title: Uptake of a fluorescent L-glucose derivative 2-NBDLG into three-dimensionally accumulating insulinoma cells in a phloretin-sensitive manner.
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.
authors.affiliation: Department of Physiology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori, 036-8562, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Sasaki
authors.affiliation: Department of Physiology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori, 036-8562, Japan.
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Nagatomo
authors.affiliation: Department of Physiology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori, 036-8562, Japan.
authors.forename: Koki
authors.initials: K
authors.lastname: Ono
authors.affiliation: Peptide Institute, Inc., Saito Research Center, Ibaraki, Osaka, 567-0085, Japan.|Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Peptide Institute, Inc., Saito Research Center, Ibaraki, Osaka, 567-0085, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Otsuka
authors.affiliation: Peptide Institute, Inc., Saito Research Center, Ibaraki, Osaka, 567-0085, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Teshima
authors.affiliation: Department of Physiology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori, 036-8562, Japan. kyamada@hirosaki-u.ac.jp.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Yamada
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D003847:Deoxyglucose / Q000031:analogs & derivatives* / Q000378:metabolism; D005453:Fluorescence; D007340:Insulinoma / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D057054:Molecular Imaging / Q000379:methods; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D010693:Phloretin / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D003847:Deoxyglucose; D009327:4-Chloro-7-nitrobenzofurazan; C098340:2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose; D010693:Phloretin
keywords: Biomarker; Cancer; L-Glucose; Spheroid; Tumor