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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2025 May 1;122(18):e2502978122. doi: 10.1073/pnas.2502978122

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

Ikki Sakuma a,b, Rafael C Gaspar a, Ali R Nasiri a, Sylvie Dufour a, Mario Kahn a, Jie Zheng a, Traci E LaMoia a, Mateus T Guerra a, Yuki Taki b, Yusuke Kawashima c, Dean Yimlamai d, Mark Perelis e, Daniel F Vatner a, Kitt Falk Petersen a, Maximilian Huttasch f,g, Birgit Knebel g,h, Sabine Kahl f,g,i, Michael Roden f,g,i, Varman T Samuel a,j, Tomoaki Tanaka b, Gerald I Shulman a,k,l,1
PMCID: PMC12067271  PMID: 40310463

Significance

Metabolic 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 5 d. Targeting Coenzyme A synthase (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 hepatic lipid droplet cholesterol reduction as a potential therapeutic target for MASH.

Keywords: cholesterol, lipid droplet, phosphatidylcholine, Coenzyme A synthase, metabolic dysfunction–associated steatohepatitis (MASH)

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 d without any changes in total liver cholesterol content. Treating mice with antisense oligonucleotides against Coenzyme A synthase (Coasy) 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 Coenzyme A synthase knockdown and bempedoic acid are therapeutic approaches to reduce liver lipid droplet cholesterol content and thereby prevent the development of MASH and liver fibrosis.

Metabolic dysfunction–associated steatosis liver disease (MASLD) is the predominant chronic liver disease in the world, affecting approximately 25% of the global population (1, 2). The prevalence of MASLD is on the rise, paralleling a rise in obesity and type 2 diabetes. Metabolic dysfunction–associated steatohepatitis (MASH) increases the risk for advanced liver diseases, including cirrhosis, hepatocellular carcinoma, and liver-related death (2). While the U.S. Food and Drug Administration has recently approved resmetirom, a liver-directed, β-selective thyroid hormone receptor agonist, as a therapy for MASH with significant fibrosis only 29% of the study participants responded to this treatment in a recent Phase III clinical trial (3), demonstrating the need for additional new targets and therapies for MASH.

One of the major unanswered questions is what triggers the conversion of “benign” fatty liver disease to MASH. Triglycerides, the predominant lipids within hepatocytes, are neutral lipids that can be exported in VLDL particles or oxidized to support hepatocellular metabolism. Triglycerides reside within the core of lipid droplets, which are surrounded by a lipid monolayer composed of various lipids, including cholesterol and phospholipids, as well as a host of proteins—such as lipases, transacylases, and cofactors—that regulate the influx and efflux of triglycerides.

Though triglycerides are an important source of cellular energy, they are considered inert in terms of modulating cellular functions. Instead, other lipid species, such as fatty acids, cholesterol, lysophosphatidic acid, lysophosphatidylcholine, and ceramides, have all been implicated in causing hepatic inflammation and fibrosis (4–7). The choline-deficient L-amino acid–defined high-fat diet (CDAHFD) model has been advanced as a mouse model that mirrors the pathological progression of MASLD (steatosis ➾ hepatitis ➾ fibrosis) (8, 9). The CDAHFD model displays MASH pathology within weeks, enabling a more expedited pathological evaluation (10). We postulated that pathogenic lipid species accumulate early in specific subcellular compartments in this model, contributing to organelle dysfunction and cellular injury. Thus, we measured candidate lipid species in key subcellular compartments in the livers of CDAHFD-fed C57BL/6J mice. We began these studies with a 5-d time course experiment of CDAHFD and observed a marked increase in cholesterol specifically in the liver lipid droplets within the first few days of treatment, which was associated with hepatic inflammation and markers of liver fibrosis. This observation informed our central hypothesis that cholesterol in liver lipid droplets triggers liver inflammation and fibrosis. We tested this hypothesis using cholesterol-lowering treatments (bempedoic acid, atorvastatin), cholesterol supplementation, and an antisense oligonucleotide (ASO) to knockdown the expression of Coenzyme A synthase (COASY). COASY generates CoA for acetyl CoA synthesis, a key substrate for cholesterol synthesis. To translate these findings to humans we examined the role of cholesterol in liver lipid droplets in humans with and without MASH/liver fibrosis as well the effects of established gene variant (HSD17B13) that have been shown to be protective against the development of MASH and liver fibrosis.

Results

Choline-Deficient L-Amino Acid–Defined High-Fat Diet Acutely Induces Hepatic Lipid Accumulation Followed by Inflammation and Fibrosis.

Male C57BL/6J mice were divided into regular chow (RC) fed or CDAHFD fed groups. CDAHFD was administered daily for a duration of 1 to 5 d (Fig. 1A). Plasma concentrations of ALT and AST increased within the first few days whereas plasma total cholesterol and triglycerides decreased in CDAHFD-fed mice (Fig. 1B). Hematoxylin, eosin (HE), and BODIPY staining demonstrated that liver lipid droplets increased in size in a time-dependent manner (Fig. 1C). CD68 staining revealed crown-like structures, which are macrophages surrounding and engulfing dying or dead hepatocytes with large lipid droplets and indicative of MASH (11). These structures were observed from day 3 of the CDAHFD onward (Fig. 1 C and D). Filipin, which stains free cholesterol but not cholesterol esters, demonstrated an accumulation of free cholesterol within the lipid droplets as early as day 2 of the CDAHFD feeding (Fig. 1C). These findings confirmed the development of steatohepatitis within 5 d of CDAHFD.

Fig. 1.

Fig. 1.

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Choline-deficient L-amino acid–defined high-fat diet acutely induces hepatic lipid accumulation followed by inflammation and fibrosis. (A) Study design. C57BL/6J mice were divided into RC fed mice or CDAHFD fed mice. CDAHFD was provided for 1, 2, 3, 4, and 5 d, respectively. (B) Plasma ALT, AST, total cholesterol, and triglycerides levels. ALT and AST increased in CDAHFD-fed mice. Plasma total cholesterol and triglycerides decreased in CDAHFD-fed mice. (C) Liver sections were stained with HE, CD68, BODIPY, and Filipin. CDAHFD induced liver lipid droplets time-dependently. CD68 staining revealed crown-like structures on day 5. BODIPY and Filipin staining demonstrated that free cholesterol was included in liver lipid droplets. (D) The number of crown-like structures increased in the liver of CDAHFD-fed mice time-dependently. (E) Total cholesterol, triglycerides, and fatty acids content in whole liver lysate. Total cholesterol did not change. Triglycerides and fatty acids increased in CDAHFD-fed mice time-dependently. (F) The levels of phospholipids in the liver. The concentration of PC in the plasma membrane exhibited a decline starting on day 1. The level of PE in the plasma membrane did not change. In liver lipid droplets, PC and PE levels increased from day 1. (G) The ratio of PC to PE in the plasma membrane and lipid droplet in the liver, respectively. PC/PE ratio in CDAHFD-fed mice was lower than RC-fed mice. (H) Total cholesterol and triglycerides in the liver lipid droplet increased in CDAHFD-fed mice from day 1. Data are presented as mean ± SEM. Groups were compared by Unpaired one-sided Student’s t test.

We next quantified candidate lipid species implicated in the pathogenesis of MASH. Triglycerides and fatty acids in whole liver lysate increased in CDAHFD-fed mice in a time-dependent manner (Fig. 1E). In contrast, total cholesterol content including free cholesterol and cholesterol esters in whole liver lysate did not change appreciably over the 5-d feeding period. Lower ratios of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) can decrease membrane integrity and contribute to liver damage (12). To examine the relative concentrations of these two phospholipids, we isolated plasma membrane and lipid droplet fractions from whole liver tissues using ultracentrifugation, as described in previous studies and as depicted in SI Appendix, Fig. S1A (13, 14). In the plasma membrane, PC concentrations declined on day 1, reached a nadir on day 3 and showed an upward trend on days 4 and 5 (Fig. 1F). The level of PE in the plasma membrane did not change (Fig. 1F). In contrast, within the liver lipid droplets, both PC and PE concentrations increased starting from day 1 (Fig. 1F). Despite the differences in PC and PE absolute concentrations in these discrete subcellular compartments, the ratio of PC to PE in CDAHFD-fed mice compared to RC-fed mice decreased in plasma membrane and lipid droplets (Fig. 1G).

We next evaluated total cholesterol and triglycerides in liver lipid droplets. Total cholesterol and triglycerides in liver lipid droplets increased in CDAHFD-fed mice from day 1 (Fig. 1H). LC-MS/MS analysis consistently verified that free cholesterol and cholesterol ester in liver lipid droplets increased (SI Appendix, Fig. S1B).

Taken together, CDAHFD feeding acutely induced hepatic lipid accumulation followed by inflammation and fibrosis. Total cholesterol in whole liver lysate did not track with inflammation and fibrosis markers, but total cholesterol in liver lipid droplets tracked with inflammation and fibrosis.

Choline-Deficient L-Amino Acid–Defined High-Fat Diet Induces Hepatocytes’ Death Possibly via Lysosomal Membrane Disruption.

Next, we compared CDAHFD-fed mice with RC-fed mice after 1 wk of feeding (Fig. 2A). Consistent with prior findings CDAHFD-fed mice did not induce obesity or hyperglycemia (8) and there were no differences in percent body fat or lean body mass as assessed by 1H NMR in (SI Appendix, Fig. S2A).

Fig. 2.

Fig. 2.

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Choline-deficient L-amino acid–defined high-fat diet induces steatosis via enhanced hepatic uptake of chylomicron remnants. (A) Study design. C57BL/6J mice were divided into RC-fed mice or CDAHFD-fed mice for 1 wk. (B) Liver sections were stained with HE, BODIPY, Filipin, Cathepsin D, Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and CD68. CDAHFD induced macrovesicular steatosis. BODIPY and Filipin staining demonstrated that free cholesterol was included in liver lipid droplets. Leakage of cathepsin D into cytoplasm indicated disruption of the lysosomal membrane. TUNEL staining revealed dead hepatocytes. CD68 staining revealed crown-like structures. (C) Representative transmission electron microscopy images of 1 wk CDAHFD-fed mice’s liver. Transmission electron microscopy demonstrated lipid-laden lysosome, disruption of lysosomal membrane, disruption of cell membrane of hepatocyte and macrophage engulfing lipid droplets. (D) RT-qPCR analysis of liver tissues. CDAHFD 1 wk feeding increased mRNA expression of MASH associated macrophage markers (Gpnmb, Trem2, and Lgals3), and fibrosis markers (αSMA, and Col1a1). (E) Study design. C57BL/6J mice were divided into a CDAHFD-fed mice, CDAHFD with 2% cholesterol-fed mice, GAN diet-fed mice. (F) Plasma ALT, AST, total cholesterol, and triglycerides levels. CDAHFD with 2% cholesterol-fed mice showed increased ALT and AST levels compared to CDAHFD-fed mice and GAN-diet fed mice. GAN diet-fed mice showed increased plasma total cholesterol and triglyceride levels compared to CDAHFD with 2% cholesterol-fed mice. (G) Liver sections were stained with HE, CD68, BODIPY, and Filipin. CDAHFD and CDAHFD with 2% cholesterol feeding for 1 wk induced steatosis. GAN diet feeding for 1 wk did not cause apparent steatosis. (H) The number of crown-like structures in the liver increased in CDAHFD with 2% cholesterol-fed mice compared to CDAHFD-fed mice and GAN diet-fed mice. (I) The metabolites levels in liver lipid droplet. Total cholesterol and triglycerides increased in CDAHFD-fed mice and CDAHFD with 2% cholesterol-fed mice compared to GAN diet-fed mice. In addition, total cholesterol in CDAHFD with 2% cholesterol-fed mice was higher than that of CDAHFD-fed mice. The level of PC and PE increased in CDAHFD-fed mice and CDAHFD with 2% cholesterol-fed mice compared to GAN diet-fed mice. The ratio of PC to PE decreased in CDAHFD-fed mice and CDAHFD with 2% cholesterol-fed mice compared to GAN diet-fed mice. Data are presented as mean ± SEM. Groups were compared by one-way ANOVA followed by Tukey’s multiple comparisons test.

CDAHFD induced macrovesicular hepatic steatosis (Fig. 2B). BODIPY and Filipin staining demonstrated that free cholesterol was included in liver lipid droplets. Leakage of cathepsin D into cytoplasm indicated disruption of the lysosomal membrane. Lysosomal rupture with cathepsin release causes various types of cell death (15) and consistent with these findings TUNEL staining revealed apoptotic hepatocytes. CD68 staining revealed crown-like structures. Transmission electron microscopy demonstrated lipid-laden lysosome, disruption of lysosomal membrane, disruption of cell membrane of hepatocyte and macrophage engulfing lipid droplets (Fig. 2C).

We next measured mRNA expression of genes associated with MASH in this 1-wk CDAHFD model. Hepatic macrophages are thought to play key roles in the pathogenesis of MASH. Resident Kupffer cells are replaced by distinct subsets of bone marrow derived macrophages (16). Previously, experiments using single-cell RNA sequencing identified MASH-associated macrophages that were associated with progression to fibrosis (17). Additional studies suggested that Trem2High-GpnmbHigh macrophages express Lgals3 (18, 19), a cytokine that promotes hepatic stellate cell activation (20). We observed that 1 wk feeding of the CDAHFD induced expression of Gpnmb, Trem2, and Lgals3. These changes were associated with increased mRNA expression of fibrosis markers (αSMA, and Col1a1) (Fig. 2D). Furthermore, the profile of mRNA changes that we observed in this 1-wk study was similar to that obtained from the livers of 12-wk CDAHFD-fed mice [Gene Expression Omnibus dataset (GSE120977)] (SI Appendix, Fig. S2B). Thus, the early changes in gene expression after 1 wk appear to be sustained with chronic CDAHFD feeding.

Cholesterol Supplementation Induces Cholesterol in Liver Lipid Droplets, Especially under Choline Deficiency.

To test our hypothesis that hepatic cholesterol accumulation in lipid droplets promotes MASH in additional mouse models of MASH, C57BL/6J mice were divided into CDAHFD-fed mice, CDAHFD with 2% cholesterol-fed mice (CDAHFD+Chol), Gubra-Amylin NASH (GAN) diet-fed mice (Fig. 2E). The GAN diet includes 40% kcal fat (palm oil, soybean oil), 22% wt fructose, sufficient choline, and 2% cholesterol. We selected a CDAHFD containing 2% cholesterol to compare with the GAN diet, which also includes 2% cholesterol. CDAHFD+Chol fed mice had an exacerbation of the MASH phenotype as reflected by higher plasma ALT and AST levels compared to CDAHFD-fed mice and GAN-diet-fed mice (Fig. 2F). GAN diet-fed mice showed increased plasma total cholesterol and triglyceride levels compared to CDAHFD with 2% cholesterol-fed mice (Fig. 2F). Interestingly while CDAHFD (with or without Chol) feeding for 1 wk resulted in hepatic steatosis, GAN diet feeding for 1 wk did not cause hepatic steatosis (Fig. 2G). Consistent with the increased hepatic inflammation, CDAHFD+Chol feeding promoted further increases in cholesterol content in liver lipid droplets. The number of crown-like structures in the liver was highest in the CDAHFD + Chol-fed mice compared to CDAHFD-fed mice and GAN diet-fed mice (Fig. 2H). Total cholesterol in liver lipid droplets was highest in the CDAHFD+Chol mice compared to both CDAHFD alone and GAN diet (Fig. 2I).

In contrast, liver lipid droplet triglyceride increased to a similar extent in both CDAHFD-fed mice and CDAHFD + Chol-fed mice compared to GAN diet-fed mice (Fig. 2I). Similarly, the concentration of PC and PE in liver lipid droplets increased in both CDAHFD and CDAHFD+Chol-fed mice compared to GAN diet-fed mice. The ratio of PC to PE decreased in both CDAHFD and CDAHFD+Chol-fed mice compared to GAN diet-fed mice. Taken together these data demonstrate that, dietary cholesterol supplementation in addition to a choline-deficient diet profoundly increased cholesterol in liver lipid droplets compared to cholesterol supplementation on choline sufficient diet conditions. This is consistent with the fact that the GAN diet induces MASH more slowly compared to CDAHFD (8). The close association between liver lipid droplet cholesterol and worsening histological changes further points to the accumulation of lipid droplet cholesterol content as being more closely tied to the development of MASH than other putative factors, such as dietary cholesterol content, plasma cholesterol concentration, or intrahepatic PC to PE ratio.

Bempedoic Acid Alleviates Liver Inflammation and Fibrosis Due to Choline-Deficient L-Amino Acid–Defined High-Fat Diet via Decreasing Cholesterol Content in Liver Lipid Droplets.

To test our hypothesis that CDAHFD leads to MASH by increasing cholesterol content in liver lipid droplets, we treated CDAHFD-fed mice with bempedoic acid (Fig. 3A). Bempedoic acid works as an ATP-citrate lyase inhibitor, which is clinically used as a plasma cholesterol-lowering drug (21). While bempedoic acid has been shown to be effective in reducing hepatic steatosis in previous studies (22–24), its effect on cholesterol content in liver lipid droplets has not yet been studied. Bempedoic acid treatment reduced plasma ALT and AST (Fig. 3B), hepatic steatosis (Fig. 3C), and the number of crown-like structures compared to the control mice (Fig. 3 C and D). BODIPY and Filipin staining demonstrated decreased free cholesterol in liver lipid droplets of bempedoic acid–treated mice (Fig. 3C). Bempedoic acid treatment also reduced triglycerides and total cholesterol content in liver lipid droplets (Fig. 3E). The level of PC in liver lipid droplets decreased in bempedoic acid–treated mice whereas PE and the ratio of PC to PE remained unchanged. Consistent with pathological findings (Fig. 3C), Quantitative real-time PCR (RT-qPCR) demonstrated a decreased mRNA expression of MASH-associated macrophage markers (Gpnmb, Trem2, and Lgals3), and fibrosis marker (Col1a1) in bempedoic acid–treated mice liver (Fig. 3F). Taken together, these data demonstrate that decreasing hepatic cholesterol synthesis by inhibiting ATP-citrate lyase reduces hepatic lipid droplet cholesterol (and triglyceride) content and attenuates the MASH phenotype in CDAHFD-fed mice.

Fig. 3.

Fig. 3.

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Bempedoic acid alleviates liver inflammation and fibrosis due to choline-deficient L-amino acid–defined high-fat diet via decreasing cholesterol content in liver lipid droplets. (A) Study design. C57BL/6J mice were divided into a control group and bempedoic acid–treated group. Both groups were fed a CDAHFD for 1 wk. (B) ALT and AST in bempedoic acid (BemA) treated mice decreased compared to control mice. (C) Liver sections were stained with HE, CD68, BODIPY, and Filipin. BemA treatment prevented steatosis. CD68 staining revealed decreased crown-like structures. BODIPY and Filipin staining demonstrated decreased free cholesterol in liver lipid droplet. (D) The number of crown-like structures decreased in BemA-treated mice liver. (E) The metabolites levels in liver lipid droplet. Total cholesterol and triglycerides decreased in BemA-treated mice. The level of PC decreased in BemA-treated mice. PE did not show a difference. The ratio of PC to PE did not show a difference. (F) RT-qPCR analysis of liver tissues demonstrated a decreased mRNA expression of MASH-associated macrophage markers (Gpnmb, Trem2, and Lgals3), and fibrosis marker (Col1a1) in BemA-treated mice. Data are presented as mean ± SEM. Groups were compared by Unpaired one-sided Student’s t test.

Atorvastatin Alleviates Liver Inflammation and Fibrosis Due to Choline-Deficient L-Amino Acid–Defined High-Fat Diet via Decreasing Cholesterol Content in Liver Lipid Droplets.

To further examine the role of liver lipid droplet cholesterol on the MASH phenotype in CDAHFD-fed mice, we evaluated whether short-term atorvastatin (statin) treatment would decrease cholesterol content in liver lipid droplets and alleviate MASH in the CDAHFD 1-wk model (SI Appendix, Fig. S3A). Statin treatment resulted in reductions of plasma ALT, AST (SI Appendix, Fig. S3B) and reduced hepatic steatosis (SI Appendix, Fig. S3C). There was a reduction in the number of crown-like structures compared to the control mice (SI Appendix, Fig. S3 C and D). BODIPY and Filipin staining demonstrated decreased free cholesterol in liver lipid droplets of statin-treated mice (SI Appendix, Fig. S3C). Consistent with BODIPY and Filipin staining, statin treatment also reduced triglycerides and total cholesterol content in liver lipid droplets (SI Appendix, Fig. S3E). The level of PC and PE in liver lipid droplets did not differ between control and statin-treated mice (SI Appendix, Fig. S3E). As a result, the ratio of PC to PE in liver lipid droplets did not show a difference. Consistent with pathological findings (SI Appendix, Fig. S3C), RT-qPCR demonstrated a decreased mRNA expression of MASH-associated macrophage markers (Gpnmb, Trem2, and Lgals3), and fibrosis markers (αSMA and Col1a1) in statin-treated mice liver (SI Appendix, Fig. S3F).

COASY Knockdown Alleviates Liver Inflammation and Fibrosis Due to Choline-Deficient L-Amino Acid–Defined High-Fat Diet via Decreasing Cholesterol in Liver Lipid Droplets.

COASY plays a crucial role in CoA production from 4′-phosphopantetheine. COASY generates CoA which is required for the synthesis of acetyl CoA, a key substrate for the cholesterol synthetic pathway (Fig. 4A). To evaluate whether Coasy knockdown decreases cholesterol in liver lipid droplets and alleviates MASH in the CDAHFD 1-wk model, we divided C57BL6 mice into a GalNAc control ASO-treated mouse group and a GalNAc Coasy ASO-treated mouse group. Both groups were subjected to a CDAHFD feeding for 1 wk (Fig. 4B).

Fig. 4.

Fig. 4.

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COASY knockdown alleviates liver inflammation and fibrosis due to Choline-deficient L-amino acid–defined high-fat diet via decreasing cholesterol in liver lipid droplets. (A) CoA biosynthesis pathway. COASY converts 4′-Phosphopantetheine into CoA by adenylation and phosphorylation. CoA subsequently catalyzes the synthesis of acetyl CoA. Acetyl CoA functions as a substrate throughout the cholesterol synthesis or fatty acids synthesis pathway. (B) Study design. C57BL/6J mice were divided into GalNAc control ASO-treated mice and GalNAc Coasy ASO-treated mice. Both groups were fed a CDAHFD for 1 wk. (C) RT-qPCR analysis of liver tissues confirmed the knockdown effect of Coasy ASO. Consistently, Coasy ASO treatment decreased hepatic CoA species, including CoA, Acetyl CoA, Malonyl CoA, and Acyl CoA. (D) ALT and AST in Coasy ASO-treated mice decreased compared to control ASO-treated mice. (E) Liver sections were stained with HE, CD68, BODIPY, and Filipin. Coasy ASO treatment alleviated steatosis. Accordingly, CD68 staining revealed decreased crown-like structures. BODIPY and Filipin staining demonstrated decreased free cholesterol in liver lipid droplets. (F) The number of crown-like structures decreased in Coasy ASO-treated mice liver. (G) The metabolites levels in liver lipid droplet. Total cholesterol and triglycerides decreased in Coasy ASO-treated mice. The level of PC and PE decreased in Coasy ASO-treated mice. The ratio of PC to PE remained unchanged. (H) RT-qPCR analysis of liver tissues demonstrated a decreased mRNA expression of MASH-associated macrophage markers (Gpnmb, Trem2, and Lgals3) and fibrosis markers (αSMA, and Col1a1) in Coasy ASO-treated mice. Data are presented as mean ± SEM. Groups were compared by Unpaired one-sided Student’s t test.

Coasy mRNA expression was decreased by >95% with Coasy ASO, confirming the efficacy of Coasy ASO (Fig. 4C). Coasy ASO treatment decreased all hepatic CoA species, including CoA, acetyl CoA, malonyl CoA, and acyl CoA (Fig. 4C). Plasma ALT and AST concentrations decreased in Coasy ASO-treated mice compared to control ASO-treated mice (Fig. 4D). HE and BODIPY staining demonstrated that Coasy knockdown decreased liver lipid droplets (Fig. 4E). The number of crown-like structures decreased in Coasy ASO-treated mouse livers (Fig. 4 E and F). BODIPY and Filipin staining demonstrated decreased free cholesterol in liver lipid droplets of Coasy ASO-treated mice (Fig. 4E). Consistent with BODIPY and Filipin staining, triglycerides and total cholesterol in liver lipid droplets decreased in Coasy ASO-treated mice (Fig. 4G). However, neither the concentration of PC and PE in liver lipid droplets nor the PC:PE ratio changed with Coasy ASO treatment (Fig. 4G). Consistent with pathological findings, RT-qPCR demonstrated decreased mRNA expression of MASH-associated macrophage markers (Gpnmb, Trem2, and Lgals3), and fibrosis markers (αSMA and Col1a1) in the Coasy ASO–treated mice liver (Fig. 4H).

It is generally accepted that there is no mouse model of MASH that manifests all of the characteristics of human MASH. To address this concern, we also examined the effect of Coasy knockdown on MASH pathogenesis in a prolonged (40 wk) GAN-diet fed mouse model of MASH (SI Appendix, Fig. S4A). Plasma ALT and AST concentrations decreased in Coasy ASO-treated mice compared to control ASO-treated mice under GAN-diet feeding (SI Appendix, Fig. S4B). The decrease in liver fibrosis in Coasy ASO-treated mice was confirmed by liver hydroxyproline assay (SI Appendix, Fig. S4B). Total cholesterol, triglycerides, PC, and PE in liver lipid droplets decreased in Coasy ASO-treated mice (SI Appendix, Fig. S4C). The PC:PE ratio increased with Coasy ASO treatment. Taken together with our prior results demonstrating a protective effect of Coasy ASO treatment in the development of MASH in a CDAHFD mouse model of MASH these results confirm the protective effects of Coasy knockdown in the liver on the progression of MASH in a well-established prolonged GAN diet fed mouse model of MASH.

Cholesterol Supplementation Negates the Protective Effect of Coasy ASO on a Choline-Deficient L-Amino Acid–Defined High-Fat Diet 1-wk Mice Model.

To test whether increases in cholesterol per se could negate the protective effect of Coasy knockdown, Coasy ASO-treated C57BL/6J mice were divided into mice fed either CDAHFD alone or CDAHFD with 2% cholesterol (Fig. 5A). As before, CDAHFD+Chol exacerbated the MASH phenotype with higher transaminase concentrations (Fig. 5B), histological evidence of increased cholesterol in liver lipid droplets (Fig. 5C) and increased number of crown-like structures (Fig. 5 C and D). Consistent with these findings, triglycerides and total cholesterol in liver lipid droplets increased in cholesterol-supplemented mice (Fig. 5E). In addition, there was a modest increase in lipid droplets PC and PE in CDAHFD+Chol-fed mice (Fig. 5E). The ratio of PC to PE in liver lipid droplets was not different between the two diet groups. RT-qPCR demonstrated an increased mRNA expression of MASH-associated macrophage markers (Gpnmb, Trem2, and Lgals3)and fibrosis markers (αSMA and Col1a1) in cholesterol-supplemented mice liver (Fig. 5F). Thus cholesterol supplementation abrogated the protective effects of Coasy ASO in CDAHFD-fed mice.

Fig. 5.

Fig. 5.

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Cholesterol supplementation negates the protective effect of Coasy ASO on a Choline-deficient L-amino acid–defined high-fat diet 1-wk mice model. (A) Study design. C57BL/6J mice were divided into CDAHFD fed mice and CDAHFD with 2% cholesterol fed mice. Both groups were injected with GalNAc Coasy ASO. (B) Cholesterol (Chol) dietary supplementation increased plasma ALT and AST levels compared to control (Ctr) mice. (C) Liver sections were stained with HE, CD68, BODIPY, and Filipin. Dietary cholesterol supplementation induced steatosis. CD68 staining revealed increased crown-like structures. BODIPY and Filipin staining demonstrated increased free cholesterol in liver lipid droplet. (D) The number of crown-like structures increased in dietary cholesterol-supplemented mice liver. (E) The metabolites levels in liver lipid droplet. Total cholesterol and triglycerides increased in cholesterol-overload mice. The level of PC and PE increased in cholesterol-supplemented mice. The ratio of PC to PE remained unchanged. (F) RT-qPCR analysis of liver tissues showed an elevated mRNA expression of MASH-associated macrophage markers (Gpnmb, Trem2, and Lgals3) as well as fibrosis markers (αSMA and Col1a1) in cholesterol-overload mice. Data are presented as mean ± SEM. Groups were compared by Unpaired one-sided Student’s t test. (G) Summary of four studies of 1-wk CDAHFD studies. (H) Enrichment analysis of comprehensive proteome analysis. Three comparisons were made: (1) CDAHFD-fed mice (n = 6) vs. RC-fed mice (n = 6); (2) Coasy ASO-CDAHFD-treated mice (n = 6) vs. Control ASO-CDAHFD-treated mice (n = 6); and (3) Coasy ASO-CDAHFD with 2% cholesterol-treated mice (n = 6) vs. Coasy ASO-CDAHFD-treated mice (n = 6). (I) Hepatic protein expression of targets of SREBP1c, MLXIPL, and SREBP2.

Comprehensive Proteome Analysis Revealed That Choline-Deficient L-Amino Acid–Defined High-Fat Diet Compensatory Suppress Targets of SREBP1c and SREBP2, and Coasy Knockdown Reverses This Alteration.

Together, these data support the hypothesis that cholesterol accumulation in liver lipid droplets plays a critical role in the rapid development of MASH in CDAHFD-fed mice (Fig. 5G). We next performed a comprehensive proteome analysis and then subjected it to enrichment analysis to identify potential pathways that may be altered in each experimental group compared to the control group. We found that Mlxipl, Srebf1, and Srebf2 pathways were significantly altered in CDAHFD-fed mice vs. RC-fed mice and Coasy ASO-treated mice vs. control ASO-treated mice (Fig. 5H). The Srebf2 pathway was the most enriched in Coasy ASO-treated cholesterol-fed mice vs. Coasy ASO-treated mice. Hepatic protein expression of targets of MLXIPL, SREBP1c, and SREBP2 were suppressed in CDAHFD-fed mice compared to RC-fed mice and activated in Coasy ASO-treated mice compared to control ASO-treated mice (Fig. 5I). The expression of SREBP2 targets was increased with Coasy ASO treatment but suppressed in Coasy ASO and cholesterol-fed mice.

Treatments that decreased intracellular cholesterol increased the expression of SREBP2 targets which likely represents a compensatory response to cholesterol depletion in Coasy ASO-treated mice. Interestingly, though Coasy ASO also increased a number of SREBP1c targets, perhaps to compensate for decreased fatty acid synthesis, cholesterol supplementation appeared to prevent upregulation of SREBP1c targets, possibly representing a degree of crosstalk between cholesterol and fatty acid biosynthetic pathways.

Hydroxysteroid 17-Dehydrogenase 13 Knockdown Prevents Liver Inflammation and Fibrosis Due to Choline-Deficient L-Amino Acid–Defined High-Fat Diet via Decreasing Cholesterol in Liver Lipid Droplets.

Recently, GWAS identified 17 loci associated with MASLD (25), including several genetically validated potential targets for treating MASH (26, 27). Patatin-like phospholipase domain-containing protein 3 (PNPLA3) I148M increases the risk (28). The hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) splice site variant (rs72613567) decreases the risk of MASH (29). Among variants associated with risk of MASLD/MASH, the effect size of PNPLA3 and HSD17B13 variants are larger than other variants (27). In addition, the HSD17B13 splice site variant (rs72613567) is found in 18% of the global population (30, 31). While HSD17B13 is a lipid droplet surface protein, it has not been shown to directly modulate the cholesterol content of lipid droplets. The loss of function variant, rs72613567:TA, in the HSD17B13 gene confers protection against both alcoholic and metabolic liver diseases (29). To evaluate whether Hsd17b13 knockdown decreases cholesterol in liver lipid droplets and alleviates MASH in the CDAHFD 1-wk model, we divided C57BL/6J mice into GalNAc control ASO treated mice, and GalNAc Hsd17b13 ASO-treated mice under CDAHFD 1 wk (Fig. 6A). RT-qPCR analysis of liver tissues confirmed the knockdown effect of Hsd17b13 ASO (Fig. 6B). Hsd17b13 ASO decreased plasma AST and ALT compared to the control ASO (Fig. 6C). HE staining demonstrated that Hsd17b13 knockdown did not significantly affect steatosis (Fig. 6D). The number of crown-like structures decreased in Hsd17b13 ASO-treated mice’s livers (Fig. 6 D and E). Total cholesterol in liver lipid droplets decreased in Hsd17b13 ASO-treated mice, while the level of triglycerides, PC and PE in liver lipid droplets did not differ between control and Hsd17b13 ASO-treated mice (Fig. 6F). As a result, the ratio of PC to PE in liver lipid droplets did not show a difference. Consistent with pathological findings, RT-qPCR demonstrated a reduced mRNA expression of MASH-associated macrophage markers (Gpnmb, Trem2, and Lgals3) and fibrosis markers (αSMA and Col1a1) in Hsd17b13 ASO-treated mice (Fig. 6G).

Fig. 6.

Fig. 6.

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HSD17B13 knockdown alleviates MASH due to CDAHFD via decreasing cholesterol in liver lipid droplets and Patatin-like phospholipase domain-containing protein 3 I148M knock-in aggravates MASH due to CDAHFD via increasing in liver lipid droplets. (A) Study design. C57BL/6J mice were divided into GalNAc control ASO-treated mice and GalNAc Hsd17b13 ASO-treated mice. Both groups were fed a CDAHFD for 1 wk. (B) RT-qPCR analysis of liver tissues confirmed the knockdown effect of Hsd17b13 ASO. (C) Plasma ALT and AST levels in Hsd17b13 ASO-treated mice decreased compared to control mice. (D) Liver sections were stained with HE, and CD68. Hsd17b13 ASO did not significantly affect steatosis. CD68 staining revealed decreased crown-like structures. (E) The number of crown-like structures decreased in Hsd17b13 ASO-treated mice liver. (F) The metabolites levels in liver lipid droplet. Total cholesterol decreased in Hsd17b13 ASO-treated mice. The level of triglycerides, PC and PE did not change. The ratio of PC to PE did not change. (G) RT-qPCR analysis of liver tissues demonstrated a decreased mRNA expression of MASH-associated macrophage markers (Gpnmb, Trem2, and Lgals3) and fibrosis markers (αSMA and Col1a1) in Hsd17b13 ASO-treated mice. (H) Study design. Patatin-like phospholipase domain-containing protein 3 (Pnpla3) I148M knock-in mice and wild-type mice were fed with a CDAHFD for 1 wk. (I) ALT and AST in Pnpla3 I148M knock-in mice increased compared to wild-type mice. (J) Liver sections were stained with HE, and CD68. Pnpla3 I148M showed macrovesicular steatosis compared to wild-type mice. CD68 staining revealed increased crown-like structures. (K) The number of crown-like structures increased in Pnpla3 I148M knock-in mice liver. (L) The metabolite levels in the liver lipid droplet. Total cholesterol and triglycerides increased in Pnpla3 I148M knock-in mice. The level of PC and PE did not change. The ratio of PC to PE did not change. (M) RT-qPCR analysis of liver tissues demonstrated an increased mRNA expression of MASH-associated macrophage markers (Gpnmb, Trem2, and Lgals3), and fibrosis markers (αSMA, and Col1a1) in Pnpla3 I148M knock-in mice. Data are presented as mean ± SEM. Groups were compared by Unpaired one-sided Student’s t test.

Cholesterol Supplementation Negates the Protective Effect of Hsd17b13 ASO on a Choline-Deficient L-Amino Acid–Defined High-Fat Diet 1-wk Mice Model.

To test whether increases in cholesterol per se could negate the protective effect of Hsd17b13 knockdown, Hsd ASO-treated C57BL/6J mice were divided into mice fed either CDAHFD alone or CDAHFD with 2% cholesterol (SI Appendix, Fig. S5A). As before, CDAHFD+Chol exacerbated the MASH phenotype with higher transaminase concentrations (SI Appendix, Fig. S5B), and increased number of crown-like structures (SI Appendix, Fig. S5 C and D). Consistent with these findings, triglycerides and total cholesterol in liver lipid droplets increased in cholesterol-supplemented mice (SI Appendix, Fig. S5E). There was no change in lipid droplets PC and PE in CDAHFD+Chol-fed mice (SI Appendix, Fig. S5E). The ratio of PC to PE in liver lipid droplets was not different between the two diet groups. RT-qPCR demonstrated an increased Gpnmb, Trem2, and Col1a1 mRNA expression in cholesterol-supplemented mice liver (SI Appendix, Fig. S5F). Thus cholesterol supplementation abrogated the protective effects of Hsd17b13 ASO in CDAHFD-fed mice.

Patatin-Like Phospholipase Domain-Containing Protein 3 I148M Knock-In Mice Increase Cholesterol in Liver Lipid Droplets.

Next we investigated the role of PNPLA3 I148M in the development of MASH. One possible mechanism is that PNPLA3 148M competes with ATGL for the interaction with CGI-58, reducing intrahepatic lipolysis (32). To evaluate whether PNPLA3 I148M increases cholesterol content in liver lipid droplets, we studied PNPLA3 I148M knock-in mice and wild-type mice under CDAHFD 1-wk treatment (Fig. 6H). Plasma ALT and AST in PNPLA3 I148M mice were higher than in wild-type mice (Fig. 6I). The number of crown-like structures increased in the liver of PNPLA3 I148M mice (Fig. 6 J and K). Total cholesterol and triglycerides in liver lipid droplets increased in PNPLA3 I148M mice (Fig. 6L). The level of PC and PE in liver lipid droplets did not differ between wild-type and PNPLA3 I148M mice (Fig. 6L). As a result, the ratio of PC to PE in liver lipid droplets did not differ. Consistent with pathological findings, RT-qPCR demonstrated an increased mRNA expression of MASH-associated macrophage markers (Gpnmb, Trem2, and Lgals3), and fibrosis marker (Col1a1) in PNPLA3 I148M mice (Fig. 6M).

We next evaluated whether bempedoic acid treatment would decrease cholesterol in liver lipid droplets and alleviate MASH in PNPLA3 I148M knock-in mice fed with CDAHFD (SI Appendix, Fig. S6A). Bempedoic acid treatment reduced plasma ALT and AST (SI Appendix, Fig. S6B), hepatic steatosis (SI Appendix, Fig. S6C), and the number of crown-like structures compared to the control mice (SI Appendix, Fig. S6 C and D). Bempedoic acid treatment also reduced triglycerides and total cholesterol content in liver lipid droplets (SI Appendix, Fig. S6E). PC and PE in liver lipid droplets did not show a difference. The ratio of PC to PE did not show a difference. RT-qPCR demonstrated a decreased mRNA expression of MASH-associated macrophage markers (Gpnmb, Trem2, and Lgals3), and fibrosis markers (αSMA and Col1a1) in bempedoic acid–treated mice liver (SI Appendix, Fig. S6F). Taken together, these data demonstrate that decreasing hepatic cholesterol synthesis by bempedoic acid reduces hepatic lipid droplet cholesterol (and triglyceride) content and attenuates the MASH phenotype in PNPLA3 I148M knock-in mice fed with CDAHFD.

Lipid Droplet Cholesterol Is a Key Factor in Human MASLD Progression Associated with Variants of PNPLA3 and HSD17B13.

To further validate the key role of liver lipid droplet cholesterol in human MASH, we evaluated metabolites in lipid droplets of human liver samples. Human liver samples were divided into five groups (no MASL, MASL without fibrosis, MASH without fibrosis, MASH and F > 0, MASL and F > 0 but no MASH) (SI Appendix, Table S1). Total cholesterol and triglycerides in liver lipid droplets were higher in MASH and F > 0, compared to other groups (Fig. 7A). Next, we evaluated these samples from the based on the relatively common PNPLA3 I148M variant and the HSD17B13 splice variant (rs72613567), which have been shown to be aggravating or protecting variants for the development of MASH, respectively (SI Appendix, Table S2). We compared the PNPLA3 I148M variant group without HSD17B13 splice site variants as the high genetic risk group to the HSD17B13 splice site variant group without PNPLA3 I148M variants as the low genetic risk group (Fig. 7B). Total cholesterol, triglycerides PC and PE in liver lipid droplets were higher in PNPLA3 I148M variants without HSD17B13 splice site variants than in HSD17B13 splice site variants without PNPLA3 I148M variants. The ratio of PC to PE in liver lipid droplets did not differ.

Fig. 7.

Fig. 7.

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Lipid droplet cholesterol is a key factor in explaining how common genetic variants both cause and protect against MASH. (A) The metabolite levels in human liver lipid droplets. Total cholesterol, triglycerides, PC and PE in liver lipid droplets were highest in human MASH with fibrosis compared to other groups. There were no differences in the ratio of PC to PE. Data are presented as mean ± SEM. Groups were compared by one-way ANOVA followed by Tukey’s multiple comparisons test. (B) Comparison of the metabolite in human liver lipid droplet levels between PNPLA3 I148M variants without HSD17B13 splice site variants (high genetic risk) and HSD17B13 splice variants (rs72613567) without PNPLA3 I148M variants (low genetic risk). (C) The CDAHFD induces the formation of large lipid droplets, including cholesterol, in the liver. Cholesterol accumulation in lipid droplets leads to lysosome-dependent hepatocyte death due to membrane leakage, which results from a relative PC deficiency on the lipid droplet surface. This process subsequently triggers macrophage crown-like formation and stellate cell activation. Cholesterol-lowering drugs, such as statins and bempedoic acid, exhibit protective effects in the CDAHFD model by reducing cholesterol levels in lipid droplets. Additionally, we identified COASY as a potential therapeutic target for decreasing cholesterol in lipid droplets. Furthermore, cholesterol accumulation in liver lipid droplets contributes to MASH and is influenced by genetic variants, including the PNPLA3 I148M variant and the HSD17B13 splice site variant.

Discussion

MASLD is the leading chronic liver disease in the world which in turn can progress to MASH and progressive liver fibrosis. However the underlying factors that promote progression from hepatic steatosis to inflammation and liver fibrosis remain unknown (33).

Several pieces of evidence support a pathogenic role for cholesterol accumulation in MASH (34–36). Increased hepatic cholesterol promotes TAZ stabilization in hepatocytes through inhibition of its proteasomal degradation, enhancing fibrogenic and inflammatory signaling (36). In addition, impaired hepatic cholesterol sensing due to LXRα mutations leads to cholesterol accumulation, inflammation, and fibrosis, even in the absence of steatosis (35). In a recent report, MC4R knockout mice fed a Western diet demonstrate that cholesterol accumulation in macrophages plays a key role in the pathological progression of MASH (37). While these previous studies suggested a role for total hepatic cholesterol content in the pathogenesis of MASH they did not examine the potential role of cholesterol lipid droplet content in this process. Total cholesterol consists of cholesterol esters and free cholesterol. Free cholesterol is highly toxic to multiple cellular processes and organelles (38). We found that total cholesterol, as well as free cholesterol, increased in liver lipid droplets, which in turn played a key role in CDAHFD-induced MASH. Accumulation of free cholesterol can form cholesterol crystals that activate inflammation (34). However, we did not observe any cholesterol crystals in liver lipid droplets by transmission electron microscopy. Furthermore, Filipin staining, which only reflects free cholesterol, showed homogenous positive staining of cholesterol in liver lipid droplets, which taken together suggests that cholesterol crystallization in lipid droplets is not required for the induction of liver inflammation and fibrosis in this mouse model of MASH.

While cholesterol excess has been shown to exacerbate MASH, the mechanism and the potential role of cholesterol in liver lipid droplets in this process has not been fully elucidated. In our study, bempedoic acid or statin treatment decreased liver lipid droplet cholesterol content, MASH-associated macrophage markers, and liver fibrosis markers (Fig. 3 and SI Appendix, Fig. S3). MASH-associated macrophages were identified using single-cell RNA sequencing (17). This macrophage subset is strongly associated with liver fibrosis (39). Recently, it has been reported that exposure to lipid droplets, released upon injury of steatotic hepatocytes, triggers MASH-associated macrophage induction followed by liver injury (40). In our study, CDAHFD induced large lipid droplets, including cholesterol and crown-like structure, within 1 wk (Fig. 1). The crown-like structures consist of macrophages surrounding or engulfing dead or dying hepatocytes in MASH; these hepatocytes contain large lipid droplets (11). MASH-associated macrophages are known to be primarily localized to crown-like structures (40) which display a profibrotic phenotype (41). Activated fibroblasts and collagen deposition are observed around crown-like structures, and the number of crown-like structures positively correlates with the fibrosis area (42). These findings suggest that liver lipid droplet cholesterol induces a MASH-associated macrophages/crown-like structure followed by fibrosis.

Statins have not been widely used in the treatment of MASLD/MASH, partly due to concerns about potential statin-induced hepatotoxicity. Although the effects of statins on liver-related outcomes in MASLD/MASH remain inconclusive, growing evidence suggests that statin use may offer beneficial effects in these conditions (43, 44). In addition recent studies have also demonstrated that bempedoic acid reduces the development of liver inflammation in mouse models of MASH (22, 45).

To further examine the role of lipid droplet cholesterol content in the pathogenesis of liver inflammation and fibrosis we assessed the effect of Coasy ASO treatment on the progression of MASH. COASY converts 4′-phosphopantetheine (PPanSH) into CoA by adenylation and phosphorylation. COASY is a bifunctional enzyme, in the 4th and 5th step of the CoA biosynthetic pathway from pantothenate (Pan) (46). Coasy knockdown decreased liver lipid droplet cholesterol, MASH-associated macrophage markers, and liver fibrosis markers similar to statin or bempedoic acid treatment (Fig. 4). Taken together, our findings (Figs. 4 and 5) indicate that the liver COASY/CoA/acetyl-CoA axis represents a potential therapeutic target to decrease hepatic cholesterol synthesis, leading to MASH and that by targeting COASY knockdown to the liver might avoid potential CNS toxicities (47).

Advances in understanding the genetic underpinnings of MASH have revealed important insights into MASH pathogenesis (27). Genetic variants in HSD17B13 have been shown to protect against the development of MASH and conversely the PNPLA3 I148M variant has been shown to promote the development of MASH (32, 48–53). Our Hsd17b13 ASO mouse studies and our PNPLA3 I148M knockin mouse studies replicated these same traits and demonstrate that cholesterol in liver lipid droplets may explain the mechanism responsible for the protective effects of the HSD17B13 variants in the development of MASH as well as the mechanism by which the PNPLA3 I148M variant promotes the development of MASH in humans.

Furthermore, we confirmed in humans that cholesterol in liver lipid droplets was higher in individuals with MASH and liver fibrosis than in individuals with MASL, MASL without fibrosis, and MASH without fibrosis using human liver biopsy samples. Furthermore, consistent with our mouse HSD17B13 gene knockdown and PNPLA3 I148M gene knock-in mouse studies, we found that total cholesterol and triglyceride content in liver lipid droplets were higher in humans with the PNPLA3 I148M variant and without the HSD17B13 splice site variants (high genetic risk group) than in HSD17B13 splice site variants without the PNPLA3 I148M variant (low genetic risk group).

Choline deficiency and reduced hepatic PC content have been shown to be associated with liver disease in a variety of conditions (54). Increased liver PC content has been shown to be protective in MASH in carriers of variants HSD17B13 rs72613567 and MARC1 A165T (55, 56) while carriers of the harmful variants PNPLA3 I148M and TM6SF2 E167K demonstrate decreased liver PC content (57, 58). Taken together these results suggest that liver PC content might play a role in the progression of MASH, however the mechanism by which liver PC content alters the pathogenesis of MASH is unknown and prior studies have not evaluated PC content in specific hepatocellular compartments. Here, we focused on PC and PE, specifically in liver lipid droplets, because the lipid droplet membrane consists of a monolayer of PC and PE, which plays a vital role in lipid droplet dynamics (59). Lipid droplets have two principal components; the amphipathic coat and the hydrophobic core. The coat primarily is composed of amphipathic lipids, including PC and PE and free cholesterol. By contrast, the hydrophobic core comprises neutral lipids, which mainly include cholesterol esters and triglycerides. PC is cylindrically shaped and would be predicted to provide good coverage of the lipid droplet surface. In contrast, PE is cone-shaped and would be expected to provide less coverage of the lipid droplet surface. We therefore hypothesized that PC deficiency might lead to packing defects on the lipid droplet monolayer (SI Appendix, Fig. S7A) which would then be filled by triglycerides and cholesterol thus exposing these lipid droplet metabolites, which are normally sequestered inside the lipid droplet, to other intracellular organelles such as the lysosomes (60). Free cholesterol accumulation in a gap of phospholipids on the lipid droplet surface might lead to cholesterol crystal formation which has been suggested to contribute to MASH pathogenesis (38). We confirmed that CDAHFD decreased the ratio of PC to PE in liver lipid droplets and increased free cholesterol content in liver lipid droplets (Fig. 2).

Cholesterol can induce phase separation in lipid monolayers composed of various phospholipids (61), and direct interactions between cholesterol and phospholipids regulate cholesterol’s accessibility for participation in signaling reactions (62). In our study, CDAHFD feeding increased the ratio of free cholesterol to [PC/PE] in liver lipid droplets compared to RC feeding (SI Appendix, Fig. S7B). These data suggest that free cholesterol accumulates on the surface of liver lipid droplets under PC deficiency in CDAHFD-fed mice.

Based on these findings, we hypothesize that free cholesterol accumulation in liver lipid droplets under PC deficiency may lead to free cholesterol leaking from the lipid droplets’ surface to other organelles, especially lysosomes, without crystal formation. Transmission electron microscopy revealed lipid droplets in lysosomes and lysosomal membrane disruption (Fig. 2). Consistently, cathepsin D immunostaining showed CDAHFD-induced leakage of cathepsin D into the cytoplasm. TUNEL staining revealed apoptotic hepatocytes in CDAHFD-fed mice liver. In general, free cholesterol is highly insoluble, cannot diffuse within the aqueous environment of the lysosomal lumen, and impairs lysosomal function (63). Abnormal cholesterol accumulation in lysosomes induces lysosomal membrane permeabilization, leading to lysosome-dependent cell death (64). It is possible that cholesterol accumulation in liver lipid droplets may cause lysosomal damage followed by lysosome-dependent hepatocyte death linked to CDAHFD-induced MASH pathogenesis.

Lgals3 mRNA expression was strongly induced by CDAHFD and suppressed by bempedoic acid, statin treatment, Coasy ASO, or Hsd17B13 ASO in our study. Cholesterol dietary supplementation or PNPLA3 I148M knock-in increased Lgals3 mRNA expression. Galectin-3 (gene name Lgals3) is a fibrogenic factor known to be secreted from activated macrophage, leading to hepatic stellate cell activation (20, 65). Galectin-3 inhibitor is now under clinical trial for treating MASH (66). Interestingly, the choline-deficient amino acid–defined diet simultaneously increases Lgals3 mRNA expression and MASH-associated macrophage markers in mouse livers (67). In addition, in vitro analysis using macrophages or smooth muscle cells demonstrates that cholesterol supplementation induces Galectin-3 expression (37, 68). CDAHFD treatment induces liver lipid droplet levels of cholesterol, triggering the development of MASH-associated macrophages, which possibly secret Galectin-3 leading to activation of hepatic stellate cells.

In conclusion, these data identify cholesterol accumulation in liver lipid droplets as a key mediator of liver inflammation and fibrosis in MASH (Fig. 7C). The buildup of cholesterol in liver lipid droplets leads to macrophage crown-like formation and stellate cell activation. This mechanism also explains the effects of the HSD17B13 variants to protect against the development of MASH and the effects of the PNPLA3I148M variant to promote MASH. Hepatic knockdown of COASY and HSD17B13 expression as well as bempedoic acid treatment represent therapeutic approaches to reduce cholesterol content in liver lipid droplets and thereby prevent the progression of hepatic steatosis to MASH and liver fibrosis.

Materials and Methods

Additional details of the materials and methods are provided in SI Appendix, Materials and Methods.

Supplementary Material

Appendix 01 (PDF)

pnas.2502978122.sapp.pdf (4.6MB, pdf)

Acknowledgments

We thank John Stack, Xiaoxian Ma, Wanling Zhu, and the Yale Histology Core Service for their excellent technical assistance. This study was supported by grants from the United States Public Health Service NIH/National Institute of Diabetes and Digestive and Kidney Diseases (F31DK126362 [T.E.L.], T32 GM007324 [T.E.L.], P30 DK34989, R01 DK119968 [G.I.S.], R01 DK113984 [G.I.S.], P30 DK045735 [G.I.S.], R01 DK133143 [G.I.S.]. I.S. was supported by the Manpei Suzuki Diabetes Foundation, Mishima Kaiun Memorial Foundation, Kowa Life Science Foundation, Japan Foundation for Applied Enzymology, Takeda Science Foundation, Ono Medical Research Foundation and the Ministry of Education, Culture, Sports, Science and Technology (Japan) Fund for the Promotion of Joint International Research [Fostering Joint International Research (A); #20KK0373].

Author contributions

I.S. and G.I.S. designed research; I.S., R.C.G., A.R.N., S.D., M.K., J.Z., T.E.L., M.T.G., Y.T., Y.K., M.P., D.F.V., M.H., B.K., S.K., and T.T. performed research; Y.K., M.P., M.H., B.K., S.K., M.R., and T.T. contributed new reagents/analytic tools; I.S., R.C.G., A.R.N., S.D., M.K., J.Z., T.E.L., M.T.G., Y.T., Y.K., D.Y., M.P., D.F.V., K.F.P., M.H., B.K., S.K., M.R., V.T.S., T.T., and G.I.S. analyzed data; and I.S., R.C.G., K.F.P., V.T.S., and G.I.S. wrote the paper.

Competing interests

M.P., an Ionis employee, develops ASO therapeutics and may own Ionis stock. All other authors declare no competing interests.

Footnotes

Reviewers: D.E.C., Brigham and Women’s Hospital; and A.R., The University of Texas Southwestern Medical Center.

Data, Materials, and Software Availability

The proteomics data was deposited in the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org/) via the jPOST partner repository (http://jpostdb.org) with the dataset identifier PXD063049 (69). All study data are included in the article and/or SI Appendix.

Supporting Information

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Appendix 01 (PDF)

pnas.2502978122.sapp.pdf (4.6MB, pdf)

Data Availability Statement

The proteomics data was deposited in the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org/) via the jPOST partner repository (http://jpostdb.org) with the dataset identifier PXD063049 (69). All study data are included in the article and/or SI Appendix.

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