Real-time resolution studies of the regulation of lactate production by hexokinases binding to mitochondria in single cells
Fig 5
Modulation of pyruvate-dependent lactate metabolism by overexpression of HKI, the catalytically inactive D657A mutant and the truncated ΔN mutant.
The FRET signal in panel A is similar to that in panel A1 Fig 4 and illustrates the time course of phases 2, 4 and 5. In this case addition of NaCN, which was applied 1min35s after exposure to pyruvate, causes an increase in lactate in both phase 4 and 5. Upon removal of lactate there is a recovery which is strongly dependent upon the presence of pyruvate. Panel B illustrates one of the effects of wild type HKI overexpression (OE) as previously reported. The first striking difference with panel A is the sharp and fast increase in lactate, which occurred within 20s following addition of pyruvate. This increase which can be assimilated to a sustained phase 1 is likely due to the transformation of pyruvate into lactate in the cytoplasm. Following this rapid increase in lactate, there was no distinctive phase 4 or 5 in response to NaCN. The histogram in panel C is a quantification of this effect. The mean phase 5 amplitude with no HKI overexpression was 64.39±8.11 (n = 63) and 16.05±1.88 (n = 54). The P value was <0.0005. Data in panel D illustrate the effects of HKI ΔN overexpression. In many cells a clear phase 5 was observed upon addition of NaCN, indicating that in this case overexpression of the HKI ΔN did not block the increase in lactate evoked by NaCN. Data in panel E illustrate the effects of overexpression of the catalytically inactive HKI D657A. In this case the results were very clear, the FRET signal was not affected. The amplitude and time course of the response resemble that obtained in the absence of HKI expression (panel A) with clear phases 2, 4 and 5. The histograms in panel F show a quantification of the effects of wild type and mutants HKI on the amplitude of phase 5. The mean phase 5 amplitude was 49.97±4.62 (n = 41) for HKI ΔN compared to 15.71±2.35 (n = 22) for HKI wt overexpression. The P value was <0.0005. The mean phase 5 amplitude was 63.55±4.02 (n = 37) for HKI D657A overexpression compared to 15.71±2.35 (n = 22) for HKI wt overexpression. In this case P<0.0005.