Ketone body-supported respiration in murine isolated brain mitochondria is augmented by alpha-ketoglutarate and is optimized by neuronal SCOT expression

Ketone bodies are increasingly examined as an alternative fuel source for the known decreases in glucose utilization that occur with neurodegeneration. Here, we established a protocol to maximize ketone body respiration in isolated brain mitochondria, while quantifying acetyl-CoA and energy charge via liquid chromatography-tandem mass spectrometry in control mice compared with mice with neuron-specific deletion of succinyl-CoA-3-oxoacid-CoA transferase (SCOT), required for CoA transfer from succinyl-CoA to acetoacetate (AcAc) to support its oxidation. Maximal ADP-dependent AcAc respiration occurred at 1 mM; however, the percent increase above basal was minimal (∼15%). Alpha-ketoglutarate (αKG) substantially increased AcAc-dependent respiration in isolated brain mitochondria, putatively through the generation of succinyl-CoA. Using mice with neuron-specific deletion of SCOT, we also examined brain mitochondrial respiration of AcAc and resulting acetyl CoA and energy charge (cellular energy availability via adenosine nucleotide ratios of ATP, ADP, and AMP). As expected, isolated brain mitochondria from SCOT-knockout (KO) mice had lower AcAc State 3 respiration than control mice. Surprisingly, we did not find differences in mitochondrial energy charge between SCOT control and neuron SCOT-KO mice despite decreased acetyl-CoA level in SCOT-KO mice when AcAc was used as the substrate. In conclusion, we show that αKG enhances ketone-supported respiration rate in isolated brain mitochondria and ketone metabolism in neurons affects acetyl-CoA level in brain mitochondria but not energy charge. Future work will determine whether diet, exercise, sex, or age impacts ketone-supported respiration rates in conjunction with differences in markers of brain health.<b>NEW & NOTEWORTHY</b> This paper established a protocol to maximize ketone body respiration in isolated brain mitochondria while quantifying acetyl-CoA and energy charge in control mice compared with mice with neuron-specific deletion of succinyl-CoA-3-oxoacid-CoA transferase (SCOT) enzyme, required for ketone body oxidation. Findings are that alpha-ketoglutarate substantially increased acetoacetate (AcAc)-dependent respiration and neuron SCOT-KO had lower AcAc state 3 respiration with a decreased acetyl-CoA level.