Adaptations of mitochondrial, autophagy and nutrient sensing pathways in the liver from long-lived mice overexpressing CYB5R3 are sex-dependent and involve inter-organ responses

Cytochrome b₅ reductase 3 (CYB5R3) overexpression mimics several metabolic benefits of calorie restriction, with sex- and tissue-specific effects. This study aimed to investigate how CYB5R3 overexpression impacts hepatic metabolism in young mice, focusing on mitochondrial biogenesis, lipid metabolism, autophagy and nutrient sensing pathways thus establishing a baseline that allows for subsequent comparisons with older animals. The accrual of CYB5R3 polypeptide exhibited marked sexual dimorphism as it was increased by transgenesis only in females with predominant microsomal targeting but mainly located in the mitochondria in males. Nevertheless, key metabolic markers, including TFAM (biogenesis), p62 and LC3 (autophagy), AKT and mTOR (nutrient sensing), SIRT1/SIRT3 (enzymatic regulation and gene expression) and ACC1/ACAA2 (lipid metabolism), were significantly altered in transgenic males. Wild-type males exhibited higher levels of mitochondrial complexes III and IV than females, and these differences were attenuated by transgenesis. TFAM was increased in transgenic mice of both sexes, indicative of enhanced mitochondrial biogenesis. Changes of lipid metabolism markers indicated reduced hepatic lipid accumulation in females while males showed changes in ACC1 and ACAA2, affecting lipid storage and oxidation. Autophagy markers (p62, LC3I/II) were altered in males, whereas mitophagy markers (PINK1, PARKIN) were upregulated in females, suggesting efficient mitochondrial turnover. In conclusion, CYB5R3 expression is regulated by post-transcriptional and post-translational mechanisms and induces sex-dependent hepatic metabolic adaptations. While females exhibit increased CYB5R3 protein and associated mitochondrial improvements, males respond with distinct metabolic reprogramming despite unchanged CYB5R3 levels, underscoring the relevance of sexual dimorphism and systemic regulatory mechanisms in the response to longevity-promoting interventions.