Abstract：
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 & NOTEWORTHY Impairment 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.
摘要：
我们之前证明了转氨酶抑制剂，氨基氧乙酸,在复合物II(琥珀酸脱氢酶，SDH）在从小鼠后肢肌肉分离的线粒体中。该效应需要膜电位降低，从而导致草酰乙酸（OAA）的积累，一种有效的SDH抑制剂。为了特别评估线粒体转氨酶的作用，谷氨酸草酰乙酸转氨酶（GOT2）对复杂II呼吸的影响，并确定完整细胞以及分离的线粒体，我们在野生型(WT)和CRISPR产生的GOT2敲低(KD)C2C12肌细胞中进行了呼吸和代谢研究.通过添加羰基氰化物-对三氟甲氧基苯基腙（FCCP）以降低电势，可以减少GOT2KD细胞对WT的完整细胞呼吸。在C2C12KD细胞的线粒体中，由1µMFCCP产生的低电位呼吸，并在复合物II处由10mM琥珀酸盐+0.5mM谷氨酸盐激发，(但不是通过复合物I底物)相对于WT线粒体减少。虽然我们无法检测到OAA,代谢物数据表明，OAA对SDH的抑制可能有助于FCCP效应。C2C12线粒体与骨骼肌线粒体的不同之处在于，添加ADP后，FCCP对复杂II呼吸的影响不明显。我们还观察到C2C12细胞，与骨骼肌不同，表达的谷氨酸脱氢酶，与GOT2竞争谷氨酸代谢。总之,GOT2KD在低电位下降低完整细胞中的C2C12呼吸。从不同的底物效应，这主要发生在复杂的II。此外，C2C12与肌肉线粒体的复合物II对ADP的敏感性不同，而谷氨酸脱氢酶的表达明显不同。
