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• W4377020132 abstract "<b>Abstract ID 18793</b> <b>Poster Board 418</b> Cellular metabolism has emerged as a key regulator of macrophage activation towards a proinflammatory M1 or a proresolution M2 phenotype. Previous studies have demonstrated that M1 macrophages upregulate glycolysis for rapid ATP production, while M2 macrophages are dependent on mitochondrial respiration. One mechanism by which metabolism is regulated is by breaking the citric acid cycle (CAC) at points, such as succinate dehydrogenase. Evaluating the balance between metabolic pathways and macrophage activation is essential for elucidating the role of macrophages in tissue injury. Such analysis has required the use of expensive metabolomics, however, here we used the Biolog MitoPlate^TM mitochondrial functional assay to assess macrophage immunometabolism by evaluating their substrate preference and respiration. RAW 264.7 mouse macrophages, cultured in Dulbecco’s modified Eagle’s medium (DMEM) with LPS (10 ng/mL) or DMEM control and bone marrow-derived macrophages (BMDMs) collected from male Wistar rats, cultured with macrophage-colony stimulating factor (MCSF, days 0, 3, and 6; 20 ng/mL/day) and LPS (day 6; 0, 10, 100 ng/mL) for 18-24 h were used. To assess mitochondrial specific metabolism, cells were permeabilized with saponin and treated with Redox MC Dye in Biolog Mitochondrial Assay Solution (BMAS). For whole cell metabolism, cells were treated with MB Dye in RPMI. Dye reduction, an indicator of substrate utilization, was measured spectrophotometrically (OD₅₉₀-OD₇₅₀, 37C) every 5 min for 4-8 h. Cellular permeabilization was required to assess mitochondrial usage of malate, a-ketoglutarate, or citrate metabolism. LPS treatment of RAW cells and BMDM resulted in increased reliance on malate, indicating a break in CAC at succinate dehydrogenase. Similar effects were observed at the whole cell level. Overall these data confirm breaks in CAC at isocitrate dehydrogenase and succinate dehydrogenase. These data demonstrate that the MitoPlate^TM is effective to evaluate specific steps in the CAC that may be modified by toxicants. Supported by NIH Grants AR055073, 5R25ES020721, and ES005022." @default.
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• W4377020132 date "2023-05-18" @default.
• W4377020132 modified "2023-09-29" @default.
• W4377020132 title "Evaluating the role of rodent macrophage immunometabolism in response to endotoxin" @default.
• W4377020132 doi "https://doi.org/10.1124/jpet.122.187930" @default.
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