Sex- and age-associated factors drive the pathophysiology of MASLD

Abstract

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is strongly associated with obesity. Sex and age affect MASLD prevalence and pathophysiology. The use of animal models fed Western-style diets is vital for investigating the molecular mechanisms contributing to metabolic dysregulation and for facilitating novel drug target identification. However, the sex-associated and age-associated mechanisms underlying the pathophysiology remain poorly understood. This knowledge gap limits the development of personalized sex-specific and age-specific drug treatments.

Methods: Young (7 wk) and aged (52 wk) male and female mice were fed a high-fat diet (HFD) or low-fat diet. Liver metabolome (>600 molecules) and transcriptome profiles were analyzed.

Results: Male and female mice fed an HFD developed obesity, glucose intolerance, and hepatic steatosis. However, fasting blood glucose, insulin, and serum alanine aminotransferase levels were higher in males fed an HFD, indicating a more severe metabolic disease. In addition, males showed significant increases in liver diacylglycerides and glycosylceramides (known mediators of insulin resistance and fibrosis), and more changes in the transcriptome: extracellular matrix organization and proinflammatory genes were elevated only in males. In contrast, no major increase in damaging lipid classes was observed in females fed an HFD. However, aging affected the liver to a greater extent in females. Acylcarnitine levels were significantly reduced, suggestive of changes in fatty acid oxidation, and broad changes in the transcriptome were observed, including reduced oxidative stress response gene expression and alterations in lipid partitioning genes.

Conclusions: Here, we show distinct responses to an HFD between males and females. Our study underscores the need for using both sexes in drug target identification studies, and characterizing the molecular mechanisms contributing to the MASLD pathophysiology in aging animals.

Graphical abstract

FIGURE 1

Experimental design, body weight, and serum chemistries. (A) Experimental design (n = 8). (B) Body weight gain over the course of 10 weeks. Bars represent SD. *p < 0.05, **p < 0.01, ^# p < 0.001. (C) Blood glucose and serum insulin (n = 8). (D) Serum triglycerides, FFA, and cholesterol (n = 7–8). Bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviation: FFA, free fatty acids.

FIGURE 2

Serum ALT and liver histology. (A) Serum ALT levels (n = 8). (B) Hematoxylin/eosin and Oil Red O staining of liver sections. NAS scores (sum of steatosis, hepatocellular ballooning, and lobular inflammation scores) are shown on the right (n = 3). Hematoxylin/eosin, ×100; Oil Red O, ×200. (C) Liver triglycerides and diacylglycerides were quantified in a targeted metabolomics assay (n = 5). Bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: ALT, alanine aminotransferase; NAS, nonalcoholic fatty liver disease activity score.

FIGURE 3

Liver acylcarnitines. (A) Levels of l-carnitine (C0). (B) Total acylcarnitines; short-chain and long-chain acylcarnitines. (C) Heatmap showing changes in acylcarnitines in males and females fed the LFD or HFD. Bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: HFD, high-fat diet; LFD, low-fat diet.

FIGURE 4

Liver sphingolipids. (A) Liver ceramides and heatmap of ceramide subclasses. (B) Liver glycosylceramides and heatmap of the classes detected. (C) Liver sphingomyelin. (D) Schematic of sphingolipid synthesis. Bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

Liver glycerophospholipids, bile acids, cholesterol esters, nucleobase-related molecules, and biogenic amines. (A) Glycerophospholipids (phosphatidylcholine plus lysophosphatidylcholine) and choline levels in liver tissue. (B) Overall levels of bile acids (TCA, TDCA, TCDCA, and TMCA) and liver cholesterol esters. (C) Xanthine and hypoxanthine, products of the catabolism of purines. (D) Levels of the biogenic amines putrescine and spermidine. Bars represent SD. *p<0.05, **p<0.01, ***p<0.001. Abbreviations: TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; TDCA, taurodeoxycholic acid; TMCA, tauro-β-muricholic acid.

FIGURE 6

Gene ontology analysis. (A) Pie plot showing the total number of genes differentially expressed (DEGs) in mice fed an HFD relative to an LFD (top) and the number of DEGs in young mice relative to aged mice (bottom). (B) Canonical pathways of DEGs between mice fed an HFD relative to an LFD. The comparison analysis between DEGs in young and aged mice was carried out using the following filters: p value cutoff of 2.5 (log10) and z score cutoff of 1.5. The bar shows the activation z score. Abbreviations: DEG, differentially expressed gene; HFD, high-fat diet; LFD, low-fat diet.

FIGURE 7

Validation of selected genes. (A) Western blot of molecules in the lipogenesis and cholesterol synthesis pathways. (B) Protein quantification of molecules in the lipogenesis pathway. (C) Protein quantification of molecules involved in cholesterol biosynthesis. Bars represent SD. *p <0.05, **p <0.01, ***p <0.001.

FIGURE 8

Gene expression changes in young versus aged mice. (A) IPA analysis of DEGs between young and aged mice fed an LFD. (B) Linear regression analysis showing a correlation between triglyceride levels (TGs, μM) and expression of Cidea, Cidec, and Fitm1 (logCPM). All groups are included (LFD and HFD; young and aged; n = 4/group). Abbreviations: DEG, differentially expressed gene; HFD, high-fat diet; IPA, ingenuity pathway analysis; LFD, low-fat diet.