Diabetologia (2012) 55:114–122 subcellular localisation of human GKRP (hGKRP) and the effect of the P446L variant on this localisation, mCherry- tagged GCKR fusion plasmids were transiently transfected into HeLa cells. HeLa cells were chosen as an established model known to exhibit efficient glucose uptake and lacking endogenous expression of GCKR and GCK [7, 23]. Both wild-type (WT)-hGKRP and P446L-hGKRP localised strongly to the nucleus, but an appreciable increase in cytoplasmic fluorescence intensity was detect- able for P446L-hGKRP (Fig. 1a,b). To quantify this apparent difference in localisation, the ratio of nuclear to whole-cell fluorescence in individual cells was calculated. Cells with very low EGFP or mCherry fluorescence intensities (<50,000 arbitrary fluorescence units) were excluded from statistical analysis. However, exclusion of these data did not change the significance of any result. WT-hGKRP (n =20 cells) was associated with significantly increased nuclear localisation when compared with P446L- hGKRP (n =21) (83.4 ±1.4% nuclear localisation compared with 65.1 ±3.1%, mean ±SEM; p <0.001; Fig. 1c). Previous studies have revealed substantial kinetic differences be- tween human and rat GKRP (rGKRP) [14]. However, no studies have compared cellular localisation between rat and human regulatory proteins. Accordingly, we also generated and transiently transfected mCherry-tagged rat Gckr. The P446L variant was also introduced into the rat Gckr sequence. There was no significant difference in the nuclear-to-whole-cell-fluorescence ratio of WT-rGKRP (62.8 ± 2.3%; n = 20) and P446L-rGKRP (56.3 ± 1.5%; n = 26; Fig. 1d). Both WT-rGKRP and P446L-rGKRP showed significantly decreased nuclear localisation com- pared with WT-hGKRP (p < 0.001; ANOVA/Bonferroni correction). Localisation of all GKRPs was independent of glucose concentration (p> 0.1; data not shown). To confirm our observations in HeLa cells in a physiologically relevant primary cell type, we assessed the 117 localisation of transiently transfected human and rat GKRPs in primary mouse hepatocytes (Fig. 1d). EYFP-WT-hGKRP (nuclear/cytoplasmic ratio 5.94 ± 0.40; n = 45 individual cells) was again significantly associated (p <0.001) with increased nuclear localisation compared with EYFP-P446L- hGKRP (4.16±0.25; n=52), EYFP-WT-rGKRP (3.37±0.17; n = 36), and EYFP-P446L-rGKRP (3.45 ± 0.18; n = 37). Localisation of all GKRPs was independent of glucose concentration. Co-transfection of human fluorescent GCK and GKRP fusion plasmids in HeLa cells Transiently transfected human EGFP-GCK localised entirely to the cytoplasm of HeLa cells (Fig. 2a). In accordance with previous in vivo studies, localisation was not affected by glucose concentra- tion (data not shown) [15]. Co-transfection of GCK and human or rat WT- and P446L-GKRP resulted in nuclear sequestration of GCK (Fig. 2b,c). Incubation for 2–16 h at a series of glucose concentrations (1–25 mmol/l) resulted in a mix of nuclear and cytoplasmic localisation for both GKRP and GCK (data not shown). Complete glucose-dependent export of GCK was not observed even at high glucose concentrations, consistent with previous observations in rat hepatocytes [17, 24]. Accordingly, quantitative image analysis was undertaken at two glucose concentrations (5.5 and 25 mmol/l glucose) which have been previously used to investigate the GCK:GKRP interaction as they are either side of the glucose S0.5 (substrate concentration needed for half maximal rate) for GCK (approximately 7.5 mmol/l) [17]. Quantitative analysis was undertaken on a total of 787 cells from two transfections per glucose concentration and from two independent plasmid preparations per variant. In the presence of GCK at 5.5 mmol/l glucose, P446L-hGKRP was again associated with decreased nuclear localisation compared with WT-hGKRP (Fig. 2d; 67.4 ± 2.5% vs 73.6 ± 1.8% Fig. 1 Localisation of GKRP fluorescent fusion proteins at 5.5 mmol/l glucose. Visualisation of mCherry-WT-hGKRP (a) or P446L-hGKRP (b) and overlap with DAPI on transient transfection into HeLa cells. c Quantification of degree of nuclear fluorescence for human and rat GKRPs in HeLa cells. Data are expressed as means± SEM of two individual experiments with a total of 20–26 cells analysed. d Quantification of nuclear/cytoplasmic ratio of human and rat GKRPs on transient transfection into primary mouse hepatocytes. Data are expressed as means±SEM of two individual experiments with a total of 36–52 cells analysed. ***p<0.001 compared with WT-hGKRP (ANOVA/Bonferroni correction)