Abstract
Assays to monitor the metabolic state or nutrient uptake capacity of immune cells at a single cell level are increasingly in demand. One assay, used by many immunologists, employs 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG), a fluorescent analogue of 2-deoxyglucose (2DG), as a substrate for glucose transporters. This molecule has been validated as a substrate for the glucose transporter Glut2 (Slc2a2) in mammalian cells but 2-NDBG selectivity for the glucose transporters expressed by T cells, Glut1 (Slc2a1) and Glut3 (Slc2a3), has never been explored. Nor has the possibility that 2-NBDG might bind to T cells that do not express glucose transporters been assessed. In this technical commentary we interrogate the specificity of 2-NBBG labelling as a readout for glucose transport in T lymphocytes. We compare flow cytometric 2-NBDG staining against well validated radiolabelled glucose transport assays in murine T cells. Our data show there can be a large discordance between glucose transport capacity and 2-NBDG labelling in T cells. We also find that 2-NBDG uptake into murine T cells is not inhibited by competitive substrates or facilitative glucose transporter inhibitors, nor can 2-NBDG competitively block glucose uptake in T cells. Collectively, these data argue that 2-NBDG uptake alone is not a reliable tool for the assessment of cellular glucose transport capacity.
Highlights
In recent years, there has been a focus on intracellular metabolic pathways, metabolites and their dynamic changes in immune cells
We find that 2-nitrobenzoxydiazoamino group (NBDG) uptake into murine T cells is not inhibited by competitive substrates or facilitative glucose transporter inhibitors, nor can 2-NBDG competitively block glucose uptake in T cells
In order for a substrate to be a reporter for glucose transporter activity, it should conform to relevant transporter dynamics
Summary
There has been a focus on intracellular metabolic pathways, metabolites and their dynamic changes in immune cells. This small reduction in fluorescence was not, specific to 2-NBDG uptake as this was present in unstained cells as well (Figure 3G) An explanation for this effect of 2DG on cell autofluorescence is that 2DG, which is known to interfere with glucose metabolism and glycolysis in T cells, could impact/perturb the NAD(P)H autofluorescence normally seen in highly glycolytic cells. This highlights another point of caution when interpreting subtle changes in fluorescence whilst using dyes that are detected in the region of cellular autofluorescence: you need to be certain that these differences are due to the amount of dye and not due to altered cellular autofluorescence These data show a disconnect between radiolabelled 2DG transport and 2-NBDG transport in T cells and argue that glucose transport and 2-NBDG uptake are not mediated by a common transporter. Further evidence for this concept comes from experiments where 2-NBDG was not able to compete 3H-2DG uptake by activated T cells (Figure 3B)
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