Abstract
Of two stereoisomers of glucose, only d- and not l-glucose is abundantly found in nature, being utilized as an essential fuel by most organisms. The uptake of d-glucose into mammalian cells occurs through glucose transporters such as GLUTs, and this process has been effectively monitored by a fluorescent d-glucose derivative 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) at the single cell level. However, since fluorescence is an arbitrary measure, we have developed a fluorescent analog of l-glucose 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-l-glucose (2-NBDLG), as a negative control substrate for more accurately identifying the stereoselectivity of the uptake. Interestingly, a small portion of mouse insulinoma cells MIN6 abundantly took up 2-NBDLG at a late culture stage (≳10 days in vitro, DIV) when multi-cellular spheroids exhibiting heterogeneous nuclei were formed, whereas no such uptake was detected at an early culture stage (≲6 DIV). The 2-NBDLG uptake was persistently observed in the presence of a GLUT inhibitor cytochalasin B. Neither d- nor l-glucose in 50 mM abolished the uptake. No significant inhibition was detected by inactivating sodium/glucose cotransporters (SGLTs) with Na+-free condition. To our surprise, the 2-NBDLG uptake was totally inhibited by phloretin, a broad spectrum inhibitor against transporters/channels including GLUTs and aquaporins. From these, a question might be raised if non-GLUT/non-SGLT pathways participate in the 2-NBDLG uptake into spheroid-forming MIN6 insulinoma. It might also be worthwhile investigating whether 2-NBDLG can be used as a functional probe for detecting cancer, since the nuclear heterogeneity is among critical features of malignancy.Electronic supplementary materialThe online version of this article (doi:10.1007/s13577-015-0125-3) contains supplementary material, which is available to authorized users.
Highlights
Mammalian cells take up D-glucose in a stereoselective manner through plasma membrane transporters, such as GLUTs, whereby only D- and not L-glucose is recognized [1]
A small portion of mouse insulinoma cells MIN6 abundantly took up 2-NBDLG at a late culture stage (Z10 days in vitro, DIV) when multi-cellular spheroids exhibiting heterogeneous nuclei were formed, whereas no such uptake was detected at an early culture stage ([6 DIV)
We have shown that 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) (Online Resource 1a), which was originally synthesized to see the viability of E. coli cells [2], is taken up into mammalian cells through GLUTs in a time, concentration and temperature-dependent manner [3, 4]
Summary
Mammalian cells take up D-glucose in a stereoselective manner through plasma membrane transporters, such as GLUTs, whereby only D- and not L-glucose is recognized [1]. We have shown that 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) (Online Resource 1a), which was originally synthesized to see the viability of E. coli cells [2], is taken up into mammalian cells through GLUTs in a time, concentration and temperature-dependent manner [3, 4]. Since fluorescence is an arbitrary measure, a control substrate has been awaited for more accurately evaluating the GLUT-mediated component [4]. A green fluorescence-emitting 2-[N-(7-Nitrobenz-2-oxa1,3-diazol-4-yl)amino]-2-deoxy-L-glucose (2-NBDLG), the mirror-image isomer of 2-NBDG, was developed as the control substrate (Online Resource 1b) [16, 17]. For evaluating an occurrence of non-specific uptake such as due to a loss of membrane integrity, we found it strongly helpful to use 2-NBDLG simultaneously with a membrane-impermeable L-glucose derivative 2-TRLG, which bears a large red fluorophore Texas red (Online Resource 1c) [17]
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