Work

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

Year: 2010

Type: article

Abstract: Glucose transport activity in mammalian cells has been monitored by radiolabeled tracers such as [14C]\n2-deoxy-D-glucose. However, due to their limited spatial and temporal resolution, measuring glucose uptake in single,\nliving cells was diffi cult. We have developed a fl uorescent D-glucose derivative, 2-[N（- 7-nitrobenz-2-oxa-1,3-diazol-4-\nyl）amino]-2-deoxy-D-glucose （2-NBDG）, that allows a more sensitive measurement of glucose uptake in live cells.\nIndeed, the uptake of 2-NBDG takes place through glucose transporters （GLUTs） in a concentration-, time-, and\ntemperature-dependent manner. Kinetic analysis revealed that apparent Km values for the uptake were similar to\nthose reported by D-glucose and the nonmetabolizable glucose analogue, 3-O-methyl-D-glucose, found in pancreatic\nislet cells. This method can be combined with Ca2+ imaging and subsequent immunocytochemical identifi cation of\ncells. So far, 2-NBDG has been used for monitoring glucose uptake into a wide variety of mammalian cells including\nastrocytes and neurons. However, it has been pointed out that 2-NBDG method requires very accurate procedures,\nsince the fl uorescence intensity is an arbitrary measure. In addition, temporal changes in health of cells in question\nshould be verifi ed throughout the experiment by an independent way such as using patch clamp. To overcome the\ndiffi culty, we synthesized transporter- unrecognizable glucose analogues as control for 2-NBDG uptake. These include\n2-[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 of\ntransporter-recognizable（ D-isomer） and unrecognizable（ L-isomer） fl uorescent analogues allows us to investigate not\nonly net stereospecifi c transport of glucose in live cells but also ligand-transporter interactions and non-transportermediated\nglucose movement.

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