organ exhibiting heterogeneity along the porto-central axis. Periportal preponderance of lipid droplet accumulation was noted in human ALD livers compared to other causes of hepatic steatosis. Using single cell multiomics, we studied transcriptional mechanisms across the hepatic lobule that could account for zonation of lipid droplets in a murine ALD model. Alcohol led to periportal zonation of lipogenesis-associated genes in mice, including Hsd17b13 and Fasn. Chromatin landscape studies demonstrated zonation of master transcription factors that led to these changes in the transcriptome. We utilized these data to provide novel insight into zone-specific HNF4α and PPARα regulation of HSD17B13. We conclude novel mechanisms underlying ALD leading to spatially distinct establishment of hepatic steatosis and provide insight into disease pathogenesis.
Data availability
The scRNA-seq and scATAC-seq data generated in this publication are available on the GEO database [https://www.ncbi.nlm.nih.gov/geo/] under accession (GSE199064, reviewer token: mxgtksespxetxuh). The RNA-seq data are available on the GEO database (GSE155926, GSE155907) and the dbGAP of the National Center for Biotechnology Information, US National Library of Medicine, Bethesda, MD (accession number phs001807.v1.p1). The ChIP-seq data are available on the GEO database (GSE166564). JASPAR database is accessible via weblink [http://jaspar.genereg.net/].
Code availability
Analysis utilized publicly available and previously published code.
REFERENCES
Sehrawat, T. S., Liu, M. & Shah, V. H. The knowns and unknowns of treatment for alcoholic hepatitis. Lancet Gastroenterol. Hepatol. 5, 494–506 (2020).
Argemi, J. et al. Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis. Nat. Commun. 10, 3126 (2019).
Arab, J. P. et al. Hepatic stellate cell activation promotes alcohol-induced steatohepatitis through Igfbp3 and SerpinA12. J. Hepatol. 73, 149–160 (2020).
Arab, J. P. et al. An open-label, dose-escalation study to assess the safety and efficacy of IL-22 agonist F-652 in patients with alcohol-associated hepatitis. Hepatology 72, 441–453 (2020).
Cao, S., Liu, M., Sehrawat, T. S. & Shah, V. H. Regulation and functional roles of chemokines in liver diseases. Nat. Rev. Gastroenterol. Hepatol. 18, 630–647 (2021).
Liu, M. et al. Super enhancer regulation of cytokine-induced chemokine production in alcoholic hepatitis. Nat. Commun. 12, 4560 (2021).
Ben-Moshe, S. & Itzkovitz, S. Spatial heterogeneity in the mammalian liver. Nat. Rev. Gastroenterol. Hepatol. 16, 395–410 (2019).
Halpern, K. B. et al. Single-cell spatial reconstruction reveals global division of labour in the mammalian liver. Nature 542, 352–356 (2017).
Itzkovitz, S. & van Oudenaarden, A. Validating transcripts with probes and imaging technology. Nat. Methods 8, S12-19 (2011).
Weichselbaum, L. et al. Epigenetic basis for monocyte dysfunction in patients with severe alcoholic hepatitis. J. Hepatol. 73, 303–314 (2020).
Zeybel, M. et al. Differential DNA methylation of genes involved in fibrosis progression in non-alcoholic fatty liver disease and alcoholic liver disease. Clin. Epigenet. 7, 25–25 (2015).
Sureshchandra, S. et al. Chronic heavy drinking drives distinct transcriptional and epigenetic changes in splenic macrophages. EBioMed. 43, 594–606 (2019).
Page, A. et al. Alcohol directly stimulates epigenetic modifications in hepatic stellate cells. J. Hepatol. 62, 388–397 (2015).
Rinella, M. E. et al. A multisociety delphi consensus statement on new fatty liver disease nomenclature. Hepatology 78, 1966–1986 (2023).
Guilliams, M. et al. Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches. Cell 185, 379-396.e338 (2022).
Ben-Moshe, S. et al. Spatial sorting enables comprehensive characterization of liver zonation. Nat. Metab. 1, 899–911 (2019).
Zhang, W., Sun, Q., Zhong, W., Sun, X. & Zhou, Z. Hepatic peroxisome proliferator-activated receptor gamma signaling contributes to alcohol-induced hepatic steatosis and inflammation in mice. Alcohol. Clin. Exp. Res. 40, 988–999 (2016).
Abul-Husn, N. S. et al. A protein-truncating HSD17B13 variant and protection from chronic liver disease. N. Engl. J. Med. 378, 1096–1106 (2018).
Elias, H. A re-examination of the structure of the mammalian liver; the hepatic lobule and its relation to the vascular and biliary systems. Am. J. Anat. 85, 379–456 (1949).
Lieber, C. S., Jones, D. P. & Decarli, L. M. Effects of prolonged ethanol intake: Production of fatty liver despite adequate diets. J. Clin. Invest. 44, 1009–1021 (1965).
Schott, M. B. et al. Lipid droplet size directs lipolysis and lipophagy catabolism in hepatocytes. J. Cell Biol. 218, 3320–3335 (2019).
Su, W. et al. Comparative proteomic study reveals 17β-HSD13 as a pathogenic protein in nonalcoholic fatty liver disease. Proc. Natl. Acad. Sci. U. S. A. 111, 11437–11442 (2014).
Wu, W. et al. Fat and carbohydrate in western diet contribute differently to hepatic lipid accumulation. Biochem. Biophys. Res. Commun. 461, 681–686 (2015).
Mashek, D. G. Hepatic lipid droplets: a balancing act between energy storage and metabolic dysfunction in NAFLD. Mol. Metab. 50, 101115 (2021).
Yu, J. & Li, P. The size matters: regulation of lipid storage by lipid droplet dynamics. Sci. China Life Sci. 60, 46–56 (2017).
Beckman, M. Great balls of fat. Science 311, 1232–1234 (2006).
Chalasani, N. et al. Relationship of steatosis grade and zonal location to histological features of steatohepatitis in adult patients with non-alcoholic fatty liver disease. J. Hepatol. 48, 829–834 (2008).
Ferri, F. et al. The propensity of the human liver to form large lipid droplets is associated with PNPLA3 polymorphism, reduced INSIG1 and NPC1L1 expression and increased fibrogenetic capacity. Int. J. Mol. Sci. 22, 6100 (2021).
Halpern, K. B. et al. Paired-cell sequencing enables spatial gene expression mapping of liver endothelial cells. Nat. Biotechnol. 36, 962–970 (2018).
Day, C. P. & James, O. F. W. Steatohepatitis: a tale of two “hits”?. Gastroenterology 114, 842–845 (1998).
Mooli, R. G. R. & Ramakrishnan, S. K. Liver steatosis is a driving factor of inflammation. Cell. Mol. Gastroenterol. Hepatol. 13, 1267–1270 (2022).
Gaul, S. et al. Hepatocyte pyroptosis and release of inflammasome particles induce stellate cell activation and liver fibrosis. J. Hepatol. 74, 156–167 (2021).
Petrasek, J. et al. IL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice. J. Clin. Invest. 122, 3476–3489 (2012).
Wree, A. et al. NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice. Hepatology 59, 898–910 (2014).
Casey, C. A. et al. Lipid droplet membrane proteome remodeling parallels ethanol-induced hepatic steatosis and its resolution. J. Lipid Res. 62, 100049 (2021).
Raeman, R. Rebuttal to: Liver steatosis is a driving factor of inflammation. Cell. Mol. Gastroenterol. Hepatol. 13, 1271–1272 (2022).
Brosch, M. et al. Epigenomic map of human liver reveals principles of zonated morphogenic and metabolic control. Nat. Commun. 9, 4150 (2018).
Islam, S. et al. Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Res. 21, 1160–1167 (2011).
Ramachandran, P. et al. Resolving the fibrotic niche of human liver cirrhosis at single-cell level. Nature 575, 512–518 (2019).
Dobie, R. et al. Single-cell transcriptomics uncovers zonation of function in the mesenchyme during liver fibrosis. Cell Rep. 29, 1832-1847.e1838 (2019).
Chen, T., Oh, S., Gregory, S., Shen, X. & Diehl, A. M. Single-cell omics analysis reveals functional diversification of hepatocytes during liver regeneration. JCI Insight 5, e141024 (2022).
Torkamani, A., Andersen, K. G., Steinhubl, S. R. & Topol, E. J. High-definition medicine. Cell 170, 828–843 (2017).
Segovia-Miranda, F. et al. Three-dimensional spatially resolved geometrical and functional models of human liver tissue reveal new aspects of NAFLD progression. Nat. Med. 25, 1885–1893 (2019).
Teutsch, H. F. The modular microarchitecture of human liver. Hepatology 42, 317–325 (2005).
Ma, Y. et al. 17-Beta hydroxysteroid dehydrogenase 13 is a hepatic retinol dehydrogenase associated with histological features of nonalcoholic fatty liver disease. Hepatology 69, 1504–1519 (2019).
Horiguchi, Y., Araki, M. & Motojima, K. 17beta-Hydroxysteroid dehydrogenase type 13 is a liver-specific lipid droplet-associated protein. Biochem. Biophys. Res. Commun. 370, 235–238 (2008).
Rotroff, D. M. et al. Genetic variants in HSD17B3, SMAD3, and IPO11 impact circulating lipids in response to fenofibrate in individuals with type 2 diabetes. Clin. Pharmacol. Ther. 103, 712–721 (2018).
Sahoo, S., Singh, D., Chakraborty, P. & Jolly, M. K. Emergent properties of the HNF4α-PPARγ network may drive consequent phenotypic plasticity in NAFLD. J. Clin. Med. 9, 870 (2020).
Ferré, P. & Foufelle, F. Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c. Diabetes Obes Metab 12(Suppl 2), 83–92 (2010).
Moslehi, A. & Hamidi-Zad, Z. Role of SREBPs in liver diseases: a mini-review. J. Clin. Transl. Hepatol. 6, 332–338 (2018).
Martinez-Jimenez, C. P., Kyrmizi, I., Cardot, P., Gonzalez, F. J. & Talianidis, I. Hepatocyte nuclear factor 4alpha coordinates a transcription factor network regulating hepatic fatty acid metabolism. Mol. Cell. Biol. 30, 565–577 (2010).
Brosch, M. et al. Epigenomic map of human liver reveals principles of zonated morphogenic and metabolic control. Nat. Commun. 9, 4150 (2018).
Marcil, V. et al. Modification in oxidative stress, inflammation, and lipoprotein assembly in response to hepatocyte nuclear factor 4alpha knockdown in intestinal epithelial cells. J Biol Chem 285, 40448–40460 (2010).
Lu, H. Crosstalk of HNF4α with extracellular and intracellular signaling pathways in the regulation of hepatic metabolism of drugs and lipids. Acta Pharm. Sin. B 6, 393–408 (2016).
Babeu, J.-P. & Boudreau, F. Hepatocyte nuclear factor 4-alpha involvement in liver and intestinal inflammatory networks. World J. Gastroenterol. 20, 22–30 (2014).
Nath, B. et al. Hepatocyte-specific hypoxia-inducible factor-1α is a determinant of lipid accumulation and liver injury in alcohol-induced steatosis in mice. Hepatology 53, 1526–1537 (2011).
Mandrekar, P., Ambade, A., Lim, A., Szabo, G. & Catalano, D. An essential role for monocyte chemoattractant protein-1 in alcoholic liver injury: Regulation of proinflammatory cytokines and hepatic steatosis in mice. Hepatology 54, 2185–2197 (2011).
Cohen, J. I., Roychowdhury, S., McMullen, M. R., Stavitsky, A. B. & Nagy, L. E. Complement and alcoholic liver disease: Role of C1q in the pathogenesis of ethanol-induced liver injury in mice. Gastroenterology 139, 664–674, 674.e661 (2010).
Bertola, A., Mathews, S., Ki, S. H., Wang, H. & Gao, B. Mouse model of chronic and binge ethanol feeding (the NIAAA model). Nat. Protoc. 8, 627–637 (2013).
Malhi, H., Bronk, S. F., Werneburg, N. W. & Gores, G. J. Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis. J. Biol. Chem. 281, 12093–12101 (2006).
Dasgupta, D. et al. IRE1A stimulates hepatocyte-derived extracellular vesicles that promote inflammation in mice with steatohepatitis. Gastroenterology 159, 1487-1503.e1417 (2020).
He, L. et al. XIAP knockdown in alcohol-associated liver disease models exhibits divergent in vitro and in vivo phenotypes owing to a potential zonal inhibitory role of SMAC. Front. Physiol. 12, 664222 (2021).
Gao, J. et al. Endothelial p300 promotes portal hypertension and hepatic fibrosis through C-C motif chemokine ligand 2–mediated angiocrine signaling. Hepatology 73, 2468–2483 (2021).
Navarro-Corcuera, A. et al. Long non-coding RNA ACTA2-AS1 promotes ductular reaction by interacting with the p300/ELK1 complex. J. Hepatol. 76, 921–933 (2022).
Kostallari, E. et al. Stiffness is associated with hepatic stellate cell heterogeneity during liver fibrosis. Am. J. Physiol. Gastrointest. Liver Physiol. 322, G234–G246 (2022).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
Halpern, K. B. et al. Paired-cell sequencing enables spatial gene expression mapping of liver endothelial cells. Nat. Biotechnol. 36, 962–970 (2018).
Cusanovich, D. A. et al. A single-cell atlas of <em>In Vivo</em> mammalian chromatin accessibility. Cell 174, 1309-1324.e1318 (2018).
Stuart, T., Srivastava, A., Madad, S., Lareau, C. A. & Satija, R. Single-cell chromatin state analysis with Signac. Nat. Methods 18, 1333–1341 (2021).
Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184, 3573-3587.e3529 (2021).
Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888-1902.e1821 (2019).
Sehrawat, T. S. et al. Circulating extracellular vesicles carrying sphingolipid cargo for the diagnosis and dynamic risk profiling of alcoholic hepatitis. Hepatology 73, 571–585 (2021).
Yaqoob, U. et al. GIPC-regulated IGFBP-3 promotes HSC migration in vitro and portal hypertension in vivo through a β1-integrin pathway. Cell. Mol. Gastroenterol. Hepatol. 10, 545–559 (2020).
Dhaher, R. et al. Network-related changes in neurotransmitters and seizure propagation during rodent epileptogenesis. Neurology 96, e2261–e2271 (2021).
Ray, A. et al. Clinico-pathological features in fatal COVID-19 infection: A preliminary experience of a tertiary care center in North India using postmortem minimally invasive tissue sampling. Expert Rev. Respir. Med. 15, 1367–1375 (2021).
Farina, M. G. et al. Small loci of astroglial glutamine synthetase deficiency in the postnatal brain cause epileptic seizures and impaired functional connectivity. Epilepsia 62, 2858–2870 (2021).
Acknowledgements
The authors thank Constantine Tzouanas, M.S. and Alex K. Shalek, Ph.D. from MIT/Harvard University, USA for their intellectual input and important discussions regarding the scATAC-sequencing analyses.
Funding
This work is supported by funding provided by the National Institutes of Health (NIH), USA grants R01 AA21171 (V.H.S.), R01 DK59615 (V.H.S.), and U01 AA21788 (H.M. and V.H.S.) and MSIT, Korea grants 2018R1A5A7059549 (Y.K.N) and 2020–0-01373 (Y.Y., Y.K.N). Mayo Clinic Center for Cell Signaling in Gastroenterology (C-SiG) provided support through the NIH, USA funding (P30DK084567). S.A.C. is a member of the Biochemistry and Molecular Biology Ph.D. Graduate Program and is supported by Mayo Clinic Graduate School of Biomedical Sciences.
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T.S.S. and S.A.C. contributed to study conception and design; acquisition, analysis, and interpretation of most of the data; and drafting of the original and finalizing the manuscript. S.A.C., Y.L., and M.K.D. contributed to the analysis and interpretation of the scRNA and scATAC-sequencing data. A.N.C. contributed to acquisition, analysis, and interpretation of data and drafting of the manuscript. R.J.S. and M.A.M contributed to acquisition of cell biology experiments related to lipid droplets. C.A.S. contributed to proteomics data. M.L. and UY contributed to animal experiments. Y.Y., S.Y., Y.K.N., and J.C.A. contributed to the machine-learning model used for lipid-droplet assessment. C.M. contributed to the machine-learning model used for lipid-droplet assessment and provided expert pathologist review and annotation of human biopsy slides. J.A. and R.A.B. contributed to bulk-RNA-sequencing studies and intellectual input. J.H.L. and T.O. contributed to scATAC-sequencing and ChIP-sequencing experiments. W.A.F. and P.S.K. contributed to interpretation of data, intellectual input, and editing of the manuscript. D.A.S. contributed to interpretation of data, intellectual input, and the machine-learning model development supervision. H.M. contributed to data analysis, interpretation, intellectual input, supervision, funding, and drafting of the manuscript. S.C. contributed to study conception and design, analysis and interpretation of data, intellectual input, and editing of the manuscript. V.H.S. contributed to study conception and design; analysis and interpretation of data; resources; funding support; drafting and editing of the manuscript; and overall study supervision.
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Sehrawat, T.S., Cooper, S.A., Navarro-Corcuera, A. et al. Single cell multiomic landscape reveals gene programs driving lipid droplet heterogeneity in hepatic steatosis. Sci Rep (2026). https://doi.org/10.1038/s41598-026-39913-6
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DOI: https://doi.org/10.1038/s41598-026-39913-6
