Abstract:
In this study, we investigated the metabolic signatures of different mitochondrial defects (two different complex I and complex V, and the one MDH2 defect) in human skin fibroblasts (HSF). We hypothesized that using a selective culture medium would cause defect specific adaptation of the metabolome and further our understanding of the biochemical implications for the studied defects. All cells were cultivated under galactose stress condition and compared to glucose-based cell culture condition. We investigated the bioenergetic profile using Seahorse XFe96 cell analyzer and assessed the extracellular metabolic footprints and the intracellular metabolic fingerprints using NMR. The galactose-based culture condition forced a bioenergetic switch from a glycolytic to an oxidative state in all cell lines which improved overall separation of controls from the different defect groups. The extracellular metabolome was discriminative for separating controls from defects but not the specific defects, whereas the intracellular metabolome suggests CI and CV changes and revealed clear MDH2 defect-specific changes in metabolites associated with the TCA cycle, malate aspartate shuttle, and the choline metabolism, which are pronounced under galactose condition.
摘要:
在这项研究中,我们研究了不同线粒体缺陷的代谢特征(两种不同的复合物I和复合物V,和人皮肤成纤维细胞(HSF)中的一个MDH2缺陷)。我们假设使用选择性培养基会导致代谢组的缺陷特异性适应,并进一步了解所研究缺陷的生化含义。在半乳糖应激条件下培养所有细胞,并与基于葡萄糖的细胞培养条件进行比较。我们使用海马XFe96细胞分析仪研究了生物能量谱,并使用NMR评估了细胞外代谢足迹和细胞内代谢指纹。基于半乳糖的培养条件迫使所有细胞系中的生物能量从糖酵解状态转换为氧化状态,从而改善了对照与不同缺陷组的整体分离。细胞外代谢组是区分对照与缺陷,但不是特定的缺陷,而细胞内代谢组提示CI和CV变化,并显示与TCA循环相关的代谢物中明显的MDH2缺陷特异性变化,苹果酸天冬氨酸穿梭,和胆碱代谢,这在半乳糖条件下很明显。