H441 cells cultured at air-liquid interface on permeable supports required altered microscope conditions for FRET acquisition, which meant that the measured FRET ratio of eCFP/Citrine was decreased compared with that observed in proliferating cells. Nevertheless, in cells exposed to 5 mM extracellular glucose the pattern of response was similar to that seen in proliferating cells. A cyclic fluctuation in FRET ratio was also observed in these cells with a full cycle taking 4.4 ± 0.6 min, in 5 mM glucose. Elevation of extracellular glucose to 15 mM resulted in an increased FRET ratio from 0.38 ± 0.007 to 0.41 ± 0.005 (P < 0.0001, n = 83 individual cells from n = 6 experiments). Addition of BrPy reduced FRET ratio to 0.34 ± 0.003 and the cycling frequency to 1.3 ± 0.23 min (P ≤ 0.001; n = 16 experiments).