Abstract
Deuterated water (2H2O), a stable isotopic tracer, provides a convenient and reliable way to label multiple cellular biomass components (macromolecules), thus permitting the calculation of their synthesis rates. Here, we have combined 2H2O labelling, GC-MS analysis and a novel cell fractionation method to extract multiple biomass components (DNA, protein and lipids) from the one biological sample, thus permitting the simultaneous measurement of DNA (cell proliferation), protein and lipid synthesis rates. We have used this approach to characterize the turnover rates and metabolism of a panel of mammalian cells in vitro (muscle C2C12 and colon cancer cell lines). Our data show that in actively-proliferating cells, biomass synthesis rates are strongly linked to the rate of cell division. Furthermore, in both proliferating and non-proliferating cells, it is the lipid pool that undergoes the most rapid turnover when compared to DNA and protein. Finally, our data in human colon cancer cell lines reveal a marked heterogeneity in the reliance on the de novo lipogenic pathway, with the cells being dependent on both ‘self-made’ and exogenously-derived fatty acid.
Highlights
The use of radioactive and stable isotope tracers to measure metabolic processes has underpinned many fundamental discoveries in biochemistry and physiology [1]
Experiment 1: C2C12 myoblasts were initially plated at 2 × 104 cells/mL in a 6-well plate in duplicate before being suspended in growth medium containing 4% 2 H2 O and harvested at 12, 24, 48, 72, 96 and 120 h
The murine C2C12 myoblast line rapidly proliferates under serum stimulated conditions, but can be induced to undergo terminal differentiation
Summary
The use of radioactive and stable isotope tracers to measure metabolic processes has underpinned many fundamental discoveries in biochemistry and physiology [1]. Unlike ‘static’ measurements of metabolite concentration, the use of tracers adds the dimension of time, allowing inferences to be made about reaction rates and metabolic fluxes [1,2,3]. This kinetic feature permits the observation and quantification of biochemical networks and biological processes, providing a detailed functional readout of the behavior of the biological system [2,3]. One would ideally use a near-universal tracer that simultaneously labels multiple cellular metabolites (which can be detected as free metabolites or constituents of macromolecules).
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