Complex I, V, and MDH2 deficient human skin fibroblasts reveal distinct metabolic signatures by 1 H HR-MAS NMR.

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.

Methods for Oxygen Determination in an NMR Bioreactor as a Surrogate Marker for Metabolomic Studies in Living Cell Cultures.

Nuclear magnetic resonance (NMR) approaches have been described as a powerful method for measuring oxygen in tissue cultures and body fluids by using relaxation time dependencies of substances on pO2. The present NMR study describes methods to longitudinally monitor global, in situ intracellular, and spatially resolved oxygen tension in culture media and 3D cell cultures using relaxation times of water without the need to use external sensors. 1H NMR measurements of water using a modified inversion recovery pulse scheme were employed for global, i.e., intra- and extracellular oxygen estimation in an NMR-bioreactor. The combination of 1H relaxation time T1 and diffusion measurements of water was employed for in situ cellular oxygen content determination. Spatially selective water relaxation time estimations were used for spatially resolved oxygen quantification along the NMR tube length. The inclusion in a study protocol of the presented techniques for oxygen quantification, as a surrogate marker of oxidative phosphorylation (OXPHOS), provides the possibility to measure mitochondrial respiration and metabolic changes simultaneously.

Targeting lactate dehydrogenase B-dependent mitochondrial metabolism affects tumor initiating cells and inhibits tumorigenesis of non-small cell lung cancer by inducing mtDNA damage.

Once considered a waste product of anaerobic cellular metabolism, lactate has been identified as a critical regulator of tumorigenesis, maintenance, and progression. The putative primary function of lactate dehydrogenase B (LDHB) is to catalyze the conversion of lactate to pyruvate; however, its role in regulating metabolism during tumorigenesis is largely unknown. To determine whether LDHB plays a pivotal role in tumorigenesis, we performed 2D and 3D in vitro experiments, utilized a conventional xenograft tumor model, and developed a novel genetically engineered mouse model (GEMM) of non-small cell lung cancer (NSCLC), in which we combined an LDHB deletion allele with an inducible model of lung adenocarcinoma driven by the concomitant loss of p53 (also known as Trp53) and expression of oncogenic KRAS (G12D) (KP). Here, we show that epithelial-like, tumor-initiating NSCLC cells feature oxidative phosphorylation (OXPHOS) phenotype that is regulated by LDHB-mediated lactate metabolism. We show that silencing of LDHB induces persistent mitochondrial DNA damage, decreases mitochondrial respiratory complex activity and OXPHOS, resulting in reduced levels of mitochondria-dependent metabolites, e.g., TCA intermediates, amino acids, and nucleotides. Inhibition of LDHB dramatically reduced the survival of tumor-initiating cells and sphere formation in vitro, which can be partially restored by nucleotide supplementation. In addition, LDHB silencing reduced tumor initiation and growth of xenograft tumors. Furthermore, we report for the first time that homozygous deletion of LDHB significantly reduced lung tumorigenesis upon the concomitant loss of Tp53 and expression of oncogenic KRAS without considerably affecting the animal's health status, thereby identifying LDHB as a potential target for NSCLC therapy. In conclusion, our study shows for the first time that LDHB is essential for the maintenance of mitochondrial metabolism, especially nucleotide metabolism, demonstrating that LDHB is crucial for the survival and proliferation of NSCLC tumor-initiating cells and tumorigenesis.

Determination of Intra- and Extracellular Metabolic Adaptations of 3D Cell Cultures upon Challenges in Real-Time by NMR.

NMR flow devices provide longitudinal real-time quantitative metabolome characterisation of living cells. However, discrimination of intra- and extracellular contributions to the spectra represents a major challenge in metabolomic NMR studies. The present NMR study demonstrates the possibility to quantitatively measure both metabolic intracellular fingerprints and extracellular footprints on human control fibroblasts by using a commercially available flow tube system with a standard 5 mm NMR probe. We performed a comprehensive 3D cell culture system characterisation. Diffusion NMR was employed for intra- and extracellular metabolites separation. In addition, complementary extracellular footprints were determined. The implemented perfused NMR bioreactor system allowed the determination of 35 metabolites and intra- and extracellular separation of 19 metabolites based on diffusion rate differences. We show the reliability and sensitivity of NMR diffusion measurements to detect metabolite concentration changes in both intra- and extracellular compartments during perfusion with different selective culture media, and upon complex I inhibition with rotenone. We also demonstrate the sensitivity of extracellular footprints to determine metabolic variations at different flow rates. The current method is of potential use for the metabolomic characterisation of defect fibroblasts and for improving physiological comprehension.