Sex-based multiomics analysis uncovers metabolic and molecular mediators linking MASH and atherosclerosis

Sandeep Das; Sumit Kumar Anand; M Peyton McKinney; Koral SE Richard; Iqbal Mahmud; Sumati Rohilla; Fabio Arias; Alia Ghrayeb; Bo Wei; Lin Tan; Zhipeng Liu; Dhananjay Kumar; Alexandra C Finney; Nilesh Pandey; Harpreet Kaur; Rajan Pandit; Xiaolu Zhang; Cyrine Ben Dhaou; Sarah P Thayer; Babak Razani; Bishuang Cai; Fei Chang; Francisco J Schopfer; Wanqing Liu; Edward A Fisher; Sridhar Radhakrishnan; Eyal Gottlieb; A Wayne Orr; Nirav Dhanesha; Arif Yurdagul, Jr; Philip L Lorenzi; Oren Rom

doi:10.1016/j.jhepr.2025.101703

. 2025 Dec 2;8(3):101703. doi: 10.1016/j.jhepr.2025.101703

Sex-based multiomics analysis uncovers metabolic and molecular mediators linking MASH and atherosclerosis

Sandeep Das ^1,^†, Sumit Kumar Anand ^1,^†, M Peyton McKinney ^1,^†, Koral SE Richard ^1,^†, Iqbal Mahmud ^2,^†, Sumati Rohilla ¹, Fabio Arias ¹, Alia Ghrayeb ^1,³, Bo Wei ², Lin Tan ², Zhipeng Liu ⁴, Dhananjay Kumar ⁵, Alexandra C Finney ¹, Nilesh Pandey ¹, Harpreet Kaur ¹, Rajan Pandit ⁵, Xiaolu Zhang ⁶, Cyrine Ben Dhaou ¹, Sarah P Thayer ⁷, Babak Razani ⁸, Bishuang Cai ⁹, Fei Chang ¹⁰, Francisco J Schopfer ¹⁰, Wanqing Liu ¹¹, Edward A Fisher ¹², Sridhar Radhakrishnan ¹³, Eyal Gottlieb ³, A Wayne Orr ^1,⁵, Nirav Dhanesha ¹, Arif Yurdagul Jr ^1,⁵, Philip L Lorenzi ^2,^⁎,^#, Oren Rom ^1,^5,^⁎,^#,^ˆ

¹Department of Pathology and Translational Pathobiology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, 71103, USA

²Metabolomics Core Facility, Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA

³Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX, 77054, USA

⁴Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN, 47907, USA

⁵Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, 71103, USA

⁶Bioinformatics and Modeling Core, Department of Microbiology and Immunology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, 71103, USA

⁷Department of Surgery, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, 71103, USA

⁸Division of Cardiology and Vascular Medicine Institute, Department of Medicine, University of Pittsburgh School of Medicine and University of Pittsburgh Medical Center, Pittsburgh, PA, 15261, USA

⁹Department of Medicine, University of California, Los Angeles, 100 Medical Plaza #205, Los Angeles, CA, 90095, USA

¹⁰Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15261, USA

¹¹Department of Pharmaceutical Sciences and Department of Pharmacology, Wayne State University, Detroit, MI, 48201, USA

¹²Department of Cell Biology, NYU Grossman School of Medicine, NY, 10016, USA

¹³Research Diets, Inc., New Brunswick, NJ, 08901, USA

^⁎

Corresponding authors. Addresses: Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, 71103, USA. (O. Rom), or The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. (P.L. Lorenzi). plorenzi@coh.orgoren.rom@lsuhs.edu

^†

These authors contributed equally

These authors share senior authorship

^ˆ

Lead contact

PMCID: PMC12950431 PMID: 41777553

Abstract

Background & Aims

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of death in patients with metabolic dysfunction-associated steatohepatitis (MASH). No therapy targets both diseases simultaneously, and a roadblock for discovering new treatments is the lack of animal models that recapitulate both diseases, especially in females.

Methods

Male and female Ldlr^-/- mice (n = 8-13) were fed a western diet (WD), modified choline-deficient high-fat diet (mCDHFD), or modified MASH-inducing diet (mMASHD) containing equivalent physiological levels of cholesterol. Comprehensive multiomics including metabolomics, lipidomics, and transcriptomics, alongside histopathological and biochemical analyses, were integrated to characterize concurrent MASH and atherosclerosis. Transcriptomics was validated in other mouse models and integrated with human data (n = 79).

Results

While mCDHFD induced MASH-fibrosis in both sexes, WD was effective only in males, whereas mMASHD primarily affected females. mCDHFD induced concurrent MASH and atherosclerosis in both sexes, while WD effectively recapitulated disease co-occurrence only in males. Correlation analyses highlighted links between MASH and atherosclerosis, identifying circulating cholesterol and C–C motif chemokine ligand 2 (CCL2) as potential predictors of coexisting disease (p <0.04). Integrated metabolomic and transcriptomic analyses identified arginine–proline, glycine–serine, glutathione, and sphingolipid metabolism (p <0.03) as key dysregulated pathways, with sphinganine emerging as a predictor of disease severity. Hepatic itaconate and lactate levels were positively correlated with disease severity, whereas glycine, carnitine, 2-aminomuconic acid, and thiamine pyrophosphate were negatively associated (p <0.04). Lipidomic analyses revealed dysregulated polyunsaturated fatty acid, steryl ester, and dihexosylceramide metabolism. Integration of mouse and human transcriptomes revealed similarities in metabolic and proinflammatory/proatherogenic pathways.

Conclusion

This sex-based multiomics analysis establishes a murine model of concurrent MASH and atherosclerosis, reveals sex-specific dietary responses, and identifies metabolic and transcriptional pathways with potential utility as biomarkers and therapeutic targets.

Impact and implications

This study addresses the critical need for an animal model that replicates both metabolic dysfunction-associated steatohepatitis (MASH) and atherosclerotic cardiovascular disease, particularly in females, to facilitate therapeutic development. Using male and female Ldlr^-/- mice, we found that different diets containing equivalent physiological levels of cholesterol induce sex-specific responses, with a modified choline-deficient high-fat diet effectively modeling both diseases in both sexes, while a western diet is effective only in males. Multiomics analyses identified key metabolic and inflammatory pathways linking MASH and atherosclerosis that mirror transcriptomic signatures found in humans, and highlight circulating cholesterol, CCL2, and sphinganine as potential biomarkers. These findings establish a translational model and reveal sex-specific metabolic pathways that will advance our understanding of the shared pathophysiology of MASH and atherosclerosis, and facilitate the development of dual therapeutic approaches, addressing an urgent unmet clinical need.

Keywords: MASLD, MASH, Atherosclerotic cardiovascular disease, Animal models, Metabolomics, Transcriptomics

Graphical abstract

Highlights

•
ASCVD is the leading cause of death in MASH, with no dual-target therapy available.
•
Ldlr^-/- mice on different diets modeled sex-specific MASH and atherosclerosis.
•
Multiomics identified key metabolic pathways and biomarkers of disease severity.
•
Inflammatory transcriptional pathways show the most overlap between mice and humans.

Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common liver disorder, affecting nearly one-third of the global population.¹ MASLD represents a spectrum of liver pathologies, ranging from simple steatosis to metabolic dysfunction-associated steatohepatitis (MASH), which is characterized by hepatocyte injury and lobular inflammation in association with hepatic fibrosis.² MASLD is closely associated with metabolic comorbidities such as obesity, insulin resistance, and type 2 diabetes, all of which increase the risk of atherosclerotic cardiovascular disease (ASCVD).²^,³ While MASH can progress to cirrhosis and hepatocellular carcinoma, thereby increasing liver-related mortality, the leading cause of death in patients with MASH is ASCVD.[4], [5], [6], [7]

ASCVD, a chronic disease of the arteries characterized by the buildup of fibrofatty plaques, is driven by dysregulated lipid homeostasis and inflammatory responses.³ The excessive accumulation of apolipoprotein B-containing lipoproteins in the arterial wall initiates a robust proinflammatory response, promoting the development of atherosclerotic plaques.³^,⁶^,⁸ As these plaques evolve, necrotic cores form due to impaired clearance of apoptotic, lipid-laden macrophages, contributing to plaque instability.³^,⁹ The rupture of these unstable plaques underlies acute cardiovascular events, including myocardial infarction and stroke.³^,⁹ Atherosclerosis is further promoted by risk factors, such as obesity and type 2 diabetes, conditions frequently present in patients with MASH.⁴ Nevertheless, MASH increases the risk of ASCVD, independent of traditional metabolic risk factors.⁵^,⁶ Despite major advances in our understanding of the pathophysiology of MASH and ASCVD, the metabolic and molecular mechanisms linking these two diseases remain unknown, hampering the development of concurrent treatments.

Statins, the most common treatment for ASCVD, are generally considered safe for patients with liver disease. However, clinical trials investigating statins as a treatment for MASH have produced inconsistent results.⁴^,¹⁰ Recently, the FDA approved resmetirom (thyroid hormone receptor beta agonist), and semaglutide (GLP-1 receptor agonist) for the treatment of MASH.¹¹ Yet, their cardiovascular consequences remain unknown. Despite significant efforts in developing drugs for MASH and ASCVD, no current therapy targets both diseases simultaneously. One major reason for this gap is the absence of animal models that effectively recapitulate both diseases concurrently. Moreover, sexual dimorphism significantly influences the development and progression of MASLD and ASCVD, and the complex roles of sex hormones and genetic predisposition further complicate therapeutic management.[12], [13], [14] Sex differences in MASLD and ASCVD are often overlooked, delaying treatments,¹² hence new translational rodent models remain essential for uncovering sex-dependent disease mechanisms. To address this gap and advance the development of therapies for both diseases while accounting for sex differences, we used male and female atheroprone mice, combined with dietary regimens containing physiologically relevant cholesterol levels, to model concurrent MASH and atherosclerosis. Using a multiomics approach that integrates metabolomics, lipidomics, and transcriptomics with histopathological and biochemical analyses, we report a murine model that concurrently features MASH and atherosclerosis. This model displays transcriptomic signatures comparable to those of human disease and reveals sexual dimorphism in response to dietary regimens. The use of this model will be instrumental in advancing our understanding of the shared pathophysiology of MASH and atherosclerosis and in facilitating the development of dual therapeutic approaches.

Materials and methods

Experimental model

All animal procedures were approved by the Institutional Animal Care & Use Committees of Louisiana State University Health Sciences Center-Shreveport (P-21-043, P-22-035, P-24-025). All animal experiments were performed according to the institutional guidelines. Mice were randomly allocated to treatment groups followed by confirmation of equal body weights between the groups prior to the initiation of the dietary regimens. Ldlr^-/-, Apoe^-/- and C57BL/6J mice were purchased from the Jackson Laboratories (stock: 002207, 002052, and 000664, respectively). Eight-week-old male and female mice were housed under controlled temperature (22 ± 2 °C) and humidity conditions (40-60%) on a 12-hour light/dark cycle and fed either a MASH-inducing diet (Research Diets, D17010103, 40% of calories from fat),¹⁵^,¹⁶^,[19], [20], [21] western diet (WD, Envigo TD.88137, 42% fat, 0.2% cholesterol), commonly used in atherosclerosis mouse studies,¹⁷^,¹⁸^,²² the modified choline-deficient high-fat diet (mCDHFD, Research Diets #D22052704, 45% fat, without added choline, 0.2% cholesterol), or a modified MASHD¹⁵^,¹⁶^,[19], [20], [21]^,²³ (mMASHD, Research Diets #D22052703, 40% fat, 0.2% cholesterol) for 12 or 24 weeks. The detailed composition of the diets, including standard diet (SD), WD, mCDHFD, and mMASHD, is provided in Table S1. Mice fed the SD (LabDiet #5053, 13% fat) served as controls.

Statistical analysis

Statistical analyses and data visualization were performed using GraphPad Prism v.10 software. All data were tested for normality and equal variance. When these assumptions were met, comparisons among more than two groups were performed using one-way ANOVA followed by Bonferroni post hoc tests. Otherwise, non-parametric tests (Mann-Whitney U test or Kruskal-Wallis test followed by Dunn’s post hoc test) were used. For comparisons between two groups, an unpaired t test (if normality was passed) or Mann-Whitney U test were used. For the metabolomic and lipidomic datasets, multivariate analysis was performed using principal component analysis (PCA)-based non-parametric PERMANOVA (permutational multivariate analysis of variance) implemented in the MetaboAnalystR package. Hierarchical clustering and heatmap visualization were conducted using the ggplot2 package in R. For the hierarchical clustering analysis, data were normalized using z-score transformation prior to visualization. Differential abundance analysis was performed with significance thresholds set at p <0.05 and fold-change >1.5. Data processing, analysis, and visualization were conducted using multiple R packages, including ggplot2, dplyr, and tidyr. Pathway enrichment analysis was performed using the Mus musculus (house mouse) KEGG database, employing a hypergeometric test to assess statistical significance of pathway enrichment. Pathway visualization was generated using ggplot2. For RNA sequencing, gene level quantification was performed using HTSeq-counts v0.6.0 based on the GRCm38.90 genome annotations. The R package DESeq2 v1.42.1 was then used to identify significantly differentially expressed genes (DEGs). A two-sided Wald test was used to identify DEGs. Genes with adjusted p values <0.05 were considered significant. The upregulated and downregulated DEGs were analyzed for significantly enriched KEGG pathways using the clusterProfiler v4.10.1 package. The significance of the enrichment was determined by the right-tailed Fisher’s exact test followed by Benjamini-Hochberg multiple testing adjustment.

Detailed methods are provided in the supplementary materials.

Results

Diet-induced MASH in male and female atheroprone mice

To establish a model for concurrent MASH and atherosclerosis, we utilized different dietary regimens in atheroprone mice. The importance of high dietary cholesterol (>1.25%) for modeling the full spectrum of MASH-fibrosis in mice and larger animals is established.[12], [13], [14], [15], [16] Nevertheless, such high levels are supraphysiological, particularly in the hyperlipidemic Ldlr^-/- mouse model. To overcome this, we designed diets with physiologically relevant cholesterol levels, including the WD (0.2% cholesterol), commonly used in atherosclerosis studies,¹⁹^,²¹^,²² the CDHFD,¹⁷ and our established MASHD,[13], [14], [15], [16] with the latter two diets modified to contain equivalent cholesterol levels as the WD (mCDHFD and mMASHD, respectively, Table S1). To study the full spectrum of MASH, mice were fed the above diets for a total of 24 weeks and compared to those fed a SD.[13], [14], [15], [16] To address sexual dimorphism in MASH and atherosclerosis,¹⁸^,[23], [24], [25] both male and female mice were included throughout (Fig. 1A).

Compared to SD, male Ldlr^-/- mice fed the WD, mCDHFD, or mMASHD showed a significant increase in body weight (Fig. S1A), and hepatomegaly (Fig. 1B), with more pronounced effects in the WD and mCDHFD groups. Liver weight and liver-to-body weight ratio (Figs 1C and S1B) followed similar trends. All three diets significantly elevated serum alanine and aspartate aminotransferase (ALT and AST, Fig. 1D,E), though AST levels were lower in mMASHD-fed mice than in WD-fed mice. Histological (Fig. 1F) and biochemical analyses revealed significantly increased hepatic steatosis, triglycerides and cholesterol in all groups (Figs 1G and S1C,D), with mMASHD inducing a milder phenotype than WD and mCDHFD. Lobular inflammation and F4/80⁺ macrophages (Figs 1F,G and S2A) were significantly higher in all groups, but lower in mMASHD than WD or mCDHFD. We further evaluated alterations in immune cell subsets. The expression of Ly6G, a marker of neutrophil infiltration,¹⁵ was significantly elevated across all groups, with the mMASHD group showing lower neutrophils compared to the WD and mCDHFD groups (Fig. S2D and E). We further assessed the expression of chemokine receptor 2 (CCR2) and triggering receptor expressed on myeloid cells 2 (TREM2) in macrophages to assess infiltration of monocyte-derived macrophages into the liver.¹⁶^,[26], [27], [28] F4/80⁺CCR2⁺ (Fig. S2D and F) and F4/80⁺TREM2⁺ (Fig. S2D and G) macrophages significantly increased in all groups, but were lower in the mMASHD group. Hepatocellular ballooning and the NAFLD activity score (NAS, Fig. 1F,G) significantly increased across all groups but were lower in mMASHD-fed mice. Picrosirius red (PSR) staining (Figs 1F and S2B) and hepatic hydroxyproline content (Fig. S2C) confirmed hepatic fibrosis in all groups, with the lowest levels in mMASHD-fed mice. Altogether, WD and mCDHFD induced severe MASH and fibrosis, whereas mMASHD led to a milder phenotype.

Male mice are more commonly used for studying MASH as females demonstrate inconsistency in disease progression.²⁴^,²⁵ Therefore, establishing a model that recapitulates MASH-fibrosis and concurrent atherosclerosis in female mice is crucial. Endpoint analyses in females showed a significant increase in body weight in the WD, mCDHFD, and mMASHD groups compared to SD, with the greatest effects observed with mCDHFD (Fig. S1E). Hepatomegaly and increased liver weight were evident in all diet groups (Figs 1H and S1F). However, unlike males, these effects were most pronounced in mMASHD-fed females, the only group showing increased liver-to-body weight ratio (Fig. 1I). Plasma ALT and AST (Fig. 1J,K) were significantly elevated in mMASHD-fed females, with comparable levels in the mCDHFD group, while WD-fed mice showed lower levels. Hepatic steatosis (Fig. 1L,M) and triglycerides (Fig. S1G) were significantly higher in mCDHFD- and mMASHD-fed mice, while hepatic cholesterol increased similarly across all diets (Fig. S1H). Lobular inflammation and F4/80⁺ macrophages (Figs 1L,M and S2H), Ly6G⁺ neutrophils (Fig. S2K,L) and monocyte-derived macrophages (F4/80⁺CCR2⁺ and F4/80⁺TREM2⁺, Fig. S2K,M,N) were increased in mCDHFD- and mMASHD-fed but not in WD-fed mice. Hepatocellular ballooning and NAS (Fig. 1M) significantly increased in the mCDHFD and mMASHD groups compared to the WD group. Hepatic fibrosis score, PSR staining, and hydroxyproline content (Figs 1L,M and S2I,J) were significantly higher in mCDHFD- and mMASHD-fed mice than in WD-fed mice. Thus, while mCDHFD induces MASH and fibrosis in both sexes, WD is effective only in males, whereas mMASHD primarily affects females. These findings highlight sex-specific differences in MASH development across dietary models.

Effects of MASH-inducing diets on atherosclerosis in atheroprone mice

We next evaluated the effects of each diet on atherosclerotic plaque size and composition in male Ldlr^-/- mice. En face analysis (Fig. 2A,B) showed a significant increase in atherosclerotic lesions along the aortic tree in WD-, mCDHFD-, and mMASHD-fed mice compared to SD. Consistent with MASH severity, lesions were lower in mMASHD-fed mice than in WD- or mCDHFD-fed mice. Comprehensive analysis of aortic sinuses (Figs 2C and S3A) using H&E, Oil red O, Van Gieson’s, and PSR staining, as well as immunofluorescence for CD68 and intercellular adhesion molecule-1 (ICAM-1) assessed in endothelial cells (von Willebrand factor positive area; vWF⁺) confirmed these findings. Plaque size, lipid content, necrotic acellular area, cap thickness, and inflammation (Figs 2D-H and S3B) significantly increased in all diet groups. WD- and mCDHFD-fed mice showed increased atherosclerotic severity in all analyses except for fibrous cap thickness. Unlike males, where MASH and atherosclerosis severity correlated across the different diets, females exhibited differing severities. A significant increase in lesion areas in aortic trees (Fig. 2I,J) and sinuses (Fig. 2K,L) was found in all groups. Nevertheless, although the mMASHD effectively induced MASH-fibrosis in females, lesion areas were significantly lower in the mMASHD group compared to the WD or mCDHFD groups. Other indices of plaque composition, including lipid content, acellular area, fibrous cap thickness, macrophage content, and ICAM1 expression in endothelial cells, were significantly increased in all groups (Figs 2M-P and S3E,F) in females. These studies indicate mCDHFD as a robust model for concurrent MASH and atherosclerosis in both sexes, while WD effectively recapitulates the co-occurrence of these diseases only in male mice.

Circulating factors linking MASH and atherosclerosis

Next, we evaluated the effects of the different diets on circulating glucose, lipids, chemokines, and adhesion molecules linking MASH and atherosclerosis.¹⁷^,[29], [30], [31], [32], [33] In males, fasting glucose was significantly increased in mice fed mCDHFD or mMASHD, but not WD (Fig. 3A), suggesting it is a poor predictor of concurrent MASH and atherosclerosis. Unlike glucose, the lipid profile closely correlated with the severity of concurrent MASH and atherosclerosis in male mice. Plasma total cholesterol and triglycerides (Fig. 3B,C) were significantly increased by all diets, but cholesterol was significantly lower in mMASHD-fed mice, in line with MASH and atherosclerosis severities. Fast protein liquid chromatography analyses revealed that cholesterol and triglycerides (Fig. 3D,E) were comparably high within the VLDL and LDL fractions in WD- and mCDHFD-fed mice, but markedly lower in mMASHD-fed mice. Similarly, circulating C–C motif ligand 2 (CCL2), CCL5, vascular cell adhesion molecule-1 (VCAM-1), and ICAM-1 (Figs 3F,G and S3C,D) were significantly elevated across all groups, but were lower in mMASHD-fed mice. To address the relationship between MASH and atherosclerosis in male mice, we next assessed the correlation between key disease indices in the liver, atherosclerotic plaque and plasma. Nearly all disease parameters were significantly and positively correlated, with plasma total cholesterol, NAS, hepatic fibrosis score, and plaque CD68⁺ macrophages showing the strongest correlations (Fig. 3H). In female mice, fasting glucose, lipid parameters, chemokine levels, and adhesion molecules were increased in all diet groups compared to SD, with no significant intergroup differences (Figs 3I-O and S3G,H). Most of the liver, atherosclerosis and plasma indices were significantly and positively correlated, with hepatic PSR and NAS, plasma total cholesterol and CCL2 showing the strongest correlations (Fig. 3P). Altogether, these findings underscore the strong link between MASH and atherosclerosis, highlighting circulating cholesterol and CCL2 as potential predictors of coexisting diseases.

Fig. 3 — Circulating factors linking MASH and atherosclerosis. (A, I) Fasting blood glucose. (B, J) Plasma total cholesterol, and (C, K) triglycerides. (D, L) Cholesterol and (E, M) triglyceride contents in lipoproteins via FPLC. (F, N) Plasma CCL2 and (G, O) CCL5. (H, P) Correlations between disease indices in liver, plasma, and atherosclerotic plaques. Data are mean ± SEM (n = 10-12/group). One-way ANOVA with Tukey’s *post hoc* test or Kruskal-Wallis with Dunn’s *post hoc* test. Pearson and Spearman correlations were used to assess linear and non-linear relationships, respectively. *p <*0.05 was considered statistically significant. MASH, metabolic dysfunction-associated steatohepatitis; mCDHFD, modified choline-deficient high-fat diet; mMASHD, modified metabolic dysfunction–associated steatohepatitis diet; SD, standard diet; WD, western diet.

Distinct metabolic signatures in concurrent MASH and atherosclerosis

Identifying dysregulated metabolic pathways in concurrent MASH and atherosclerosis is essential for understanding pathophysiology and discovering therapeutic targets. Using unbiased metabolomics via ion chromatography-mass spectrometry and hydrophilic interaction liquid chromatography-mass spectrometry, we detected 216 liver and 126 plasma polar metabolites. In males, PCA of the liver and plasma metabolomes (Fig. 4A,B) showed a clear separation between the groups. We next performed KEGG-based pathway analysis on liver and plasma metabolomes (Fig. 4C,D). Most metabolic pathways were significantly downregulated, including arginine–proline, glycine–serine, and glutamate metabolism. Pyrimidine and purine metabolism were among the top significantly downregulated pathways in WD- and mCDHFD-fed mice, but not in mMASHD-fed mice. We employed heatmap and volcano plot representation to identify prominent metabolic changes (Figs 4E and S4A-D). The hepatic metabolome of WD- and mCDHFD-fed mice was highly similar, whereas mMASHD-fed mice were more comparable to SD-fed mice. Certain metabolites, including carnitine, glycine, ornithine, and glycerol 3-phosphate, were significantly decreased across all diets, while purine and pyrimidine metabolites (UTP, UDP, dCDP) significantly declined in WD and mCDHFD-fed mice but not in mMASHD-fed mice (Figs 4E and S4A-D). Conversely, hepatic glucose, fructose 1-phosphate, sphinganine, oleic acid, itaconate, oxalate, and lactate significantly increased in all diet groups, but sphinganine decreased in livers from mMASHD-fed mice compared to WD- or mCDHFD-fed mice (Figs 4E and S4A-D). In plasma, glycine and serine metabolism, along with arginine–proline metabolism, were significantly downregulated across all diets. The citric acid cycle pathway was significantly downregulated in WD- and mCDHFD-fed mice but upregulated in mMASHD-fed mice. In contrast, glycolysis was the most significantly upregulated pathway in WD-fed mice, but not in mCDHFD or mMASHD (Fig. 4D). In line with these altered pathways, arginine significantly decreased while ornithine and dimethylglycine increased across all diets. Malonate significantly decreased in WD- and mCDHFD-fed mice but not in mMASHD-fed mice. Glucose, oleic acid, and sphinganine significantly increased across all diets, although sphinganine was lower in mMASHD-fed compared to WD-fed mice (Figs 4F, S5A-D).

In females, PCA revealed distinct liver and plasma metabolomes across the diets (Fig. 4G,H). Similar to males, glycine–serine metabolism, arginine–proline metabolism, and pyrimidine metabolism pathways were significantly downregulated in livers of WD-, mCDHFD-, or mMASHD-fed females. Pyrimidines (uridine, CTP, and CDP), carnitine and glycine were significantly decreased in all groups (Figs 4K and S6A-D). While oleic acid and palmitoleic acid were significantly increased across all groups, itaconate and sphinganine were elevated only in mCDHFD-fed females (Fig. S6B–D). In female plasma, glycine–serine metabolism, and arginine–proline metabolism, were the most significantly downregulated pathways across all diets (Fig. 4J), as found in males. Unlike males, females exhibited consistent glycolysis upregulation and citric acid cycle downregulation across all groups. Plasma arginine significantly decreased, while ornithine and dimethylglycine were markedly increased (Figs 4L and S7A-D). Notably, sphinganine was significantly increased only in mCDHFD-fed females (Fig. S7B–D), which showed the strongest co-occurrence of MASH and atherosclerosis. Collectively, these findings highlight arginine–proline metabolism and glycine–serine metabolism as critical pathways implicated in concurrent MASH and atherosclerosis, and indicate sphinganine as a potential predictor of disease severity.

The hepatic metabolome links MASH and atherosclerosis

To identify metabolites as potential predictors or therapeutic targets in concurrent MASH and atherosclerosis, we examined the relationship between the hepatic metabolome, MASH, and atherosclerosis indices. Focusing on the top 50 dysregulated hepatic metabolites in males, sphinganine showed the strongest and most consistent positive association with all disease indices across the liver, plasma, and atherosclerotic plaques. Other metabolites with significant positive associations with MASH severity (NAS and fibrosis score), plasma markers (total cholesterol, ALT, and CCL2), and atherosclerosis (lesion area and macrophage content) included itaconate, glucose, oleic acid, succinylcarnitine, fructose, and lactate. In contrast, 2-aminomuconic acid, pyridoxal 5′-phosphate, glycerol 3-phosphate, thiamine pyrophosphate, carnitine, and glycine showed significant negative correlations with these indices (Fig. 5A). In females, oleic acid showed significant positive correlations with most MASH, plasma, and atherosclerosis indices. Sphinganine was positively associated with plasma cholesterol and atherosclerosis indices, with a similar trend for NAS. Itaconate was positively correlated with NAS, plasma cholesterol, lesion area and macrophages in aortic sinuses. Conversely, 2-aminomuconic acid, glycerol 3-phosphate, thiamine pyrophosphate, carnitine, and glycine showed significant negative correlations with NAS, plasma cholesterol and CCL2, and aortic sinus lesion area (Fig. 5B).

Fig. 5 — Correlations between the hepatic metabolome and MASH and atherosclerosis indices. Pearson and Spearman correlations were computed to assess relationships between the top 50 dysregulated liver metabolites and MASH, plasma, and atherosclerosis disease markers in (A) male and (B) female *Ldlr*^-/- mice (n = 4-5/group). ALT, alanine aminotransferase; AST, aspartate aminotransferase; MASH, metabolic dysfunction–associated steatohepatitis; NAS, nonalcoholic fatty liver disease activity score; PSR, picrosirius red; TC, total cholesterol; TG, triglyceride.

We further validated the hepatic metabolites most significantly correlated with disease severity in different murine mouse models of MASH and atherosclerosis established in our lab[15], [16], [17], [18], [19], [20], [21] including C57BL/6J mice on the MASH diet (24 weeks; Fig. S8A and D) and Apoe^-/- mice on the WD (12 weeks; Fig. S8G). Of those, we observed that sphinganine, itaconate, fructose, lactate, thiamine pyrophosphate, 2-aminomuconic acid, carnitine, and pyridoxal 5’-phosphate were consistent in at least one other mouse model of MASH and atherosclerosis (Fig. S8A–H). Interestingly, 2-aminomuconic acid was the only hepatic metabolite which was consistently decreased in the Ldlr^-/-, C57BL/6J, and Apoe^-/- mouse models (Figs. S8B,E,H). Sex differences also became evident with this analysis as many hepatic metabolites were only consistent in males, including sphinganine, fructose, and lactate, which differed from our Ldlr^-/- model where sphinganine was the metabolite most correlated with all disease parameters independent of sex. Furthermore, we also validated the most significantly altered metabolites in the plasma of our Ldlr^-/- models including sphinganine, ornithine, and arginine (Fig. 4F,L), in males and females of an additional mouse disease model (Fig. S8C and F). In the plasma, arginine was the only consistent metabolite between both male and female C57BL/6J mice, which was significantly decreased, consistent with our Ldlr^-/- models. Altogether, correlation analysis and validation in additional disease models confirmed that hepatic sphinganine, itaconate, fructose, and lactate are positively associated, while thiamine pyrophosphate, 2-aminomuconic acid, carnitine, and pyridoxal 5’-phosphate are negatively associated with MASH and atherosclerosis severity.

To validate the causal and functional relevance of the hepatic metabolites identified, as both correlating with disease severity in our Ldlr^-/- mice (Fig. 5) and other disease models (Fig. S8), lipid-loaded primary hepatocytes were treated with key metabolites overnight, after which the conditioned media was isolated. From the same mice, bone marrow-derived macrophages were isolated and loaded with oxidized LDL (oxLDL, to mimic a proatherogenic setting in vitro) and treated overnight with conditioned media from the metabolite-treated primary hepatocytes. In hepatocytes, treatment with key upregulated metabolites, sphinganine, fructose, and lactate, significantly lowered fatty acid β-oxidation (Cpt1a and Acadm), and increased inflammatory (Ccl2 and Ccl5) gene expression (Figs. S9A,E,I), accompanied by a significant increase in lipid accumulation as determined by Nile red staining (Figs. S9C,G,K). In macrophages, treatment with conditioned media from metabolite-treated hepatocytes significantly enhanced inflammatory gene expression (Il1b and Tnf; Figs. S9B,F,J) and oxLDL uptake (Fig. S9D and H,L). Hepatocyte treatment with key downregulated metabolites, thiamine pyrophosphate and pyridoxal 5’-phosphate, significantly lowered inflammatory gene expression in hepatocytes and macrophages, along with lowering oxLDL accumulation in macrophages (Fig. S9M-T). Interestingly, while there was no effect on fatty acid β-oxidation genes, lipid accumulation was significantly decreased in primary hepatocytes. Altogether, correlation analysis, followed by validation in various disease models in vivo, and substantial functional validation in vitro using a co-culture system, indicate a causative role of sphinganine, fructose, and lactate, as well as a protective role of thiamine pyrophosphate and pyridoxal 5’-phosphate in MASH and atherosclerosis.

Distinct lipidomic signatures in concurrent MASH and atherosclerosis

Given the central role of lipid metabolism in MASH and atherosclerosis, we further extended our investigation to include untargeted lipidomics. We identified 1,245 liver and 1,281 plasma lipid species across the different groups. In males, PCA of the liver lipidome (Fig. 6A) revealed a clear separation between the groups, whereas the plasma lipidome showed no distinct clustering (Fig. S10A), leading us to focus on the liver. Pathway analysis indicated a significant downregulation of the polyunsaturated fatty acid (PUFA) pathway across all groups, most notably in WD-fed mice. While the monounsaturated fatty acid pathway was upregulated in all groups, the saturated fatty acid pathway was specifically increased in WD. Additionally, the steryl esters pathway was significantly upregulated in WD- and CDHFD-fed mice, with a milder increase in mMASHD. The dihexosylceramide pathway was significantly elevated in WD- and CDHFD-fed mice, but not mMASHD-fed mice (Fig. 6B). Volcano plots and heatmap representation highlighted specific lipids associated with these pathways. Triglycerides with varying fatty acid chain lengths and saturation levels were among the most differentially regulated lipid species across all diets. Triglycerides (TG) containing PUFA (e.g. TG(18:2/18:2/22:5), TG(18:1/18:2/22:6)) were significantly decreased, whereas those containing saturated fatty acids (e.g. TG(18:0,18:1,18:1), TG(20:0,18:1,18:2)) were significantly increased (Figs 6C and S10B-D). Cholesteryl esters were most elevated in WD-fed mice, followed by mCDHFD-fed mice, with a milder or no increase in mMASHD-fed mice (Fig. 6D). A similar pattern was observed in sphingomyelins, particularly species containing saturated and monounsaturated fatty acids (Fig. 6E). While diacylglycerols and ceramides (Fig. S10E and F) did not show a clear correlation with disease severity, increased acylcarnitine levels were found in WD- and mCDHFD-fed, but not in mMASHD-fed mice (Fig. S10G).

Fig. 6 — Lipidomic signatures in concurrent MASH and atherosclerosis. Untargeted lipidomics of livers from male and female *Ldlr*^-/- mice (n = 4-5 per group). PCA of liver lipidomes in males (A) and females (F). KEGG-based pathway analysis in males (B) and females (G). Heatmaps of altered triglyceride (C, H), cholesteryl ester (D, I), and sphingomyelin (E, J) species. Significance thresholds *p <*0.05 and fold-change >1.5. PERMANOVA. Pathway enrichment significance assessed by a hypergeometric test. MASH, metabolic dysfunction-associated steatohepatitis; mCDHFD, modified choline-deficient high-fat diet; mMASHD, modified metabolic dysfunction–associated steatohepatitis diet; PCA, principal component analysis; SD, standard diet; WD, western diet.

Similar to males, a distinct separation between the dietary regimens in females was noted in the liver, but not plasma lipidomes (Figs 6F and S10H). The PUFA pathway was the most significantly downregulated across all groups. Although the steryl esters and dihexosylceramides pathways were among the most upregulated, no differences were observed between diets (Fig. 6G), unlike in males. Triglyceride species showed no clear pattern in fatty acid length or saturation. However, hepatic triglycerides were notably elevated in mMASHD-fed females (Figs 6H and S10I-K), aligning with greater hepatic steatosis. Hepatic cholesteryl esters and sphingomyelins increased across all diets (Fig. 6I,J), unlike in males, where they correlated with disease severity. Lipid species related to diacylglycerols, ceramides, and mitochondrion pathways did not correlate with disease severity (Fig. S10L-N). These findings uncover PUFA, steryl esters, and dihexosylceramides as shared pathways dysregulated in concurrent MASH and atherosclerosis in both males and females, while indicating sex-based differences in the correlation between lipid pathways and disease severity.

Distinct transcriptomic signatures in concurrent MASH and atherosclerosis

To determine transcriptional dysregulation in concurrent MASH and atherosclerosis, we conducted unbiased RNA sequencing of livers. In males, PCA showed a clear separation of the hepatic transcriptome between mice fed WD, mCDHFD, or mMASHD compared to SD (Fig. 7A). The transcriptomes of WD- and mCDHFD-fed mice overlapped, while mMASHD clustered more closely with SD. Volcano plots demonstrated major alterations in DEGs in all groups. In WD-fed mice, 2,224 DEGs were identified, with 1,818 upregulated and 406 downregulated (Fig. 7B). Pathway analysis identified numerous upregulated proatherogenic and inflammatory pathways: lipid and atherosclerosis, efferocytosis, chemokine signaling, cytokine-cytokine receptor interaction, NF-kB, TNF, and toll-like receptor signaling. Conversely, pathways related to fatty acid and cholesterol metabolism, including fatty acid degradation and peroxisome pathways, were significantly downregulated (Fig. 7C, Table S2). Key inflammatory (Ccl2, Trem2, Gpnmb, Cd68, Lgals3) and fibrotic (Mmp12, Itgax, Timp1) genes were significantly upregulated, while genes regulating lipid metabolism (Cyp51, Gm15441) were significantly suppressed in WD-fed mice. The hepatic transcriptome of mCDHFD-fed mice closely resembled WD-fed mice, with 2,264 DEGs (1,782 upregulated, 482 downregulated, Fig. 7D) and similar pathways altered (Fig. 7E, Table S3). In mMASHD-fed mice, 1,739 DEGs were identified (1,444 upregulated, 295 downregulated, Fig. 7F). While proatherogenic/proinflammatory pathways were upregulated in mMASHD-fed mice, the fatty acid degradation and peroxisome pathways were unaltered (Fig. 7G, Table S4) in line with the milder phenotype observed in this model.

Fig. 7 — Distinct transcriptomic signatures in concurrent MASH and atherosclerosis. RNA-sequencing of liver samples from male and female *Ldlr*^-/- mice (n = 4-5 per group). PCA of liver transcriptomes in males (A) and females (H). Volcano plots of DEGs in males (B, D, F) and females (I, K, M) comparing WD, mCDHFD, or mMASHD to SD. Pathway analysis of upregulated (red) and downregulated (blue) pathways in males (C, E, G) and females (J, L, N). Two-sided Wald test for significant DEGs. Significance of enrichment determined by right-tailed Fisher’s exact test and Benjamini-Hochberg multiple testing adjustment. DEGs, differentially expressed genes; mCDHFD, modified choline-deficient high-fat diet; mMASHD, modified metabolic dysfunction–associated steatohepatitis diet; PCA, principal component analysis; SD, standard diet; WD, western diet.

While hepatic transcriptomes correlated with MASH and atherosclerosis severity in males, PCA revealed overlapping transcriptomes across the diets in females (Fig. 7H). Additionally, females showed fewer DEGs. In WD-fed females, 756 DEGs were identified (644 upregulated, 112 downregulated, Fig. 7I). While some proinflammatory pathways (cytokine-cytokine receptor interaction) were upregulated, only three pathways – spliceosome, basal transcription factors, and ribosome biogenesis – were downregulated (Fig. 7J, Table S5), none of which were altered in males. A larger number of DEGs was observed in mCDHFD-fed females (1,033 total: 833 upregulated, 200 downregulated, Fig. 7K). Aligning with more pronounced MASH and atherosclerosis, proatherogenic/proinflammatory pathways (toll-like receptor signaling, efferocytosis, and chemokine signaling) were significantly enriched (Fig. 7L, Table S6). In mMASHD-fed females, 779 DEGs were identified (658 upregulated, 121 downregulated, Fig. 7M), with inflammatory pathways such as IL-17 and toll-like receptor signaling significantly enriched (Fig. 7N, Table S7). Notably, Trem2 and Mmp12 were the most significantly upregulated genes across all diets (Fig. 7I,K,M), indicating heightened inflammatory and fibrotic responses.

To validate our unbiased transcriptomics, we performed quantitative reverse-transcription PCR for key DEGs related to lipid metabolism (Gm15441, Cyp51), inflammation (Trem2, Lgals3), and fibrosis (Mmp12, Itgax; Fig. S11A and B). Moreover, we also compared these results to additional well-established murine MASH (Fig. S11C and D) and atherosclerosis (Fig. S11E) models.[15], [16], [17], [18], [19], [20], [21] Notably, Trem2 expression was consistently upregulated and Gm15441 was consistently downregulated across our Ldlr^-/- models, and all three additional cohorts independent of sex. Mmp12 was the most significantly upregulated gene across all our cohorts (except for a mild trend in the female C57BL/6J model), but was more significantly increased in males, while Trem2 was more significantly increased in females (Fig. 7B,D,F,I,K,M), which was consistent across our additional mouse models (Fig. S11) revealing novel sex-specific transcriptomic candidates driving disease pathogenesis in mice. Overall, our unbiased transcriptomic analyses, together with validation of key transcriptomic signatures in complementary murine model systems, underscore upregulated hepatic inflammatory pathways in concurrent MASH and atherosclerosis in both sexes, while indicating sex-specific differences in the relationship between the hepatic transcriptome and disease severity.

Integrated multiomics highlight common pathways linking MASH and atherosclerosis in mice and humans

To further explore metabolic and molecular mechanisms linking MASH and atherosclerosis, we integrated hepatic transcriptomics and metabolomics followed by pathway analyses. In males, several integrated pathways were significantly enriched across all groups. These included the sphingolipid metabolism, glycine, serine, and threonine metabolism, glutathione metabolism, glycolysis/gluconeogenesis, glycerophospholipid metabolism, and valine, leucine, and isoleucine metabolism. In contrast, oxidative phosphorylation, fatty acid degradation, and pyrimidine metabolism were specifically enriched in WD- and mCDHFD-fed mice (Fig. 8A), which showed pronounced MASH and atherosclerosis. In females, the sphingolipid metabolism, glutathione metabolism, pyrimidine metabolism, and glycerophospholipid metabolism pathways were significantly enriched across all groups. The glycolysis/gluconeogenesis, and cysteine and methionine metabolism pathways were specifically enriched by mCDHFD (Fig. 8B), which showed the strongest disease phenotypes.

Fig. 8 — Integrated multiomics analysis highlights common pathways linking MASH and atherosclerosis in mice and humans. Network analysis using MetaboAnalyst 6.0 for integration of metabolites and DEGs using KEGG metabolic networks in male (A) and female (B) *Ldlr*^-/- mice fed SD, WD, mCDHFD, or mMASHD for 24 weeks (n = 4-5/group). KEGG pathways with *p <*0.05 were considered significantly enriched. Heatmap of KEGG pathways comparing RNA-sequencing of livers from male (C) and female (D) mice (n = 4 group) to RNA-sequencing of liver samples obtained from patients with MASH compared to controls. GSE130970 (n = 7 control and n = 17 MASH), GSE239422 (n = 12 control and n = 13 MASH), GSE126848 (n = 14 control and n = 16 MASH). Pathways enriched in the upregulated or downregulated DEGs are plotted in red or blue, respectively. Scale bars represents -log₁₀(p value) ∗sign(NES). Right-tailed Fisher’s exact test followed by Benjamini-Hochberg multiple testing adjustment. DEGs, differentially expressed genes; MASH, metabolic dysfunction–associated steatohepatitis; mCDHFD, modified choline-deficient high-fat diet; mMASHD, modified metabolic dysfunction–associated steatohepatitis diet; NES, normalized enrichment score; PCA, principal component analysis; SD, standard diet; WD, western diet.

Additionally, we integrated our transcriptomics data with the only publicly available transcriptomics dataset (GSE10934) of a combined MASH and atherosclerosis model, Ldlr^-/-.Leiden model,³⁷ which has been studied exclusively in males (Fig. S12). Our integration revealed a dramatically strong overlap, particularly in upregulated inflammatory pathways like phagosome, cell adhesion molecules, NF-κB signaling, lipid/atherosclerosis, cytokine-cytokine receptor interaction, chemokine signaling, toll-like receptor, IL-17, and TNF pathways, along with associated DEGs, further emphasizing the robustness of our model. To establish translational relevance, we integrated liver transcriptomes of our Ldlr^-/- mice with those of patients with MASH (GSE130970,³³ GSE239422,³⁴ and GSE126848 ³⁵). Pathway analysis revealed that independent of sex, inflammatory pathways, particularly chemokine, toll-like receptor, TNF, and IL-17 signaling pathways in our Ldlr^-/- mouse models highly overlap with those of patients with MASH (Fig. 8C,D). In contrast, while in mice ribosomal pathways were decreased, in patients with MASH these pathways were increased, independent of sex. Consistent with overlapping murine and human inflammatory pathways, the top 100 DEG analysis revealed prominent overlap in genes associated with inflammation (TREM2, LGALS3, and CD52), fibrosis (ITGAX) and lipid metabolism (LPL; Fig. S13A and B). Interestingly, while Mmp12 was one of the most consistently upregulated genes in our mouse models, this was not consistent in patients with MASH, despite similar inflammatory expression profiles. Next, to analyze sex-specific differences, we additionally compared our male and female mice to the same human cohorts which we stratified by sex as per availability in public datasets (Figs. S14 and S15). These analyses revealed that while several inflammatory pathways remained consistent in both sexes, sphingolipid metabolism and cytokine-cytokine receptor interaction pathways were only consistent in males. DEG analysis further emphasized this difference as several genes were only consistent in males (LGALS3 and ITGAX). Altogether, these unbiased, integrative studies highlight hepatic proinflammatory pathways shared across mice and humans, males and females, underscoring the relevance of these murine models of concurrent MASH and atherosclerosis to human disease.

Discussion

While MASH is clearly associated with liver-related mortality, ASCVD is the leading cause of death in this disease.[4], [5], [6], [7] Currently, no pharmacological therapy targets both MASH and atherosclerosis concurrently. A major hurdle in identifying new therapeutic targets is the absence of animal models that simultaneously feature both diseases, particularly in females. Here, we utilized male and female atheroprone mice under different dietary regimens, integrating multiomics approaches (metabolomics, lipidomics, and transcriptomics) with histopathological and biochemical analyses to establish a murine model featuring both MASH and atherosclerosis. Our findings reveal sexual dimorphism in disease progression and highlight metabolic and molecular mediators linking MASH and atherosclerosis. Importantly, the high similarity observed between the mouse and human transcriptomes underscores the translational value of this mouse model.

In humans, major sex differences exist in the prevalence and severity of both MASH and ASCVD.¹⁴^,²⁵ Such differences are rarely addressed in preclinical studies, as MASH progression in female mice is inconsistent, and male mice are more commonly used.²⁶^,²⁷ To date, only a few studies have focused on developing a concurrent model of MASH and atherosclerosis, using solely the Ldlr^-/-.Leiden mouse.³⁶^,³⁷ Those studies were limited by the exclusive use of males and specific diets. Here, we addressed these limitations by using both male and female, widely available, Ldlr^-/- mice, and by testing various dietary approaches. High dietary cholesterol (>1.25%) is an established driver of MASH-fibrosis that replicates the human disease in mice and non-human primates;¹⁵^,¹⁶^,¹⁹^,²¹^,²⁴ however, such levels are supraphysiological, particularly in hyperlipidemic/atheroprone mice. To develop a physiologically relevant model, we modified commonly used CDHFD²² and MASHD¹⁵^,¹⁶^,¹⁹^,²¹ to contain lower cholesterol levels (0.2%), similar to those in the WD used in atherosclerosis research.¹⁷^,¹⁸^,²³ Unlike previous Ldlr^-/-.Leiden studies reporting extreme hypercholesterolemia (∼1,300-1,600 mg/dl) and hypertriglyceridemia (∼425-735 mg/dl),³⁶^,³⁷ none of our dietary regimens led to plasma cholesterol and TG levels exceeding 1,000 mg/dl and 300 mg/dl, respectively.

Previous studies on concurrent MASH and atherosclerosis provided limited characterization of disease severity and only in male mice.³⁶^,³⁷ Here, we conducted a comprehensive, blinded histopathological analysis, corroborated by a range of biochemical assays. Surprisingly, our extensive investigation revealed that the commonly used atherogenic WD¹⁷^,¹⁸^,²³ is not only sufficient but also highly effective in inducing advanced MASH-fibrosis in male mice. Additionally, WD led to the most severe atherosclerosis phenotype, characterized by extensive lesions throughout the aortic tree and sinuses, and increased plaque complexity. While mCDHFD also effectively induced MASH, hepatic fibrosis, and atherosclerosis in males, mMASHD resulted in milder phenotypes. Given that all diets contained equal cholesterol levels but differed in fat sources, our findings suggest that milkfat in WD, along with lard and choline deficiency in mCDHFD, are potent inducers of concurrent MASH and atherosclerosis, at least in male mice. Our findings revealed unrecognized sexual differences in concurrent MASH and atherosclerosis in response to different diets. Unlike males, MASH and atherosclerosis severities in females showed no clear correlation. Notably, mMASHD, the least potent diet in males, uniquely induced MASH-fibrosis in females, aligning with prior evidence that females exhibit greater MASLD severity in response to high-fructose diets.³⁸ Nevertheless, atherosclerosis was significantly lower in mMASHD-fed females compared to WD or mCDHFD. Moreover, circulating total cholesterol, VLDL- and LDL-cholesterol, as well as CCL2 and CCL5, strongly correlated with MASH and atherosclerosis severity in males but not in females. Yet, our correlation analysis revealed a strong link between MASH and atherosclerosis in both sexes, highlighting circulating cholesterol and CCL2 and hepatic sphinganine as potential disease predictors.

Our study presents the first comprehensive and unbiased integrated metabolomic and transcriptomic analysis of concurrent MASH and atherosclerosis in mice. Our metabolomics analysis identified arginine–proline metabolism, and glycine–serine metabolism as being consistently downregulated across all groups and tissues. Moreover, glycine–serine metabolism, and glutathione metabolism were highlighted by integrating hepatic metabolomics and transcriptomics, along with sphingolipid and energy metabolism pathways (oxidative phosphorylation, fatty acid degradation, and glycolysis). Specific metabolites within these pathways may serve as potential predictors or therapeutic targets in concurrent MASH and atherosclerosis. Sphinganine is formed by the condensation of serine with palmitoyl-CoA by serine palmitoyltransferase, the first step in sphingolipid/ceramide biosynthesis,³⁹^,⁴⁰ and we found it to be most significantly correlated with disease indices in the liver, plasma and atherosclerotic plaques. Accordingly, sphingolipids/ceramides are known to be implicated in MASH and ASCVD, and serine palmitoyltransferase inhibition was recently reported to ameliorate both hepatic steatosis and atherosclerosis in mice.⁴⁰ The consistent downregulation of the glycine–serine and glutathione pathways was driven by decreased glycine, which was inversely correlated with MASH and atherosclerosis indices. These findings support previous studies that established dysregulated glycine–serine and glutathione metabolism in both MASH and atherosclerosis, a causative role for lower glycine, and therapeutic properties of glycine-based treatments.¹⁵^,¹⁶^,¹⁸^,¹⁹^,²¹^,³⁵^,[41], [42], [43]

Key metabolites involved in energy metabolism – including the citric acid cycle metabolite itaconate and the glycolysis-related metabolites glucose and lactate – were positively correlated with indices of MASH and atherosclerosis. Interestingly, increased itaconate levels were reported in both MASH and atherosclerosis, and its administration ameliorated both diseases in mice.⁴⁴^,⁴⁵ Moreover, lactate was reported to promote hepatic fibrosis via hepatocyte apoptosis and hepatic stellate cell activation.⁴⁶^,⁴⁷ Yet, whether hepatic lactate metabolism can be targeted for treating MASH and atherosclerosis remains unknown. Conversely, carnitine, which is essential for the transfer of long-chain fatty acids across the mitochondrial membrane for subsequent β-oxidation, was inversely associated with MASH and atherosclerosis indices, supporting the notion that hepatic fatty acid β-oxidation may be targeted for concurrent treatment of MASH and ASCVD.⁶ Moreover, we identified metabolites that have not been associated with MASH or atherosclerosis previously. These include 2-aminomuconic acid and thiamine pyrophosphate, which were inversely correlated with MASH and atherosclerosis parameters in both sexes. Notably, 2-aminomuconic acid was the only metabolite consistent in all other MASH and atherosclerosis mouse models we tested. In our co-culture in vitro system, thiamine pyrophosphate treatment decreased hepatocyte lipid accumulation without altering fatty acid β-oxidation gene expression. This may be due to the role of thiamine pyrophosphate as a cofactor for fatty acid β-oxidation,⁴⁸ though future studies are needed to explore the lipid-lowering properties of this metabolite. Whether these metabolites play causative roles in concurrent MASH and atherosclerosis warrants further investigation.

Our untargeted lipidomics aligns with previous studies of liver biopsies from patients with MASH,⁴⁹ and MASH mouse models,⁵⁰ revealing the PUFA pathway as the most significantly downregulated in both sexes. Conversely, the steryl esters pathway was the most upregulated, reinforcing the critical role of cholesterol accumulation in MASH progression.²⁵ While hepatic steryl esters correlated with disease severity in males, females exhibited increased levels across all groups, independent of disease severity. Additionally, higher hepatic sphingomyelin levels, consistent with increased sphinganine and enhanced sphingomyelin synthesis in MASH,⁵¹ correlated with more severe MASH and atherosclerosis, particularly in males. These findings highlight sex-specific differences in hepatic lipid metabolism linking MASH and atherosclerosis.

Our integrative analysis of metabolomics with hepatic transcriptomes, and the integration of hepatic transcriptomics in humans and mice, supported by plasma chemokine profiling, underscores the role of exacerbated inflammation in concurrent MASH and atherosclerosis. Although the fat sources differed, as WD and mCDHFD are enriched in saturated fatty acids and mMASHD contains trans fats, all diets activated similar proinflammatory and cytotoxic signaling pathways. Integration of transcriptomics with metabolomics highlighted that fatty acid degradation and oxidative phosphorylation pathways were significantly downregulated in WD- and mCDHFD-compared to mMASHD-fed mice, which may be attributed to the different fat sources, as trans fats are known to enhance lipogenesis.⁵² RNA sequencing also revealed sex-specific differences in downregulated pathways, primarily associated with fatty acid and cholesterol metabolism, consistent with sex-based differences in hepatic lipid metabolism discussed above. Conversely, proinflammatory/proatherogenic pathways were consistently upregulated in both sexes and in human MASH, though more prominently in males, highlighting the critical need for future murine models that recapitulate the human disease in women specifically. Interestingly, while the most significantly upregulated genes in our mouse models were Trem2 and Mmp12, across all diets and independent of sex, Mmp12 was not consistent in patients with MASH. Across species and sexes, proinflammatory genes including Trem2, Ccl2, and Cd52 were upregulated in hepatic tissues. While TREM2 and CCL2 have established roles in MASH and atherosclerosis,²⁹^,³⁰^,[53], [54], [55], [56] the role of CD52 remains unexplored. Future research is encouraged to validate if these altered genes are potential therapeutic targets for concurrent MASH and atherosclerosis.

Although this study establishes a link between MASH and atherosclerosis using a multiomics approach, it has several limitations. While we provided mechanistic validation using in vitro studies with hepatocytes and macrophages, more comprehensive in vivo studies are needed using genetic or pharmacological manipulation to determine the therapeutic potential of these targets for MASH and atherosclerosis treatment. In particular, key genes such as Trem2 and Lpl, both significantly altered across all dietary groups irrespective of sex and consistent with human transcriptomic data, require further validation to assess their therapeutic potential against both diseases. Another important limitation is the possible influence of gut microbiota differences across dietary groups on MASH and atherosclerosis outcomes. Given that our diets differ in fat sources, which may shape gut microbiota composition, this aspect warrants further investigation in future studies. Addressing these limitations will facilitate the development of effective therapeutic strategies for concurrent MASH and atherosclerosis.

In summary, through a sex-based multiomics and integrative analysis of concurrent MASH and atherosclerosis, we developed a translational murine model that simultaneously features both diseases. We uncover sexual dimorphism in response to different dietary regimens, and unique metabolic and transcriptional pathways that could serve as biomarkers and therapeutic targets.

Abbreviations

ALT, alanine aminotransferase; ASCVD, atherosclerotic cardiovascular disease; AST, aspartate aminotransferase; CCR2, chemokine receptor 2; CCL2, C–C motif ligand 2; CCL5, C–C motif ligand 5; CDHFD, choline-deficient high-fat diet; DEGs, differentially expressed genes; LPL, lipoprotein lipase; MASLD, metabolic dysfunction-associated steatotic liver disease; MASH, metabolic dysfunction-associated steatohepatitis; MASHD, MASH-inducing diet; mCDHFD, modified choline-deficient high-fat diet; mMASHD, modified MASH diet; NAS, NAFLD activity score; PCA, principal component analysis; PERMANOVA, nonparametric permutational multivariate analysis of variance; PSR, Picrosirius red; PUFA, polyunsaturated fatty acids; SD, standard diet; TG, triglyceride; TREM2, triggering receptor expressed on myeloid cells 2; WD, western diet.

Authors’ contributions

Conception: O. Rom; Experimental design: S. Das, S. K. Anand, and O. Rom; Investigation: S. Das, S.K. Anand, M. P. McKinney, I. Mahmud, S. Rohilla, K. S. E. Richard, F. Arias, B. Wei, L. Tan, Z. Liu, D. Kumar, A.C. Finney, N. Pandey, H. Kaur, R. Pandit, C. B. Dhaou, N. Dhanesha, A. Yurdagul Jr., P. L. Lorenzi, and O. Rom. Methodology: S. Das, S.K. Anand, M. P. McKinney, I. Mahmud, S. Rohilla, K. S. E. Richard, A. Ghrayeb, F. Arias, B. Wei, L. Tan, Z. Liu, D. Kumar, A.C. Finney, N. Dhanesha, A. Yurdagul Jr., P. L. Lorenzi, and O. Rom. Resources: S. P. Thayer, S. Radhakrishnan, E. Gottlieb, A. W. Orr, W. Liu, N. Dhanesha, A. Yurdagul Jr., P. L. Lorenzi, and O. Rom. Formal analysis: S. Das, S.K. Anand, M. P. McKinney, I. Mahmud, S. Rohilla, K. S. E. Richard, F. Arias, B. Wei, L. Tan, Z. Liu, X. Zhang, N. Dhanesha, A. Yurdagul Jr., P. L. Lorenzi, and O. Rom. Critical review & discussion: I. Mahmud, B. Razani, B. Cai, F. Chang, F. J. Schopfer, E. A. Fisher, A. W. Orr, W. Liu, N. Dhanesha, A. Yurdagul Jr, P. L. Lorenzi, and O. Rom. Funding acquisition: O. Rom. All authors read and approved the manuscript.

Data availability

All raw omics data generated in this study were deposited in publicly available databases. The raw metabolomics and lipidomics data were deposited in Zenodo (accession number: 10.5281/zenodo.14025183 and https://zenodo.org/records/17187056). RNA sequencing data were deposited in NCBI’s GEO database (accession number: GSE280888). All data will be made available upon the manuscript's acceptance for publication.

Financial support

This study was partially supported by the National Institutes of Health grants DK136685, DK134011, and HL150233 (O. Rom), DK137711 (O. Rom, F. Chang, F.J. Schopfer), HL145131 and HL167758 (A. Yurdagul Jr.), HL158546 (N. Dhanesha), HL133497 and HL173972 (A.W. Orr), GM125944 (F.J. Schopfer), GM134974 (X. Zhang, CAIPP CoBRE Core: RRID_SCR_024779), National Science Foundation grant 2537597 (O. Rom and A.W. Orr), the American Heart Association grants 24POST1196650 (S. Das), 24POST1199805 (S.K. Anand), 23POST1026505 (A.C. Finney), and 20CDA35260123 (N. Dhanesha), the Collaborative Intramural Research Program (CIRP, LSUHS and Ochsner Clinic Foundation, A. Yurdagul Jr., and O. Rom), the LSUHS Chancellor's Pathways Research Award (A. Yurdagul Jr., and O. Rom), the Chancellor's Aim High Research Award (N. Dhanesha, and O. Rom), and the LSUHS Center for Cardiovascular Diseases and Sciences Malcolm Feist Postdoctoral Fellowships (S. Das, S. Rohilla, and C. Ben Dhaou).

Conflicts of interest

F.J. Schopfer and F. Chang have financial interests in Creegh Pharma Inc., and Furanica Inc. O. Rom is a scientific advisor at Diapin Therapeutics LLC.

Please refer to the accompanying ICMJE disclosure forms for further details.

Footnotes

Author names in bold designate shared co-first authorship

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jhepr.2025.101703.

Contributor Information

Philip L. Lorenzi, Email: plorenzi@coh.org.

Oren Rom, Email: oren.rom@lsuhs.edu.

Supplementary data

The following are the Supplementary data to this article:

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Data Availability Statement

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[bib31] 31.Braunersreuther V., Steffens S., Arnaud C., et al. A novel RANTES antagonist prevents progression of established atherosclerotic lesions in mice. Arterioscler Thromb Vasc Biol. 2008;28:1090–1096. doi: 10.1161/ATVBAHA.108.165423. [DOI] [PubMed] [Google Scholar]

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[bib35] 35.Cherubini A., Ostadreza M., Jamialahmadi O., et al. Interaction between estrogen receptor-α and PNPLA3 p.I148M variant drives fatty liver disease susceptibility in women. Nat Med. 2023;29:2643–2655. doi: 10.1038/s41591-023-02553-8. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Sex-based multiomics analysis uncovers metabolic and molecular mediators linking MASH and atherosclerosis

Sandeep Das

Sumit Kumar Anand

M Peyton McKinney

Koral SE Richard

Iqbal Mahmud

Sumati Rohilla

Fabio Arias

Alia Ghrayeb

Bo Wei

Lin Tan

Zhipeng Liu

Dhananjay Kumar

Alexandra C Finney

Nilesh Pandey

Harpreet Kaur

Rajan Pandit

Xiaolu Zhang

Cyrine Ben Dhaou

Sarah P Thayer

Babak Razani

Bishuang Cai

Fei Chang

Francisco J Schopfer

Wanqing Liu

Edward A Fisher

Sridhar Radhakrishnan

Eyal Gottlieb

A Wayne Orr

Nirav Dhanesha

Arif Yurdagul Jr

Philip L Lorenzi

Oren Rom

Abstract

Background & Aims

Methods

Results

Conclusion

Impact and implications

Graphical abstract

Highlights

Introduction

Materials and methods

Experimental model

Statistical analysis

Results

Diet-induced MASH in male and female atheroprone mice

Fig. 1.

Effects of MASH-inducing diets on atherosclerosis in atheroprone mice

Fig. 2.

Circulating factors linking MASH and atherosclerosis

Fig. 3.

Distinct metabolic signatures in concurrent MASH and atherosclerosis

Fig. 4.

The hepatic metabolome links MASH and atherosclerosis

Fig. 5.

Distinct lipidomic signatures in concurrent MASH and atherosclerosis

Fig. 6.

Distinct transcriptomic signatures in concurrent MASH and atherosclerosis

Fig. 7.

Integrated multiomics highlight common pathways linking MASH and atherosclerosis in mice and humans

Fig. 8.

Discussion

Abbreviations

Authors’ contributions

Data availability

Financial support

Conflicts of interest

Footnotes

Contributor Information

Supplementary data

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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