Characterization of Sex-Dependent Differences in Brain Microvascular Bioenergetics

Background: Women experience increased incidence of stroke-related deaths and lifetime risk of Alzheimer's disease (AD). Microvascular dysfunction has been implicated in the pathogenesis of ischemic stroke and AD. Our objective is to investigate the sex-dependent differences in bioenergetics of young mouse brain microvessels (BMVs). Methods: BMVs were isolated from young male and female mice (3-4 months), using a combination of gradient centrifugation and filtration (300μm and 40μm filters) methods. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of BMVs were measured utilizing the Agilent Seahorse XFe 24 analyzer. The levels of proteins and mRNAs of mitochondrial respiratory complexes were measured by western blot and RT-PCR, respectively. Citrate synthase (CS) enzyme activity and ATP levels were determined in lysates of BMVs. Results: Real-Time ATP Rate assay demonstrated a decreased total ATP production rate and ATP contribution from both glycolysis and oxidative phosphorylation (OxPhos) in BMVs from females compared to males. Mitochondrial stress test assay revealed significantly lower values of several mitochondrial respiratory parameters, including basal respiration, ATP production, maximal respiration, spare respiratory capacity, and proton leak in BMVs from females compared to males. Glycolytic Rate assay uncovered reduced basal glycolysis, proton efflux rate (PER) originating from basal glycolysis, and the mitoOCR/glycoPER ratio in BMVs from females compared to males. Interestingly, no sex-dependent differences were observed in basal PER and post-2-DG acidification rates in BMVs. Mito Fuel Flex Test assay showed no sex-dependent difference in the utilization of glucose, glutamine, and long-chain fatty acids as fuel substrates in BMVs. ATP measurements confirmed the reduced ATP levels in BMVs from females compared to males. Measurements of CS activity revealed no sex-dependent difference in the BMVs. Lastly, Western blot and RT-PCR experiments indicated no differences in either protein or mRNA levels of various mitochondrial complex protein levels.Conclusions: Female BMVs showed reduced mitochondrial respiratory and glycolytic parameters compared to male BMVs although relative contribution of various substrates for energy production were not different. These sex-dependent differences involving glycolysis and OxPhos make the microvasculature vulnerable to injury in females. Funding sources: NIH grants AG074489 and NS114286 (PVGK and RM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.