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Proceedings of the National Academy of SciencesPathologyPeer-reviewed journal article

Liver lipid droplet cholesterol content is a key determinant of metabolic dysfunction-associated steatohepatitis

Ikki SakumaiD, Rafael Calais GaspariD, Ali R. NasiriiD, Sylvie DufouriD, Mario KahniD, Jie Zheng, Traci E. LaMoiaiD, Mateus T. GuerraiD, Yuki TakiiD, Yusuke KawashimaiD, Dean YimlamaiiD, Mark PerelisiD, Daniel F. VatneriD, Kitt Falk PeterseniD, Maximilian HuttaschiD, Birgit KnebeliD, SABINE KAHLiD, Michael RodeniD, Varman T. SamueliD, Tomoaki TanakaiD, Gerald I. ShulmaniD

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Abstract

Metabolic dysfunction-associated steatohepatitis (MASH) represents a progressive form of steatotic liver disease which increases the risk for fibrosis and advanced liver disease. The accumulation of discrete species of bioactive lipids has been postulated to activate signaling pathways that promote inflammation and fibrosis. However, the key pathogenic lipid species is a matter of debate. We explored candidates using various dietary, molecular, and genetic models. Mice fed a choline-deficient L-amino acid-defined high-fat diet (CDAHFD) developed steatohepatitis and manifested early markers of liver fibrosis associated with increased cholesterol content in liver lipid droplets within 5 days without any changes in total liver cholesterol content. Treating mice with antisense oligonucleotides (ASOs) against Coenzyme A synthase (Cosay) or treatment with bempedoic acid or atorvastatin decreased liver lipid droplet cholesterol content and prevented CDAHFD-induced MASH and the fibrotic response. All these salutary effects were abrogated with dietary cholesterol supplementation. Analysis of human liver samples demonstrated that cholesterol in liver lipid droplets was increased in humans with MASH and liver fibrosis and was higher in PNPLA3 I148M (variants rs738409) than in HSD17B13 variants (rs72613567). Together, these data identify cholesterol in liver lipid droplets as a critical mediator of MASH and demonstrate that COASY knockdown and bempedoic acid are novel therapeutic approaches to reduce liver lipid droplet cholesterol content and thereby prevent the development of MASH and liver fibrosis. Significance StatementMetabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease linked to fibrosis. The role of specific lipid species in its pathogenesis remains debated. Using dietary, molecular, and genetic models, we found that mice on a choline-deficient, high-fat diet (CDAHFD) developed steatohepatitis and early fibrosis, marked by increased cholesterol in liver lipid droplets within five days. Targeting COASY with antisense oligonucleotides or treating with bempedoic acid or atorvastatin reduced lipid droplet cholesterol and prevented MASH. However, dietary cholesterol supplementation negated these effects. Human liver samples confirmed elevated lipid droplet cholesterol in MASH and fibrosis, especially in PNPLA3 I148M carriers. These findings highlight cholesterol reduction as a potential MASH therapy.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (1 Mar 2025)

Cholesterol accumulation in hepatic lipid droplets drives inflammation and fibrosis in mouse and human MASH and is reversed by inhibiting cholesterol synthesis or knocking down Coasy or Hsd17b13.

Only 29% of phase III trial participants with fibrotic metabolic dysfunction-associated steatohepatitis (MASH) responded to resmetirom, and the specific lipid species triggering progression from steatosis to MASH remain debated. Subcellular fractionation in mouse models and human liver biopsies pinpoints lipid-droplet cholesterol as a modifiable driver of macrophage crown-like structures and hepatic fibrogenesis.

Key results

  1. ShownCholine-deficient high-fat diet (CDAHFD) feeding in mice causes rapid free and total cholesterol accumulation specifically inside liver lipid droplets within 1 to 5 days, preceding macrophage crown-like structures and fibrosis gene induction.

    In male C57BL/6J mice fed CDAHFD for 1-5 days and 1 week, whole-liver total cholesterol was unchanged while lipid-droplet total and free cholesterol (Filipin, LC-MS/MS) and triglycerides rose from day 1 with a lower PC:PE ratio, lysosomal cathepsin D leakage, TUNEL+ hepatocyte apoptosis, CD68+ crown-like structures from day 3, and elevated Gpnmb, Trem2, Lgals3, alphaSMA, and Col1a1 mRNA. · Choline-deficient L-amino acid-defined high-fat…; Choline-deficient L-amino acid-defined high-fat…; Figure legends

  2. ShownInhibiting cholesterol synthesis with bempedoic acid or atorvastatin, or liver-targeted antisense knockdown of Coenzyme A synthase (Coasy), lowers lipid-droplet cholesterol and prevents MASH in mice.

    In 1-week CDAHFD mice, bempedoic acid, atorvastatin, or GalNAc-Coasy ASO (>95% Coasy mRNA knockdown depleting hepatic CoA, acetyl-CoA, malonyl-CoA, and acyl-CoA), plus Coasy ASO in a 40-week GAN-diet model (lowering hepatic hydroxyproline), reduced lipid-droplet cholesterol and triglycerides, ALT/AST, crown-like structures, and fibrosis markers; 2% dietary cholesterol abrogated Coasy ASO protection. · Bempedoic acid alleviates liver inflammation and…; Atorvastatin alleviates liver inflammation and…; Coenzyme A synthase knockdown alleviates liver…; Cholesterol supplementation negates the…

  3. ShownKnockdown of Hsd17b13 protects against CDAHFD-induced MASH by selectively reducing lipid-droplet cholesterol without altering triglycerides or steatosis, whereas Pnpla3 I148M knock-in elevates lipid-droplet cholesterol and worsens MASH.

    In 1-week CDAHFD mice, GalNAc-Hsd17b13 ASO lowered lipid-droplet total cholesterol, ALT/AST, crown-like structures, and Gpnmb, Trem2, Lgals3, alphaSMA, and Col1a1 mRNA without changing triglycerides, PC:PE ratio, or steatosis (negated by 2% dietary cholesterol), whereas Pnpla3 I148M knock-in mice had higher lipid-droplet cholesterol, triglycerides, ALT/AST, and fibrosis markers rescued by bempedoic acid. · Hydroxysteroid 17-dehydrogenase 13 knockdown…; Cholesterol supplementation negates the…; Patatin-like phospholipase domain-containing…

  4. ShownHuman liver biopsies confirm elevated lipid-droplet cholesterol in patients with fibrotic MASH and in carriers of the high-risk PNPLA3 I148M genotype.

    Across 50 bariatric surgery liver biopsies from the BARIA_DDZ cohort (NCT01477957) stratified into 5 histological groups, lipid-droplet total cholesterol and triglycerides were highest in MASH with fibrosis (F > 0) and were significantly higher in PNPLA3 I148M carriers lacking the protective HSD17B13 rs72613567 splice variant than in HSD17B13 rs72613567 carriers lacking PNPLA3 I148M. · Lipid droplet cholesterol is a key factor in…; Study population; Figure legends

Methods, data and reproducibility

The authors isolated liver lipid droplets and plasma membranes by ultracentrifugation from C57BL/6J and Pnpla3 I148M mice fed CDAHFD (1-8 days, +/- 2% cholesterol) or a 40-week GAN diet and treated with bempedoic acid, atorvastatin, or GalNAc-ASOs against Coasy or Hsd17b13. They paired lipidomics, histology, EM, RT-qPCR, and DIA-MS proteomics with analysis of 50 human liver biopsies.

  • Subcellular liver lipid-droplet and plasma-membrane lipidomics, histology, and RT-qPCR in C57BL/6J and Pnpla3 I148M mice on CDAHFD or GAN diets with cholesterol-lowering or ASO treatments · mouse · n = Multiple cohorts (1-5 day time course, 1-week CDAHFD, 40-week GAN studies)
  • Non-targeted data-independent acquisition mass spectrometry (DIA-MS) liver proteomics on Orbitrap Exploris 480 across mouse experimental groups · mouse · n = 96 samples across 16 groups (n = 6 per group; n = 5 in pathway comparisons) · UP000000589
  • Human liver biopsy lipid-droplet metabolite profiles, histology, and TaqMan SNP genotypes from obese Caucasian bariatric surgery patients (BARIA_DDZ cohort) · human · n = 50 individuals across 5 histological groups and PNPLA3/HSD17B13 genotypes · NCT01477957
  • Published 12-week CDAHFD mouse liver RNA-seq dataset from Gene Expression Omnibus · mouse · GSE120977
Methods & tools
Orbitrap Exploris 480 (DIA-MS), Scaffold DIA (v3.0.1), DIA-NN (v1.8.1), TCC-GUI, Metascape, GREIN, ImageJ
Reagents & environment
Scaffold DIA v3.0.1, DIA-NN v1.8.1, TCC-GUI, Metascape, GREIN, NIH ImageJ
Reproducibility gaps
Raw mouse DIA-MS proteomics files and individual mouse and human lipid-droplet metabolite tables are not deposited in a public repository.
Limitations (3)
  • CDAHFD rapidly induces steatohepatitis without obesity or hyperglycemia, and no single mouse model reproduces all clinical and metabolic features of human MASH. · stated by the authors
  • Whole-body COASY loss of function causes an autosomal recessive neurodegenerative disorder, requiring liver-selective delivery (such as GalNAc-ASO) and further study of long-term hepatic safety. · stated by the authors
  • Cholesterol crystals were not detected in liver lipid droplets by transmission electron microscopy or Filipin staining in the 1-week CDAHFD model, leaving the exact physical mechanism of lipid-droplet-to-lysosome cholesterol transfer to be clarified. · stated by the authors

Read this if you investigate MASH pathogenesis, hepatic lipid droplet biology, or therapeutic strategies targeting cholesterol synthesis, COASY, HSD17B13, and PNPLA3.

Citations
46
FWCI
31.60
Field percentile
top 1%

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