Uptake of fluorescent D- and L-glucose analogues, 2-NBDG and 2-NBDLG, into human osteosarcoma U2OS cells in a phloretin-inhibitable manner

Abstract

Mammalian cells take in D-glucose as an essential fuel as well as a carbon source. In contrast, L-glucose, the mirror image isomer of D-glucose, has been considered merely as a non-transportable/non-metabolizable control for D-glucose. We have shown that 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), a D-glucose analogue combining a fluorophore NBD at the C-2 position, is useful as a tracer for monitoring D-glucose uptake through glucose transporters (GLUTs) into mammalian cells. To more precisely evaluate the stereoselectivity of 2-NBDG uptake, we developed an L-glucose analogue 2-NBDLG, the mirror-image isomer of 2-NBDG. Interestingly, 2-NBDLG was taken up into mouse insulinoma MIN6 cells showing nuclear heterogeneity, a cytological feature of malignancy, while remaining MIN6 cells only exhibited a trace amount of 2-NBDLG uptake. The 2-NBDLG uptake into MIN6 cells was abolished by phloretin, but persisted under blockade of major mammalian glucose transporters. Unfortunately, however, no such uptake could be detected in other tumor cell lines. Here we demonstrate that human osteosarcoma U2OS cells take in 2-NBDLG in a phloretin-inhibitable manner. The uptake of 2-NBDG, and not that of 2-NBDLG, into U2OS cells was significantly inhibited by cytochalasin B, a potent GLUT inhibitor. Phloretin, but neither phlorizin, an inhibitor of sodium-glucose cotransporter (SGLT), nor a large amount of D/L-glucose, blocked the 2-NBDLG uptake. These results suggest that a phloretin-inhibitable, non-GLUT/non-SGLT, possibly non-transporter-mediated yet unidentified mechanism participates in the uptake of the fluorescent L-glucose analogue in two very different tumor cells, the mouse insulinoma and the human osteosarcoma cells.

Keywords: Imaging; L-glucose; Sarcoma; Transport; Tumor.

Fig. 1

Chemical structures of 2-NBDG (a), 2-NBDLG (b) and 2-TRLG (c)

Fig. 2

Representative images of U2OS cells subjected to 2-NBDG and 2-TRLG for 5 min followed by washout on the stage of confocal microscope at 7 DIV. a, b Differential interference contrast (DIC) images taken before administration (a) and 6 min after washout (b) of KRB solution containing a mixture of 200 μM 2-NBDG, which emits a large fluorescence in 500–580 nm (green channel) and a small fluorescence in 580–740 nm (red channel), and 20 μM 2-TRLG, which emits fluorescence solely in 580–740 nm (red channel) and was added for detecting non-specific entry of the fluorescent tracers due to a loss of membrane integrity. c, d Similar to (a, b), but fluorescence images taken in the green channel. e, f Similar to (c, d), but that in the red channel. g Superimposed images of (c) and (e). h Similar to (g), but for (d) and (f). An asterisk denotes small debris. The bar is common to all panels. Note heterogeneity in the fluorescence intensity among cells

Fig. 3

Similar to Fig. 2, but for administration of KRB solution containing 200 μM of 2-NBDLG and 20 μM of 2-TRLG

Fig. 4

Quantitative evaluation of the 2-NBDG and 2-NBDLG uptake into U2OS cells examined by a laser confocal microscope. a Changes in the mean fluorescence intensities of U2OS cells examined at 7 DIV before and after administration of KRB solution containing 200 μM of 2-NBDG or 2-NBDLG. Data are the means ± SD of total fluorescence intensity of individual ROIs. b Net increase in the fluorescence in (a) was expressed as percent increase in the fluorescence relative to that for 2-NBDG administration. Numbers in parenthesis show the number of ROIs that were assigned as much as possible (14–24) for each of 7 pre-determined areas of the cover slip. Similar results were obtained in three separate experiments

Fig. 5

Pharmacological characteristics of 2-NBDG and 2-NBDLG uptake in U2OS cells evaluated by a fluorescent microplate reader. a Relative increase in the fluorescence intensity of U2OS cells for administration of 200 μM of 2-NBDG or 2-NBDLG examined at 7 DIV. Similar results were obtained in three separate experiments. b Effect of a GLUT inhibitor cytochalasin B (10 μM, CB) on the uptake of 2-NBDG and 2-NBDLG examined at 8 DIV. Similar results were obtained in two independent experiments. c Effect of phloretin (150 μM, PHT) on the uptake of 2-NBDG and 2-NBDLG examined at 7 DIV. Results were confirmed in experiments done in triplicate. Data are the means ± SD of percent increase in the fluorescence relative to the fluorescence increase for 2-NBDG administration on the same 96-well plate. Numbers in parenthesis show the number of ROIs that were assigned for each well of the same condition in the same 96-well plate as described in the Methods