β-Hydroxybutyrate Oxidation Promotes the Accumulation of Immunometabolites in Activated Microglia Cells

Metabolic regulation of immune cells has arisen as a critical set of processes required for appropriate response to immunological signals. While our knowledge in this area has rapidly expanded in leukocytes, much less is known about the metabolic regulation of brain-resident microglia. In particular, the role of alternative nutrients to glucose remains poorly understood. Here, we use stable-isotope (C-13) tracing strategies and metabolomics to characterize the oxidative metabolism of beta-hydroxybutyrate (BHB) in human (HMC3) and murine (BV2) microglia cells and the interplay with glucose in resting and LPS-activated BV2 cells. We found that BHB is imported and oxidised in the TCA cycle in both cell lines with a subsequent increase in the cytosolic NADH:NAD(+)ratio. In BV2 cells, stimulation with LPS upregulated the glycolytic flux, increased the cytosolic NADH:NAD(+)ratio and promoted the accumulation of the glycolytic intermediate dihydroxyacetone phosphate (DHAP). The addition of BHB enhanced LPS-induced accumulation of DHAP and promoted glucose-derived lactate export. BHB also synergistically increased LPS-induced accumulation of succinate and other key immunometabolites, such as alpha-ketoglutarate and fumarate generated by the TCA cycle. Finally, BHB upregulated the expression of a key pro-inflammatory (M1 polarisation) marker gene,NOS2, in BV2 cells activated with LPS. In conclusion, we identify BHB as a potentially immunomodulatory metabolic substrate for microglia that promotes metabolic reprogramming during pro-inflammatory response.