Intracellular glucose concentration of H441 cells and HBECs.
A dose-response curve for FRET ratio was generated for the three different cell/growth conditions using the individual imaging conditions used. An exemplar dose-response curve for proliferating H441 cells is shown (Fig. 4B). This was then used to interpolate the data points shown in Fig. 4A to calculate the intracellular concentration of glucose. The mean intracellular glucose concentration for proliferating H441 cells in 5 mM glucose was 0.23 ± 0.05 mM. Raising the glucose concentration to 15 mM glucose resulted in a decrease in intracellular glucose to 0.05 ± 0.04 mM. Pretreatment with BrPy increased intracellular glucose concentration to 0.49 ± 0.01 mM in 5 mM glucose and 0.46 ± 0.03 in 15 mM glucose (P < 0.0001 compared with control, respectively; n = 117 individual cells; Fig. 6A).
Fig. 6.  Intracellular glucose concentration calculated from Förster resonance energy transfer ratio dose-response curves. A: calculated intracellular glucose concentration in H441 cells grown on plastic and exposed to 5 mM (●) or 15 mM d-glucose (15 mM; hyperglycemia, ▲) or exposed to the same conditions in the presence of 3-bromopyruvic acid (BrPy; ○ or △, respectively). Values were calculated using the dose-response curve shown in Fig. 4D. Individual data points are shown with mean ± SD; n = 117 individual cells. ****P < 0.0001 between groups as indicated. B: calculated intracellular glucose concentration for H441 cells grown at air-liquid interface in either 5 mM glucose (●), 15 mM glucose (▲), or 5 mM glucose in the presence of BrPy (○). Individual data points are shown with mean ± SD; n = 83 individual cells. ****P < 0.0001 between groups as indicated. C: calculated intracellular glucose for human bronchial epithelial cells (HBECs) cultured at air-liquid interface in either 5 mM glucose (●) or 15 mM glucose (▲). Individual data points are shown with mean ± SD; n = 150 individual cells. ****P < 0.0001 between groups as indicated. Interpolation of data from H441 cells cultured at air-liquid interface indicated that these cells had a mean intracellular glucose concentration of 0.36 ± 0.005 mM in 5 mM basolateral glucose and this decreased to 0.26 ± 0.003 mM when basolateral glucose concentration was increased to 15 mM. Addition of BrPy in the presence of 5 mM basolateral glucose increased intracellular glucose concentration to 0.72 ± 0.003 mM (P ≤ 0.0001; n = 83 individual cells; Fig. 6B).
A similar pattern was seen in HBECs grown at air-liquid interface. Intracellular glucose concentration was 0.09 ± 0.002 mM in 5 mM glucose, and this decreased to 0.03 ± 0.001 mM when basolateral glucose concentration was raised to 15 mM (n = 150 individual cells; Fig. 6C).