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 i ts effect on the various intracellular functions in single, living mammalia n 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 glu cose transporter GLUT2 show significantly greater 2-NBDG uptake than mock t ransfected cells. Using GLUT2-abundant mouse insulin-secreting clonal MING 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 con centrations of D-glucose in a dose-dependent manner and also was almost com pletely inhibited by 10 mu M cytochalasin B. We then measured both glucose uptake and the intracellular calcium concentration ([Ca2+](i)) in single, l iving pancreatic islet cells. 2-NBDG and fura-2 were used as the tracer of glucose and indicator of intracellular calcium, respectively. All of the ce lls that showed an increase in [Ca2+](i) in response to a high concentratio n of glucose (16.8 mM) rapidly incorporated significant 2-NBDG, Immunocytoc hemical examination confirmed these cells to be insulin-positive beta-cells , All of the cells that showed no significant, rapid 2-NBDG uptake lacked s uch glucose responsiveness of [Ca2+](i), indicating that these cells were n on-beta-cells such as glucagon-positive alpha-cells. These results show the uptake of glucose causing a concomitant increase of [Ca2+](i) in beta-cell s, Because 2-NBDG is incorporated into mammalian cells through glucose tran sporters, it should be useful for the measurement of glucose uptake togethe r with concomitant intracellular activities in many types of single, living mammalian cells.