, 15:32 | Cite as

Selective galactose culture condition reveals distinct metabolic signatures in pyruvate dehydrogenase and complex I deficient human skin fibroblasts

  • Damian Hertig
  • Andrea Felser
  • Gaëlle Diserens
  • Sandra Kurth
  • Peter Vermathen
  • Jean-Marc NuofferEmail author
Original Article



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.


Galactose 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.

Author contribution

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.

Ethical approval

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

Informed consent was obtained from all individual participants included in the study.

Supplementary material

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Supplementary material 1 (DOCX 27 KB)
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Supplementary material 2 (DOCX 18 KB)
11306_2019_1497_MOESM3_ESM.pptx (81 kb)
Supplementary material 3 (PPTX 81 KB)
11306_2019_1497_MOESM4_ESM.pptx (458 kb)
Supplementary material 4 (PPTX 458 KB)


  1. Aguer, C., et al. (2011). Galactose enhances oxidative metabolism and reveals mitochondrial dysfunction in human primary muscle cells. PLoS ONE, 6, e28536. Scholar
  2. Aguilar, J. A., Nilsson, M., Bodenhausen, G., & Morris, G. A. (2012). Spin echo NMR spectra without J modulation. Chemical Communications (Camb), 48, 811–813. Scholar
  3. Baykal, A. T., Jain, M. R., & Li, H. (2008). Aberrant regulation of choline metabolism by mitochondrial electron transport system inhibition in neuroblastoma cells. Metabolomics, 4, 347–356. Scholar
  4. Beckonert, O., et al. (2010). High-resolution magic-angle-spinning NMR spectroscopy for metabolic profiling of intact tissues. Nature Protocols, 5, 1019–1032. Scholar
  5. Bluml, S., Seymour, K. J., & Ross, B. D. (1999). Developmental changes in choline- and ethanolamine-containing compounds measured with proton-decoupled (31)P MRS in in vivo human brain. Magnetic Resonance in Medicine, 42, 643–654.CrossRefGoogle Scholar
  6. Brown, G. K., Otero, L. J., LeGris, M., & Brown, R. M. (1994). Pyruvate dehydrogenase deficiency. Journal of Medical Genetics, 31, 875–879.CrossRefGoogle Scholar
  7. 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. Scholar
  8. DiMauro, S. (2004). Mitochondrial diseases. Biochimica et Biophysica Acta, 1658, 80–88. Scholar
  9. Diserens, G., et al. (2017). Metabolic stability of cells for extended metabolomical measurements using NMR. A comparison between lysed and additionally heat inactivated cells. Analyst, 142, 465–471. Scholar
  10. Gropman, A. L. (2013). Neuroimaging in mitochondrial disorders. Neurotherapeutics, 10, 273–285. Scholar
  11. Harper, A. E., Miller, R. H., & Block, K. P. (1984). Branched-chain amino acid metabolism. Annual Review of Nutrition, 4, 409–454. Scholar
  12. Jansen, P. H., van der Knaap, M. S., & de Coo, I. F. (1996). Leber’s hereditary optic neuropathy with the 11 778 mtDNA mutation and white matter disease resembling multiple sclerosis: Clinical, MRI and MRS findings. Journal of the Neurological Sciences, 135, 176–180.CrossRefGoogle Scholar
  13. Johnson, M. T., Yang, H. S., & Patel, M. S. (2000). Targeting E3 component of alpha-keto acid dehydrogenase complexes. Methods in Enzymology, 324, 465–476.CrossRefGoogle Scholar
  14. Kirby, D. M., Crawford, M., Cleary, M. A., Dahl, H. H., Dennett, X., & Thorburn, D. R. (1999). Respiratory chain complex I deficiency: An underdiagnosed energy generation disorder. Neurology, 52, 1255–1264.CrossRefGoogle Scholar
  15. Koopman, W. J., Distelmaier, F., Smeitink, J. A., & Willems, P. H. (2013). OXPHOS mutations and neurodegeneration. EMBO Journal, 32, 9–29. Scholar
  16. Koopman, W. J., Willems, P. H., & Smeitink, J. A. (2012). Monogenic mitochondrial disorders. New England Journal of Medicine, 366, 1132–1141. Scholar
  17. Li, T., et al. (2017). Defective branched-chain amino acid catabolism disrupts glucose metabolism and sensitizes the heart to ischemia-reperfusion injury. Cell Metabolism, 25, 374–385. Scholar
  18. Li, Y., et al. (2016). PDHA1 gene knockout in prostate cancer cells results in metabolic reprogramming towards greater glutamine dependence. Oncotarget, 7, 53837–53852. Scholar
  19. Loeffen, J. L., et al. (2000). Isolated complex I deficiency in children: Clinical, biochemical and genetic aspects. Human Mutation, 15(200002), 123–134.CrossRefGoogle Scholar
  20. Lynch, C. J., & Adams, S. H. (2014). Branched-chain amino acids in metabolic signalling and insulin resistance. Nature Reviews Endocrinology, 10, 723–736. Scholar
  21. 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. Scholar
  22. Mastorodemos, V., Zaganas, I., Spanaki, C., Bessa, M., & Plaitakis, A. (2005). Molecular basis of human glutamate dehydrogenase regulation under changing energy demands. Journal of Neuroscience Research, 79, 65–73. Scholar
  23. Mitochondrial Medicine Society’s Committee on, D., et al. (2008). The in-depth evaluation of suspected mitochondrial disease. Molecular Genetics and Metabolism, 94, 16–37. Scholar
  24. Moestue, S., Sitter, B., Bathen, T. F., Tessem, M. B., & Gribbestad, I. S. (2011). HR MAS MR spectroscopy in metabolic characterization of cancer. Current Topics in Medicinal Chemistry, 11, 2–26.CrossRefGoogle Scholar
  25. Morvan, D., & Demidem, A. (2018). NMR metabolomics of fibroblasts with inherited mitochondrial Complex I mutation reveals treatment-reversible lipid and amino acid metabolism alterations. Metabolomics, 14, 55. Scholar
  26. 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. Scholar
  27. Robinson, B. H. (1998). Human complex I deficiency: Clinical spectrum and involvement of oxygen free radicals in the pathogenicity of the defect. Biochimica et Biophysica Acta, 1364, 271–286.CrossRefGoogle Scholar
  28. Robinson, B. H., MacKay, N., Chun, K., & Ling, M. (1996). Disorders of pyruvate carboxylase and the pyruvate dehydrogenase complex. Journal of Inherited Metabolic Diseases, 19, 452–462.CrossRefGoogle Scholar
  29. Robinson, B. H., Petrova-Benedict, R., Buncic, J. R., & Wallace, D. C. (1992). Nonviability of cells with oxidative defects in galactose medium: A screening test for affected patient fibroblasts. Biochemical Medicine and Metabolic Biology, 48, 122–126.CrossRefGoogle Scholar
  30. Rossignol, R., Gilkerson, R., Aggeler, R., Yamagata, K., Remington, S. J., & Capaldi, R. A. (2004). Energy substrate modulates mitochondrial structure and oxidative capacity in cancer cells. Cancer Research, 64, 985–993.CrossRefGoogle Scholar
  31. Smeitink, J. A. (2003). Mitochondrial disorders: Clinical presentation and diagnostic dilemmas. Journal of Inherited Metabolic Diseases, 26, 199–207.CrossRefGoogle Scholar
  32. Smeitink, J. A., Zeviani, M., Turnbull, D. M., & Jacobs, H. T. (2006). Mitochondrial medicine: A metabolic perspective on the pathology of oxidative phosphorylation disorders. Cell Metabolism, 3, 9–13. Scholar
  33. Udhane, S. S., et al. (2017). Combined transcriptome and metabolome analyses of metformin effects reveal novel links between metabolic networks in steroidogenic systems. Scientific Reports, 7, 8652. Scholar
  34. Vafai, S. B., & Mootha, V. K. (2013). Medicine. A common pathway for a rare disease? Science, 342, 1453–1454. Scholar
  35. Vermathen, M., Diserens, G., Vermathen, P., & Furrer, J. (2017). Metabolic profiling of cells in response to drug treatment using (1)H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. Chimia, 71, 124–129. Scholar
  36. 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. Scholar
  37. Wishart, D. S., et al. (2013). HMDB 3.0—the human metabolome database in 2013. Nucleic Acids Research, 41, D801–D807. Scholar
  38. Yang, C., et al. (2014). Glutamine oxidation maintains the TCA cycle and cell survival during impaired mitochondrial pyruvate transport. Molecular Cell, 56, 414–424. Scholar
  39. Zhou, X., & Thompson, J. R. (1996). Regulation of glutamate dehydrogenase by branched-chain amino acids in skeletal muscle from rats and chicks. The International Journal of Biochemistry and Cell Biology, 28, 787–793.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019
corrected publication 2019

Authors and Affiliations

  1. 1.Departments of BioMedical Research and RadiologyUniversity of BernBernSwitzerland
  2. 2.Institute of Clinical Chemistry, InselspitalUniversity Hospital BernBernSwitzerland
  3. 3.Graduate School for Cellular and Biomedical SciencesUniversity of BernBernSwitzerland
  4. 4.Department of Paediatrics, InselspitalUniversity Hospital BernBernSwitzerland

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