Testosterone deficiency aggravates diet-induced non-alcoholic fatty liver disease by inducing hepatocyte ferroptosis via targeting BMAL1 in mice

Abstract

Background

Testosterone deficiency is linked to an increased prevalence of non-alcoholic fatty liver disease (NAFLD), although the mechanisms underlying this association are not fully understood. Ferroptosis, a regulated cell death pathway driven by iron-dependent lipid peroxidation, has been suggested to play a role in NAFLD pathogenesis. Since testosterone deficiency is associated with lipid disorders and iron deposition, we hepothesize that ferroptosis may be involved in the pathogenesis of diet-induced NAFLD exacerbated by testosterone deficiency.

Methods

Apolipoprotein E (APOE^−/−) mice were subjected to sham surgery or bilateral castration and subsequently fed a high-fat diet for 16 weeks. Liver gene expression was analyzed using RNA sequencing. Additional assessments included blood analysis, histological staining, measurement of iron and antioxidant enzyme levels, quantitative real-time PCR, Western blotting, and electron microscopy. The effects of testosterone on ferroptosis induced by free fatty acids (FFAs) and Erastin were further investigated in HepG2 cells in vitro.

Results

Testosterone deficiency resulted in increased hepatic lipid accumulation and macrovesicular steatosis in high-fat diet-fed APOE^−/− mice, accompanied by hepatic inflammation, fibrosis, and elevated liver enzyme levels. Transcriptomic analysis revealed that testosterone deficiency affects ferroptosis and circadian rhythm-related signaling pathways. Castrated APOE^−/− mice exhibited significantly higher hepatic iron deposition, lipid peroxidation, and expression of key ferroptosis-related proteins, along with decreased Brain and muscle ARNT-like gene 1 (BMAL1) protein expression. In vitro, testosterone treatment reduced lipid and iron accumulation and lipid peroxidation in HepG2 cells subjected to FFAs and Erastin. Moreover, BMAL1 knockdown negated the protective effects of testosterone against ferroptosis in hepatocytes.

Conclusion

Our study demonstrated that testosterone deficiency exacerbates NAFLD induced by a high-fat diet by promoting hepatocyte ferroptosis through modulation of the circadian protein BMAL1.