Sex dimorphism and substrate dependency of liver mitochondrial bioenergetics and H₂O₂ production

Mitochondrial bioenergetics and hydrogen peroxide (H₂O₂) production play a central role in maintaining liver metabolic function and redox balance. Understanding sex dimorphism and substrate dependency in these mitochondrial processes is crucial for elucidating the regulatory mechanisms that govern male versus female differences in liver physiology in health and disease. This study aimed at investigating sex-specific and substrate-dependent alterations in liver mitochondrial respiratory rates (Jo₂), membrane potential (ΔΨ), and H₂O₂ production and their metabolic regulation. Liver mitochondria were isolated from adult male and female Sprague-Dawley rats. Four substrate combinations-pyruvate + malate (PM), glutamate + malate (GM), succinate, and succinate with complex I inhibitor rotenone-were used to determine their impact on the activities of the electron transport chain (ETC) and TCA cycle complexes. Adenosine diphosphate (ADP) was added to determine the influence of substrates on oxidative phosphorylation (OxPhos). Jo₂ and ΔΨ were measured simultaneously using an Oroboros Oxygraph-2k respirometer with the cationic rhodamine dye tetramethylrhodamine methyl ester. H₂O₂ production was measured spectrofluorometrically using the Amplex Red and horseradish peroxidase assay. Our results show that male and female liver mitochondria displayed distinct respiratory patterns for different substrates. GM and succinate yielded higher Jo₂, whereas PM yielded the lowest Jo₂. Notably, female mitochondria exhibited higher Jo₂ than males across all substrates. Both ΔΨ and H₂O₂ production showed substrate-dependent patterns, with females exhibiting higher values than males across all substrates. These findings reveal sex-specific differences in liver mitochondrial function, driven by substrate-dependent engagement of the ETC and TCA cycle complexes toward OxPhos, with females showing higher respiratory capacity and H₂O₂ production.NEW & NOTEWORTHY We examined sex-specific and substrate-dependent differences in liver mitochondrial function of adult SD rats. Liver mitochondria preferentially use GM over PM for respiration, whereas PM produces more H₂O₂. Female mitochondria exhibited higher respiration and H₂O₂ production than males across all substrates, likely driven by hormonal factors and sex-specific regulatory pathways. These findings highlight the importance of both substrate and sex in shaping liver mitochondrial bioenergetics and redox function, offering insight into intrinsic metabolic differences.