Nitric oxide dictates the reprogramming of carbon flux during M1 macrophage polarization

Abstract

Abstract Classical pro-inflammatory activation of macrophages is characterized by profound intracellular metabolic changes, with increased glycolytic usage of carbon, away from Oxidative Phosphorylation (OXPHOS). We previously demonstrated that Nitric Oxide (NO) levels induced in Bone Marrow Derived Macrophages (BMDMs) from Wild Type (WT) mice are necessary and sufficient for the repression of OXPHOS. Here we demonstrate that NO is also responsible for the “break” in the mitochondrial TCA cycle and citrate accumulation during LPS/IFNγ stimulation; macrophages that lack NO maintain indeed substantial levels of Oxygen Consumption Rates (OCR) and TCA cycle intermediates. Carbon tracing experiments in the presence of U13C-glucose show almost undetected labelled α-ketoglutarate from citrate in WT but a conserved pattern of heavy carbon fate in Nos2−/− macrophages. Moreover we found that mitochondrial respiration elicited through citrate was decreased in WT M1 macrophages, but isocitrate was a full substrate for complex I-dependent OCR suggesting suppression of metabolism at mitochondrial Aconitase (ACO2). Consistent with this data, we found ACO2 enzymatic activity blunted in WT vs Nos2−/−. In addition we observed that M1 macrophages reroute pyruvate away from Pyruvate Dehydrogenase (PDH) in an NO dependent manner since only WT show halted flux through PDH. Surprisingly, we demonstrate this mechanism to be independent on the activation of Hif1α and its suggested effect on limiting acetyl-coA for the TCA. With these data together we hypothesize that NO orchestrates macrophage metabolism during inflammation inhibiting OXPHOS by blocking Krebs Cycle, therefore depriving of substrates the mitochondrial electron transport chain.