118 Fig. 2 Localisation of co- transfected human GCK and GKRP in HeLa cells at 5.5 mmol/l glucose. a Visual- isation of human EGFP-GCK and DAPI. b,c Co-transfection of mCherry-WT-hGKRP and EGFP-GCK (b) or mCherry- P446L-hGKRP and EGFP-GCK (c): GKRP signal, upper left; GCK signal, upper right; DAPI, lower left; overlay, lower right. d Quantification of degree of nuclear fluorescence of GKRP and GCK. Data are expressed as means ±SEM of two individual experiments with a total of 77– 98 cells analysed. *p<0.05; ***p<0.001 (t test). Black bars, GKRP; white bars, GCK Diabetologia (2012) 55:114–122 nuclear fluorescence; p= 0.04). Human GCK showed signif- icantly decreased nuclear localisation in the presence of P446L-hGKRP (Fig. 2d; 57.5±2.1% vs 80.4±1.8% nuclear fluorescence; p< 0.001). The nuclear localisation of GCK remained significantly reduced in the presence of P446L- hGKRP compared with WT-hGKRP when individual cells were matched for mCherry, EGFP or mCherry:EGFP fluorescence intensity (p< 0.05 for all groups). There was no significant difference in GCK sequestration between high and low glucose for either human variant (Fig. 3). The differences in GCK sequestration between WT- hGKRP and P446L-hGKRP observed at 5.5 mmol/l glucose were also detectable at 25 mmol/l glucose (p< 0.001 for GKRP and GCK localisation). By comparison, transient co- transfection of rGKRP proteins with human GCK resulted in no difference in GCK sequestration between WT-rGKRP and P446L-rGKRP (p> 0.1 for both glucose concentrations). However, there was a significant glucose dependence in GCK sequestration by both rGKRPs, with increased GCK nuclear localisation observed at low glucose (Fig. 3; p< 0.05 for both WT-rGKRP and P446L-rGKRP). These findings are not likely due to differences in rat and human GCK, as previous kinetic studies have extensively characterised the interaction between rat and human GKRP and either rat or human GCK, showing no difference in interaction of GKRP with either rat or human GCK [14]. Fig. 3 Glucose dependence of GCK localisation in HeLa cells. Degree of nuclear localisation of human GCK transiently transfected in the presence of WT-hGKRP, P446L-hGKRP, WT-rGKRP or P446L-rGKRP at 5.5 mmol/l and 25 mmol/l glucose. Data are expressed as means ± SEM of two individual experiments with a total of 18–98 cells analysed. *p<0.05; ***p<0.001 (ANOVA/Bonferroni correction). White bars, 5.5 mmol/l glucose; black bars, 25 mmol/l glucose