ght intensity between two intracellular regions of interest with and without a high concentration of mitochondria. Background light was subtracted in both cases. Images were acquired using an Olympus IX70 inverted microscope fitted with an Olympus plan apo 660. 1.4 N.A. oil immersion objective and a cooled CCD camera. Imaging Workbench software was used for data acquisition and analysis. YFP and CFP filter cubes were purchased from Omega Optical Inc.. insertion into the plasma membrane, we used constructs of 23370967 GLUT1 and GLUT4 linked to GFP. In both cases, there was only faint GFP fluorescence detected in the plasma membrane, with most of the fluorescence associated with intracellular membrane compartments. However, as shown in Panels B and C, even this low level of plasma membrane GLUT insertion had dramatic effects on glucose transport. With GLUT1 over expression, the rate of glucose entry, illustrated by a decrease in FRET ratio, increased dramatically, with the time constant t dropping from 34.5+/25 s to 1.9+/20.6 s . The rate of glucose clearance, indicated by an increase in FRET ratio also increased, with a t1/2 approaching 10 s. With GLUT4 over expression, the rate of glucose uptake decreased by almost 14-fold, from 34.5 s to 2.5 s. The rate of glucose clearance also increased in this case, although to a lesser level than with GLUT1, the time to reach half clearance being around 12.5+/26.5 s. In panels 1, 3 and 5 the rate 
of glucose uptake was fitted to an exponential decay, in panels 2, 4 and 6 glucose clearance was fitted to a sigmoidal function. These data suggest that increased insertion of GLUTs in 15703812 the plasma membrane increase both glucose influx and efflux. Glucose-6-Phosphate assay We used a commercial G-6-P assay kit. CHO cells were transfected with GLUT1. Two days later cells were washed in ice cold PBS and frozen in situ in dryice ethanol. 200 ml of 6% perchloric acid added and the cells scraped while frozen. Cells were homogenized via a ��Qiashredder��column at 4uC and the homogenate neutralized with 500 mM ethanolamine and 10 M KOH. Samples were then centrifuged to remove insoluble material 50 ml samples were transferred into wells of a 96-well plate and brought to a volume of 100 ml with assay buffer. Absorbance at 450 nm was measured, using a POLARstar Omega microplate reader. Background samples were introduced to estimate the NADH/NADPH levels prior to G-6-P conversion. Finally, to estimate the G-6-P levels a curve was generated with G-6-P standards containing 0, 2, 4, 6, 8, 10, 20 nmol/well. presence of Cyto B in CHO cells. Panel A shows data obtained with a CHO cell exposed to Cyto B in the continuous presence of 10 mM glucose. The right hand side plots show the rate of glucose clearance following removal of glucose in the absence of Cyto B and in the presence of Cyto B. The closeness of the two ts suggests that, with cells exhibiting a slow glucose uptake phenotype, GLUT-mediated glucose MedChemExpress CHIR99021 efflux has little effect in glucose clearance. This trace also illustrates that the effect of Cyto B is readily reversible, as glucose entry resumed, albeit at a lower rate, 30 s after Cyto B removal. In B data were obtained with a CHO cell transfected with GLUT1, which exhibited a high rate of glucose uptake. In this case, Cyto B dramatically reduced the rate of clearance as compared to that recorded in the absence of Cyto B and after outside glucose removal. These data suggest that GLUTmediated efflux plays a role in glucose cleara
