Abstract
Metabolism changes extensively during the normal proliferation and differentiation of mammalian cells, and in cancer and inflammatory diseases. Since changes in the metabolic network reflect interactions between genetic, epigenetic and environmental changes, it is helpful to study the flow of label from isotopically labelled precursors into other metabolites rather than static metabolite levels. For this Nuclear Magnetic Resonance (NMR) spectroscopy is an attractive technique as it can quantify site-specific label incorporation. However, for applications using human cells and cell lines, the challenge is to optimize the process to maximize sensitivity and reproducibility. Here we present a new framework to analyze metabolism in mammalian cell lines and primary cells, covering the workflow from the preparation of cells to the acquisition and analysis of NMR spectra. We have applied this new approach in hematological and liver cancer cell lines and confirm the feasibility of tracer-based metabolism in primary liver cells.
Highlights
The metabolism of a cell changes as a net downstream response to the cellular environment
Cells must be grown within a controlled environment, with identical cell numbers, and typically a final cell number of 10–20 million cells must be obtained in order to acquire 1H,13C-HSQC and other Nuclear Magnetic Resonance (NMR) spectra in a reasonable time frame
We found that uridine diphosphate (UDP) can be used to determine relative amounts of oxidative vs non-oxidative phosphate pathway (PPP) branch activities[2]
Summary
The metabolism of a cell changes as a net downstream response to the cellular environment. The advantage of tracer-based analyses is that changes in metabolites can be assigned to particular mechanisms. For tracer-based analyses, different isotopically labelled precursors have been used as starting points to determine the intermediates and products of metabolism in cells. A tracer-based metabolic analysis can assign a product to one particular or multiple pathways, or even describe the contribution of different pathways which is often not possible based on static metabolite concentrations. We present a workflow for efficiently using NMR in the context of tracer-based metabolism using mammalian cell lines or even primary cells under physiologically relevant conditions. This includes methods of preparing cells, along with NMR methods suitable for such analyses. We show applications in cancer cell lines and in primary liver cells
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