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

Combined transcriptome and metabolome analyses of metformin effects reveal novel links between metabolic networks in steroidogenic systems.

Metformin is an antidiabetic drug, which inhibits mitochondrial respiratory-chain-complex I and thereby seems to affect the cellular metabolism in many ways. It is also used for the treatment of the polycystic ovary syndrome (PCOS), the most common endocrine disorder in women. In addition, metformin possesses antineoplastic properties. Although metformin promotes insulin-sensitivity and ameliorates reproductive abnormalities in PCOS, its exact mechanisms of action remain elusive. Therefore, we studied the transcriptome and the metabolome of metformin in human adrenal H295R cells. Microarray analysis revealed changes in 693 genes after metformin treatment. Using high resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS-NMR), we determined 38 intracellular metabolites. With bioinformatic tools we created an integrated pathway analysis to understand different intracellular processes targeted by metformin. Combined metabolomics and transcriptomics data analysis showed that metformin affects a broad range of cellular processes centered on the mitochondrium. Data confirmed several known effects of metformin on glucose and androgen metabolism, which had been identified in clinical and basic studies previously. But more importantly, novel links between the energy metabolism, sex steroid biosynthesis, the cell cycle and the immune system were identified. These omics studies shed light on a complex interplay between metabolic pathways in steroidogenic systems

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

Live monitoring of cellular metabolism and mitochondrial respiration in 3D cell culture system using NMR spectroscopy.

BACKGROUND Because of the interplay between mitochondrial respiration and cellular metabolism, the simultaneous monitoring of both cellular processes provides important insights for the understanding of biological processes. NMR flow systems provide a unique window into the metabolome of cultured cells. Simplified bioreactor construction based on commercially available flow systems increase the practicability and reproducibility of bioreactor studies using standard NMR spectrometers. We therefore aim at establishing a reproducible NMR bioreactor system for metabolic 1H-NMR investigations of small molecules and concurrent oxygenation determination by 19F-NMR, with in depth description and validation by accompanying measures. METHODS We demonstrate a detailed and standardized workflow for the preparation and transfer of collagen based 3D cell culture of high cell density for perfused investigation in a 5 mm NMR tube. Self-constructed gas mixing station enables 5% CO2 atmosphere for physiological pH in carbon based medium and is perfused by HPLC pump. RESULTS & DISCUSSION Implemented perfused bioreactor allows detection of perfusion rate dependent metabolite content. We show interleaved dynamic profiling of 26 metabolites and mitochondrial respiration. During constant perfusion, sequential injection of rotenone/oligomycin and 2-deoxy-glucose indicated immediate activation and deactivation of glycolytic rate and full inhibition of oxygen consumption. We show sensitivity to detect substrate degradation rates of major mitochondrial fuel pathways and were able to simultaneously measure cellular oxygen consumption

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

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

INTRODUCTION 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. OBJECTIVES 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. METHODS 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. RESULTS 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. CONCLUSION 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

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

The original version of this article contained an error in Table 2. The text in the second header line should read "GAL supernatant" and "GAL Medium" instead of "GLC supernatant" and "GLC Medium". The corrected Table 2 is given below. The original article has been corrected

Improving the metabolic stability of cultured cells during extended HR-MAS NMR measurements by prior heating

Introduction: 1HHRMAS NMR is an established tool for metabolic characterization of biological samples. However, the accuracy of biomarker detection depends on the sample stability over measurement time. Previously, Duarte et al. found enhanced metabolite visibility and different lipid profiles in lysed compared to frozen cells [1]. Here we investigated effects of shorttime heating prior to HRMAS measurements on metabolite stability of cells. We hypothesized that cell heating has only minor effects on initial metabolite contents (i.e. initially similar spectra with and without heating) and results in increased temporal metabolite stability due to reduced enzymatic activity. Methods: The metabolite content of six lysed nonheated (LFB) and six lysed heated fibroblastsamples (LHFB) was measured as a function of time. Additionally, one sample each of nonheated and heated lysed adrenal cells (LAD & LHAD), which are less robust than fibroblasts, were measured. After lysis according to [1], half of the samples were heated (70°C) for 20min. 1HPROJECT [2] spectra were obtained on a Bruker AvanceII spectrometer (500.13MHz, 277K, MAS=3kHz) at different times over 9 hours. Individual peak analysis was performed investigating temporal changes. For fibroblasts, statistical methods were applied including PCA. Results and Discussion: PCA results and individual peak analysis clearly confirmed our hypothesis: The temporal metabolite stability for LHFB was largely maintained in contrast to LFB. Average temporal variation of all peaks was 14.4% for LHFB compared to 43.1% for LFB. Additionally, the PCA plot demonstrated close clustering for LHFB, whereas LFB were spreading out over time. Also LHAD showed temporal metabolic stability, while LAD exhibited strong changes. Our results suggest using cell lysis in combination with heating for extended longterm HRMAS NMR measurements, in order to minimize metabolite content modifications over measurement time. References: 1. Duarte IF, et al. Anal. Chem. 2009;81:5023. 2. Aguilar JA, et al. Chemical Communications 2012;48:811

Argininosuccinate neurotoxicity and prevention by creatine in argininosuccinate lyase deficiency: An in vitro study in rat three-dimensional organotypic brain cell cultures

The urea cycle disorder (UCD) argininosuccinate lyase (ASL) deficiency, caused by a defective ASL enzyme, exhibits a wide range of phenotypes, from life-threatening neonatal hyperammonemia to asymptomatic patients, with only the biochemical marker argininosuccinic acid (ASA) elevated in body fluids. Remarkably, even without ever suffering from hyperammonemia, patients often develop severe cognitive impairment and seizures. The goal of this study was to understand the effect on the known toxic metabolite ASA and the assumed toxic metabolite guanidinosuccinic acid (GSA) on developing brain cells, and to evaluate the potential role of creatine (Cr) supplementation, as it was described protective for brain cells exposed to ammonia. We used an in vitro model, in which we exposed three-dimensional (3D) organotypic rat brain cell cultures in aggregates to different combinations of the metabolites of interest at two time points (representing two different developmental stages). After harvest and cryopreservation of the cell cultures, the samples were analyzed mainly by metabolite analysis, immunohistochemistry, and western blotting. ASA and GSA were found toxic for astrocytes and neurons. This toxicity could be reverted in vitro by Cr. As well, an antiapoptotic effect of ASA was revealed, which could contribute to the neurotoxicity in ASL deficiency. Further studies in human ASL deficiency will be required to understand the biochemical situation in the brain of affected patients, and to investigate the impact of high or low arginine doses on brain Cr availability. In addition, clinical trials to evaluate the beneficial effect of Cr supplementation in ASL deficiency would be valuable