A framework for tracer-based metabolism in mammalian cells by NMR

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.


Supplementary Figure 1: Using 1D and TOCSY spectra for tracer-based metabolism.
1D and TOCSY spectra have previously been used to analyse labelled metabolite extracts from cells 1 by Lane and Fan, who also list the metabolites and pathways probed. The use of TOCSY spectra is somewhat limited by overlap. While this can be reduced by decoupling 13 C in the incremented dimension, we still find this to be a limiting factor. Here, we show some examples where TOCSY spectra can be used. These include lactic acid, threonine, alanine, glutamic acid and glutamine as well as aspartic acid. Panel a shows the region of the 1 H NOESY spectrum with overlapped doublets from threonine H4 and lactic acid H3. Panels b-f show sections from the 1 H, 1 H-TOCSY; 1=lactic acid H3,H2; 2=threonine H4,H3; 3=alanine H3,H2; 4=glutamic acid H2,H4; 5=glutamine H2,H4; 6=glutathione H2,H4; 7=gluta* H2,H3; 8=aspartic acid; 9=UDP H5/H6; * denotes 1 H with attached 13 C. gluta* is glutathione/glutamine/glutamate.

Supplementary Figure 2:
HSQC data for 1 H-13 C-HSQC spectrum acquired with 16k increments (red) vs 4k increments (blue) with a 25% non-uniformly incremented schedule (NUS). Panel a shows an overview and panel b shows subspectra for glutamic acid. The resulting spectra are virtually indistinguishable; differences are mainly in the noise. NUS spectra were processed using Hybert's 2 hmsIST module in nmrPipe 3 .   1.2 When pyrimidines are synthesized from aspartate, 2,3-13 C labelling in aspartate is transferred to C5-C6 in the pyrimidine base ring. However, 3,4-13 C labelling in aspartate gives rise to an isolated 13 C at C6 as the carbon, C4 of aspartate is lost. Therefore, the ratio of the doublet to singlet resonances at C6 gives an indication of the PC/PDH ratio. Nitrogens in purine and pyrimidine bases result from transamination reactions using Gln or Glu either directly or indirectly via incorporation of Gly or Asp [5]. C8 and C14 resonances of AXP 2-13 C guanidoacetate is synthesised from arginine and 2-13 C glycine. The produced 2-13 C guanidoacetate is converted to 2-13 C creatine. 8,14-13 C AXP results from the synthesis of N 10 -formyl-tetrahydrofolate and N 5 ,N 10 -methenyl-tetrahydrofolate where the transferable carbon is 13 Clabelled from one-carbon metabolism.
[4] These approaches to probing PDH vs PC activity were used in our previous work on AML K562 cells. We observed only the doublet for the malonate carbonate in the 13 C spectrum of the [1,2-13 C]glucose-labelled AML cell extract suggesting high PC activity in this cell-line 7 . Furthermore, we observed a shift from PC to PDH as flux time increased. In recent spectra using the hsqcphprsp sequence we were able to resolve the PDH and PC products separately (Figure 3e&f). [5] The following coupled spins can potentially be seen in labelled samples and all were seen with [U-15 N]ATP or [U-15 N]UTP samples: In purines: N1/H2*, N3/H2*, N7/H8, N9/H2*, N9/H8 * adenine only In pyrimidines: N1/H5*, N1/H6, N3/H5*, N3/H6, N1/H1(of ribose) * cytosine and uracil only Source of the atoms in nucleotide bases.
Furthermore, all these resonances were seen in 1 H-15 N spectra of cell extracts from TALL cell-lines cultured in 15 N-glutamine-labelled media except N3/H5 in pyrimidines and N7/H8 in purines.
[6] The g-glutamyl cycle can be analysed without labeling 5 . This can however not be resolved in 1D spectra. The greater resolution of the 2D spectra was essential for following the changing ratios of the g-glutamyl cycle intermediates.