Departments of BioMedical Research and Radiology, University of Bern, Bern, Switzerland.
Institute of Clinical Chemistry, Inselspital, University Hospital Bern, 3010, Bern, Switzerland.
Metabolomics. 2019 Feb 28;15(3):32. doi: 10.1007/s11306-019-1497-2.
A decline in mitochondrial function represents a key factor of a large number of inborn errors of metabolism, which lead to an extremely heterogeneous group of disorders.
To gain insight into the biochemical consequences of mitochondrial dysfunction, we performed a metabolic profiling study in human skin fibroblasts using galactose stress medium, which forces cells to rely on mitochondrial metabolism.
Fibroblasts from controls, complex I and pyruvate dehydrogenase (PDH) deficient patients were grown under glucose or galactose culture condition. We investigated extracellular flux using Seahorse XF24 cell analyzer and assessed metabolome fingerprints using NMR spectroscopy.
Incubation of fibroblasts in galactose leads to an increase in oxygen consumption and decrease in extracellular acidification rate, confirming adaptation to a more aerobic metabolism. NMR allowed rapid profiling of 41 intracellular metabolites and revealed clear separation of mitochondrial defects from controls under galactose using partial least squares discriminant analysis. We found changes in classical markers of mitochondrial metabolic dysfunction, as well as unexpected markers of amino acid and choline metabolism. PDH deficient cell lines showed distinct upregulation of glutaminolytic metabolism and accumulation of branched-chain amino acids, while complex I deficient cell lines were characterized by increased levels in choline metabolites under galactose.
Our results show the relevance of selective culture methods in discriminating normal from metabolic deficient cells. The study indicates that untargeted fingerprinting NMR profiles provide physiological insight on metabolic adaptations and can be used to distinguish cellular metabolic adaptations in PDH and complex I deficient fibroblasts.
线粒体功能下降是许多先天性代谢错误的一个关键因素，这些错误导致了一组非常异质的疾病。
为了深入了解线粒体功能障碍的生化后果，我们使用半乳糖应激培养基在人皮肤成纤维细胞中进行了代谢组学研究，该培养基迫使细胞依赖于线粒体代谢。
在葡萄糖或半乳糖培养条件下培养来自对照、复合物 I 和丙酮酸脱氢酶（PDH）缺陷患者的成纤维细胞。我们使用 Seahorse XF24 细胞分析仪研究细胞外通量，并使用 NMR 光谱法评估代谢组指纹图谱。
将成纤维细胞在半乳糖中孵育会导致耗氧量增加和细胞外酸化率降低，这证实了对更需氧代谢的适应。NMR 允许快速分析 41 种细胞内代谢物，并使用偏最小二乘判别分析（PLS-DA）在半乳糖下从对照中清楚地区分线粒体缺陷。我们发现了经典的线粒体代谢功能障碍标志物的变化，以及氨基酸和胆碱代谢的意外标志物。PDH 缺陷细胞系表现出明显的谷氨酰胺分解代谢上调和支链氨基酸的积累，而复合物 I 缺陷细胞系在半乳糖下以胆碱代谢物水平升高为特征。
我们的结果表明选择性培养方法在区分正常和代谢缺陷细胞方面的相关性。该研究表明，非靶向指纹图谱 NMR 图谱提供了对代谢适应的生理见解，并可用于区分 PDH 和复合物 I 缺陷成纤维细胞的细胞代谢适应。
