Nobuya Inagaki

Measurement of glucose uptake and intracellular calcium concentration in single, living pancreatic beta-cells.

There has been no method previously to measure both glucose transport and its effect on the various intracellular functions in single, living mammalian cells. A fluorescent derivative of d-glucose, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), that we have developed has made such measurements possible. COS-1 cells that overexpress the human glucose transporter GLUT2 show significantly greater 2-NBDG uptake than mock transfected cells. Using GLUT2-abundant mouse insulin-secreting clonal MIN6 cells, we found that 2-NBDG was incorporated into the cells in a time- and concentration-dependent manner. The 2-NBDG uptake was inhibited by high concentrations of d-glucose in a dose-dependent manner and also was almost completely inhibited by 10 μm cytochalasin B. We then measured both glucose uptake and the intracellular calcium concentration ([Ca2+] i ) in single, living pancreatic islet cells. 2-NBDG and fura-2 were used as the tracer of glucose and indicator of intracellular calcium, respectively. All of the cells that showed an increase in [Ca2+] i in response to a high concentration of glucose (16.8 mm) rapidly incorporated significant 2-NBDG. Immunocytochemical examination confirmed these cells to be insulin-positive β-cells. All of the cells that showed no significant, rapid 2-NBDG uptake lacked such glucose responsiveness of [Ca2+] i , indicating that these cells were non-β-cells such as glucagon-positive α-cells. These results show the uptake of glucose causing a concomitant increase of [Ca2+] i in β-cells. Because 2-NBDG is incorporated into mammalian cells through glucose transporters, it should be useful for the measurement of glucose uptake together with concomitant intracellular activities in many types of single, living mammalian cells.