Dataset Information

Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2-deoxyglucose using pulsed stable isotope-resolved metabolomics.

ABSTRACT:

Background

Cellular metabolism is highly dynamic and continuously adjusts to the physiological program of the cell. The regulation of metabolism appears at all biological levels: (post-) transcriptional, (post-) translational, and allosteric. This regulatory information is expressed in the metabolome, but in a complex manner. To decode such complex information, new methods are needed in order to facilitate dynamic metabolic characterization at high resolution.

Results

Here, we describe pulsed stable isotope-resolved metabolomics (pSIRM) as a tool for the dynamic metabolic characterization of cellular metabolism. We have adapted gas chromatography-coupled mass spectrometric methods for metabolomic profiling and stable isotope-resolved metabolomics. In addition, we have improved robustness and reproducibility and implemented a strategy for the absolute quantification of metabolites.

Conclusions

By way of examples, we have applied this methodology to characterize central carbon metabolism of a panel of cancer cell lines and to determine the mode of metabolic inhibition of glycolytic inhibitors in times ranging from minutes to hours. Using pSIRM, we observed that 2-deoxyglucose is a metabolic inhibitor, but does not directly act on the glycolytic cascade.

SUBMITTER: Pietzke M

PROVIDER: S-EPMC4101711 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Publications

Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2-deoxyglucose using pulsed stable isotope-resolved metabolomics.

Pietzke Matthias M Zasada Christin C Mudrich Susann S Kempa Stefan S

Cancer & metabolism 20140630

<h4>Background</h4>Cellular metabolism is highly dynamic and continuously adjusts to the physiological program of the cell. The regulation of metabolism appears at all biological levels: (post-) transcriptional, (post-) translational, and allosteric. This regulatory information is expressed in the metabolome, but in a complex manner. To decode such complex information, new methods are needed in order to facilitate dynamic metabolic characterization at high resolution.<h4>Results</h4>Here, we des ...[more]

PMID: 25035808

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Project description:Conventional two-dimensional (2D) cell cultures are grown on rigid plastic substrates with unrealistic concentration gradients of O?, nutrients, and treatment agents. More importantly, 2D cultures lack cell?cell and cell?extracellular matrix (ECM) interactions, which are critical for regulating cell behavior and functions. There are several three-dimensional (3D) cell culture systems such as Matrigel, hydrogels, micropatterned plates, and hanging drop that overcome these drawbacks but they suffer from technical challenges including long spheroid formation times, difficult handling for high throughput assays, and/or matrix contamination for metabolic studies. Magnetic 3D bioprinting (M3DB) can circumvent these issues by utilizing nanoparticles that enable spheroid formation and growth via magnetizing cells. M3DB spheroids have been shown to emulate tissue and tumor microenvironments while exhibiting higher resistance to toxic agents than their 2D counterparts. It is, however, unclear if and how such 3D systems impact cellular metabolic networks, which may determine altered toxic responses in cells. We employed a Stable Isotope-Resolved Metabolomics (SIRM) approach with 13C?-glucose as tracer to map central metabolic networks both in 2D cells and M3DB spheroids formed from lung (A549) and pancreatic (PANC1) adenocarcinoma cells without or with an anti-cancer agent (sodium selenite). We found that the extent of 13C-label incorporation into metabolites of glycolysis, the Krebs cycle, the pentose phosphate pathway, and purine/pyrimidine nucleotide synthesis was largely comparable between 2D and M3DB culture systems for both cell lines. The exceptions were the reduced capacity for de novo synthesis of pyrimidine and sugar nucleotides in M3DB than 2D cultures of A549 and PANC1 cells as well as the presence of gluconeogenic activity in M3DB spheroids of PANC1 cells but not in the 2D counterpart. More strikingly, selenite induced much less perturbation of these pathways in the spheroids relative to the 2D counterparts in both cell lines, which is consistent with the corresponding lesser effects on morphology and growth. Thus, the increased resistance of cancer cell spheroids to selenite may be linked to the reduced capacity of selenite to perturb these metabolic pathways necessary for growth and survival.

Project description:BackgroundStable isotope tracing with ultra-high resolution Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS) can provide simultaneous determination of hundreds to thousands of metabolite isotopologue species without the need for chromatographic separation. Therefore, this experimental metabolomics methodology may allow the tracing of metabolic pathways starting from stable-isotope-enriched precursors, which can improve our mechanistic understanding of cellular metabolism. However, contributions to the observed intensities arising from the stable isotope's natural abundance must be subtracted (deisotoped) from the raw isotopologue peaks before interpretation. Previously posed deisotoping problems are sidestepped due to the isotopic resolution and identification of individual isotopologue peaks. This peak resolution and identification come from the very high mass resolution and accuracy of FT-ICR-MS and present an analytically solvable deisotoping problem, even in the context of stable-isotope enrichment.ResultsWe present both a computationally feasible analytical solution and an algorithm to this newly posed deisotoping problem, which both work with any amount of 13C or 15N stable-isotope enrichment. We demonstrate this algorithm and correct for the effects of 13C natural abundance on a set of raw isotopologue intensities for a specific phosphatidylcholine lipid metabolite derived from a 13C-tracing experiment.ConclusionsCorrection for the effects of 13C natural abundance on a set of raw isotopologue intensities is computationally feasible when the raw isotopologues are isotopically resolved and identified. Such correction makes qualitative interpretation of stable isotope tracing easier and is required before attempting a more rigorous quantitative interpretation of the isotopologue data. The presented implementation is very robust with increasing metabolite size. Error analysis of the algorithm will be straightforward due to low relative error from the implementation itself. Furthermore, the algorithm may serve as an independent quality control measure for a set of observed isotopologue intensities.

Dataset Information

Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2-deoxyglucose using pulsed stable isotope-resolved metabolomics.

Background

Results

Conclusions

Publications

Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2-deoxyglucose using pulsed stable isotope-resolved metabolomics.

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