Effect of the Mitochondrial Transaminase (GOT2) on Membrane Potential Sensitive Respiration in Mitochondria of Differentiated C2C12 Muscle Cells.
Effect of the Mitochondrial Transaminase (GOT2) on Membrane Potential Sensitive Respiration in Mitochondria of Differentiated C2C12 Muscle Cells.
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线粒体转氨酶 (GOT2) 对分化 C2C12 肌细胞线粒体膜电位敏感呼吸的影响。
DOI：
10.1152/ajpcell.00576.2023
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Sivitz,WilliamI
中科院分区：
文献类型：
--
作者：
Som,Ritu;Fink,BrianD;Yu,Liping;Sivitz,WilliamI
我们之前的研究表明，转氨酶抑制剂氨基氧乙酸能减少小鼠后肢肌肉线粒体中复合体II(琥珀酸脱氢酶，SDH)的呼吸。这种作用需要降低膜电位，从而导致草酰乙酸酯(OAA)的积累，草酰乙酸酯是SDH的有效抑制剂。为了具体评估线粒体转氨酶谷草转氨酶(GOT2)对复合体II呼吸的影响，并确定在完整细胞以及分离的线粒体中的影响，我们对野生型(WT)和CRISPR产生的GOT2基因敲除(KD)C2C12心肌细胞进行了呼吸和代谢研究。加入对三氟甲氧基苯肼(FCCP)降低GOT2KD细胞的完整细胞呼吸。在C2C12KD细胞的线粒体中，1µM FCCP产生的低电位呼吸和10 mM琥珀酸+0.5 mM谷氨酸(但不是通过复合I底物)在复合体II产生的低电位呼吸作用比WT线粒体减少。虽然我们无法检测到OAA，但代谢产物数据表明，OAA对SDH的抑制可能参与了FCCP的作用。C2C12线粒体与骨骼肌线粒体的不同之处在于，在添加ADP的情况下，FCCP对复合体II呼吸的影响不明显。我们还观察到，与骨骼肌不同，C2C12细胞表达谷氨酸脱氢酶，该酶与GOT2竞争谷氨酸代谢。综上所述，GOT2 KD在低电势下减少了完整细胞的C2C12呼吸。从不同的底物效应来看，这主要发生在复合体II上。此外，C2C12和肌肉线粒体在复合体II对ADP的敏感性上存在差异，在谷氨酸脱氢酶的表达上也存在显著差异。线粒体转氨酶GOT2的缺失降低了复合体II(琥珀酸脱氢酶，SDH)供能的C2C12心肌细胞的呼吸。这仅在低内膜电位时发生，并与SDH的抑制一致。顺便说一句，我们观察到C2C12线粒体与肌肉组织线粒体相比，在复合体II呼吸对ADP的敏感性和谷氨酸脱氢酶的表达方面存在差异。
We previously showed that the transaminase inhibitor, aminooxyacetic acid, reduced respiration energized at complex II (succinate dehydrogenase, SDH) in mitochondria isolated from mouse hindlimb muscle. The effect required a reduction in membrane potential with resultant accumulation of oxaloacetate (OAA), a potent inhibitor of SDH. To specifically assess the effect of the mitochondrial transaminase, glutamic oxaloacetic transaminase (GOT2) on complex II respiration, and to determine the effect in intact cells as well as isolated mitochondria, we performed respiratory and metabolic studies in wildtype (WT) and CRISPR-generated GOT2 knockdown (KD) C2C12 myocytes. Intact cell respiration by GOT2KD cells versus WT was reduced by adding carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to lower potential. In mitochondria of C2C12 KD cells, respiration at low potential generated by 1 µM FCCP and energized at complex II by 10 mM succinate + 0.5 mM glutamate (but not by complex I substrates) was reduced versus WT mitochondria. Although we could not detect OAA, metabolite data suggested that OAA inhibition of SDH may have contributed to the FCCP effect. C2C12 mitochondria differed from skeletal muscle mitochondria in that the effect of FCCP on complex II respiration was not evident with ADP addition. We also observed that C2C12 cells, unlike skeletal muscle, expressed glutamate dehydrogenase, which competes with GOT2 for glutamate metabolism. In summary, GOT2 KD reduced C2C12 respiration in intact cells at low potential. From differential substrate effects, this occurred largely at complex II. Moreover, C2C12 versus muscle mitochondria differ in complex II sensitivity to ADP and differ markedly in expression of glutamate dehydrogenase.NEW & NOTEWORTHYImpairment of the mitochondrial transaminase, GOT2, reduces complex II (succinate dehydrogenase, SDH)-energized respiration in C2C12 myocytes. This occurs only at low inner membrane potential and is consistent with inhibition of SDH. Incidentally, we observed that C2C12 mitochondria compared with muscle tissue mitochondria differ in sensitivity of complex II respiration to ADP and in the expression of glutamate dehydrogenase.
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