Selective galactose culture condition reveals distinct metabolic signatures in pyruvate dehydrogenase and complex I deficient human skin fibroblasts
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.
KeywordsGalactose Complex I Pyruvate dehydrogenase NMR Mitochondrial dysfunction
We thank André Schaller (Division of Human Genetics and Department of Paediatrics, Inselspital, Bern) for providing the genetic characteristics of the patient fibroblasts.
DH, AF, GD, SK, PV, and JMN conceived the work and designed the experiments; DH, AF, and SK cultured cells, performed metabolic flux experiments and analyzed the data. DH and GD performed NMR analysis of cells or supernatant and analyzed the data. PV and JMN provided experimental advice and overall guidance. DH, AF, GD, SK, PV and JMN wrote the manuscript or revised it critically for important intellectual content. All authors approved the final manuscript.
This work was supported by a grant from the Batzebär foundation of the children’s university hospitals Bern to JMN.
Compliance with ethical standards
Conflict of interest
The authors have declared no conflicts of interest.
All procedures performed in studies involving human participants were in accordance with the 1964 Helsinki declaration and its later amendments and approved by the Ethics Committee of the University Hospital of Bern.
Informed consent was obtained from all individual participants included in the study.
- Dieterle, F., Ross, A., Schlotterbeck, G., & Senn, H. (2006). Probabilistic quotient normalization as robust method to account for dilution of complex biological mixtures. Application in 1H NMR metabonomics. Analytical Chemistry, 78, 4281–4290. https://doi.org/10.1021/ac051632c.CrossRefPubMedGoogle Scholar
- Harper, A. E., Miller, R. H., & Block, K. P. (1984). Branched-chain amino acid metabolism. Annual Review of Nutrition, 4, 409–454. https://doi.org/10.1146/annurev.nu.04.070184.002205.CrossRefPubMedGoogle Scholar
- Marroquin, L. D., Hynes, J., Dykens, J. A., Jamieson, J. D., & Will, Y. (2007). Circumventing the Crabtree effect: Replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants. Toxicological Sciences, 97, 539–547. https://doi.org/10.1093/toxsci/kfm052.CrossRefPubMedGoogle Scholar
- Nicholson, J. K., Lindon, J. C., & Holmes, E. (1999). ‘Metabonomics’: understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica, 29, 1181–1189. https://doi.org/10.1080/004982599238047.CrossRefPubMedGoogle Scholar
- Vermathen, M., Paul, L. E., Diserens, G., Vermathen, P., & Furrer, J. (2015). 1H HR-MAS NMR based metabolic profiling of cells in response to treatment with a hexacationic ruthenium metallaprism as potential anticancer drug. PLoS ONE, 10, e0128478. https://doi.org/10.1371/journal.pone.0128478.CrossRefPubMedPubMedCentralGoogle Scholar