Davis Xin C, McCoin Colin S, Morris E Matthew, Allen Julie, Stierwalt Harrison D, Franczak Edziu, Queathem Eric D, Fulghum Kyle L, Puchalska Patrycja, Crawford Peter A, Thyfault John P
Department of Cell Biology & Physiology, University of Kansas Medical Center, Kansas City, Kansas, United States.
Kansas Center for Metabolism and Obesity Research, Kansas City, Kansas, United States.
Am J Physiol Endocrinol Metab. 2025 Jun 1;328(6):E822-E832. doi: 10.1152/ajpendo.00058.2025. Epub 2025 Apr 17.
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. 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.
随着神经退行性变过程中已知的葡萄糖利用减少，酮体作为一种替代燃料来源越来越受到关注。在此，我们建立了一种方案，以最大化分离的脑线粒体中的酮体呼吸作用，同时通过液相色谱 - 串联质谱法对对照小鼠与神经元特异性缺失琥珀酰辅酶A - 3 - 氧代酸 - 辅酶A转移酶（SCOT）的小鼠中的乙酰辅酶A和能量电荷进行定量分析，该酶是辅酶A从琥珀酰辅酶A转移至乙酰乙酸（AcAc）以支持其氧化所必需的。最大的依赖ADP的AcAc呼吸作用发生在1 mM浓度时；然而，相对于基础水平的增加百分比很小（约15%）。α - 酮戊二酸（αKG）显著增加了分离的脑线粒体中依赖AcAc的呼吸作用，推测是通过生成琥珀酰辅酶A实现的。利用神经元特异性缺失SCOT的小鼠，我们还研究了AcAc的脑线粒体呼吸作用以及由此产生的乙酰辅酶A和能量电荷（通过ATP、ADP和AMP的腺苷核苷酸比率反映的细胞能量可用性）。正如预期的那样，来自SCOT基因敲除（KO）小鼠的分离脑线粒体的AcAc状态3呼吸作用低于对照小鼠。令人惊讶的是，尽管在以AcAc为底物时SCOT - KO小鼠中的乙酰辅酶A水平降低，但我们并未发现SCOT对照小鼠和神经元SCOT - KO小鼠之间线粒体能量电荷存在差异。总之，我们表明αKG增强了分离的脑线粒体中酮体支持的呼吸速率，并且神经元中的酮体代谢会影响脑线粒体中的乙酰辅酶A水平，但不影响能量电荷。未来的工作将确定饮食、运动、性别或年龄是否会结合脑健康标志物的差异影响酮体支持的呼吸速率。本文建立了一种方案，以最大化分离的脑线粒体中的酮体呼吸作用，同时对对照小鼠与神经元特异性缺失酮体氧化所需的琥珀酰辅酶A - 3 - 氧代酸 - 辅酶A转移酶（SCOT）酶的小鼠中的乙酰辅酶A和能量电荷进行定量分析。研究结果表明，α - 酮戊二酸显著增加了乙酰乙酸（AcAc）依赖的呼吸作用，并且神经元SCOT - KO小鼠的AcAc状态3呼吸作用较低，乙酰辅酶A水平降低。
