Sex-Specific and Substrate-Dependent Mitochondrial Bioenergetics and H₂O₂Emission in Liver

Rationale: Identifying and investigating sex differences in physiological phenomena and understanding the underlying regulatory mechanisms is crucial for precision medicine. Oxidative stress is implicated in various diseases, including ischemia-reperfusion injury (IRI) with liver transplantation. Understanding sex-specific and substrate-dependent mitochondrial bioenergetics and H 2 O 2 emission in the liver is important for developing mechanism-based therapeutic strategies to mitigate IRI in liver transplantation. The aim of this study was therefore to investigate sex-specific and substrate-dependent changes in mitochondrial respiration (oxygen consumption rate; OCR), membrane potential (dPsi), and H 2 O 2 emission in the liver and their metabolic regulations. Methods: Mitochondria were freshly isolated using differential centrifugation method from adult Sprague-Dawley rat livers (n=5/group; both sexes). To assess the electron transport chain (ETC) and TCA cycle dependencies of liver mitochondria, four different substrate combinations, namely, pyruvate+malate (PM), glutamate+malate (GM), succinate, and succinate in the presence of the complex I inhibitor rotenone (SR) were used. A single saturated dose of ADP was added to mitochondria to determine how different substrates influence mitochondrial OCR and dPsi during oxidative phosphorylation. Mitochondrial OCR and dPsi were measured simultaneously using a dual chamber Oroboros Oxygraph-2k instrument coupled to a fluorometer using the cationic rhodamine dye TMRM. The H 2 O 2 emission was measured spectrofluorometrically using the amplex red and horseradish peroxidase in the respiration buffer, resulting in resorufin fluorescence with the non-H 2 O 2 -mediated resorufin fluorescence inhibited by PMSF (phenylmethylsulfonyl fluoride), a serine protease inhibitor. Results: Male and female liver mitochondria exhibited distinct respiratory patterns in the presence of different substrates. The respiratory rates were higher when GM and succinate were utilized as substrates, whereas the use of PM showed comparatively lower respiration. Interestingly, female mitochondria exhibited significantly higher OCR than male mitochondria irrespective of the substrate utilized. Similar sex-specific and substrate-dependent responses were observed in mitochondrial dPsi and H 2 O 2 emission. PMSF effectively inhibited the non-H 2 O 2 -mediated resorufin fluorescence, implying a potential role of serine proteases in modulating oxidative processes and redox signaling in liver mitochondria. Conclusion: Sex differences were observed when mitochondria were fueled with distinct respiratory substrates that selectively activate specific complexes of the ETC and TCA cycle enzymes. Female liver mitochondria demonstrated greater capacity and efficiency in respiratory function and H 2 O 2 emission, suggesting that sex-specific differences may arise from regulatory pathways associated with respiratory substrate utilization in liver mitochondria. NIH R01 HL151587 and NSF DMS 2153387. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.