<Symposium I>A new imaging method of glucose transport in live cells


Glucose transport activity in mammalian cells has been monitored by radiolabeled tracers such as [14C]2-deoxy-D-glucose. However, due to their limited spatial and temporal resolution, measuring glucose uptake in single,living cells was diffi cult. We have developed a fl uorescent D-glucose derivative, 2-[N(- 7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), that allows a more sensitive measurement of glucose uptake in live cells.Indeed, the uptake of 2-NBDG takes place through glucose transporters (GLUTs) in a concentration-, time-, andtemperature-dependent manner. Kinetic analysis revealed that apparent Km values for the uptake were similar tothose reported by D-glucose and the nonmetabolizable glucose analogue, 3-O-methyl-D-glucose, found in pancreaticislet cells. This method can be combined with Ca2+ imaging and subsequent immunocytochemical identifi cation ofcells. So far, 2-NBDG has been used for monitoring glucose uptake into a wide variety of mammalian cells includingastrocytes and neurons. However, it has been pointed out that 2-NBDG method requires very accurate procedures,since the fl uorescence intensity is an arbitrary measure. In addition, temporal changes in health of cells in questionshould be verifi ed throughout the experiment by an independent way such as using patch clamp. To overcome thediffi culty, we synthesized transporter- unrecognizable glucose analogues as control for 2-NBDG uptake. These include2-[N(- 7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-L-glucose [2-NBDLG], the antipode of 2-NBDG. A combined use oftransporter-recognizable( D-isomer) and unrecognizable( L-isomer) fl uorescent analogues allows us to investigate notonly net stereospecifi c transport of glucose in live cells but also ligand-transporter interactions and non-transportermediatedglucose movement.


MATSUOKA HIDEAKI Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology
WATANABE Seiji Department of Dermatology, Kanazawa Medical University
Matsuoka Hideaki Department Of Biotechnology And Life Science Faculty Of Technology Tokyo University Of Agriculture A
Yamamoto Toshihiro Saito Res. Center Peptide Institute Inc. Ibaraki Osaka
Suga Sechiko Center Res. & Edu. Lifelong Learning Hirosaki Univ.
Yamada Katsuya Dept. Physiol. Hirosaki Univ. Grad. Sch. Med.
Nishiuchi Yuji Saito Res. Center Peptide Institute Inc. Ibaraki Osaka
TESHIMA Tadashi Peptide Institute, Protein Research Foundation
Teshima Tadashi Saito Research Center, Peptide Institute, Inc., Ibaraki
Teshima T Saito Res. Center Peptide Institute Inc. Ibaraki Osaka
Watanabe Seiji Department Of Anatomical Science Hirosaki Univ. Grad. Sch. Med.
Teshima Tadashi Saito Research Center Peptide Institute Inc.
Teshima Tadashi Department Of Chemistry Faculty Of Science Osaka University
Teshima Tadashi Peptide Institute Protein Research Foundation
Watanabe Seiji Department Of Agricultural Chemistry Faculty Of Agriculture Tohoku University:(present Address) Show
Teshima Tadashi SAITO Res. Center, Peptide Institute, Inc., Ibaraki,Osaka