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.
Highlights
Introduction d-glucose, the minimum unit of starch, is one of the most fundamental nutrients for living things
We show in the present study that human osteosarcoma U2OS cells take up 2-NBDLG into cells with pharmacological properties very similar to those we reported in MIN6 cells
We demonstrated that human osteosarcoma U2OS cells took in a fluorescent analogue of l-glucose 2-NBDLG abundantly as well as its mirror image isomer 2-NBDG, a widely used fluorescent analogue of d-glucose
Summary
Osteosarcoma U2OS (HTB-96, ATCC) cells were cultured using RPMI 1640 medium (11875-093, Gibco) containing 10% Fetal Bovine Serum (26140-079, Gibco) and 1% Penicillin–Streptomycin (15140-122, Gibco). Confocal microscopy was conducted to visualize difference in the uptake of the fluorescent d- and l-glucose tracers in U2OS cells. Cells used for the measurement were cultured for 7 days in vitro (DIV) when an adequate number of cells maintaining healthy condition were obtained for each coverslip at the concentration seeded. The tracer administration and image acquisition were conducted by modifying a method reported previously (see Online Resource 4 for details) [7, 27]. Details in the culturing and experimental procedures were similar to those reported previously (see Online Resource 4) [18]. 100 μM of carbenoxolone (C4790, Sigma) was routinely added to exclude non-specific permeation of the fluorescent tracers through gap junctions/hemichannels [28]. All the values represent mean fluorescence intensity, and are expressed as mean ± SD
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