Diabetologia (2012) 55:114–122 Direct interaction of human GCK and GKRP in HeLa cells FRETN was used to detect the interaction between ECFP-GCK and EYFP, EYFP-WT-hGKRP, or EYFP- P446L-hGKRP in the nucleus of HeLa cells. FRETN measures the transfer of energy from ECFP to EYFP on excitation of ECFP and occurs when these proteins are within close proximity (<10 nm) because of the overlap of the emission spectrum of ECFP and the excitation spectrum of EYFP [22]. There was a significant reduction in interaction with GCK for the P446L-hGKRP variant protein compared with WT-hGKRP at 5.5 mmol/l glucose (Fig. 4). WT-hGKRP showed significantly reduced interac- tion with GCK at 25 mmol/l glucose compared with 5.5 mmol/l. There was no significant glucose dependence of interaction for P446L-hGKRP. Direct interaction and cellular localisation of human GCK and GKRP in primary mouse hepatocytes To compare these results with those with primary cells, we assessed the localisation and interaction of transiently transfected human GCK and hGKRP in mouse hepatocytes. The importance of endogenous mouse GCK and GKRP in this cell type was emphasised by our observation that transfected ECFP-GCK localises strongly to the nucleus at 5.5 mmol/l glucose and translocates to the cytoplasm in a glucose-dependent manner in the absence of transfected hGKRP (Fig. 5a). Translocation of ECFP-GCK was not abolished by the presence of EYFP-WT-hGKRP. However, EYFP-P446L- hGKRP abolished glucose-dependent translocation of Fig. 4 Direct interaction between human GCK and WT-hGKRP or P446L-hGKRP in HeLa cells. HeLa cells were transiently transfected with plasmids encoding ECFP-GCK and EYFP, EYFP-WT-hGKRP or EYFP-P446L-hGKRP as indicated and cultured in medium with 5.5 or 25 mmol/l glucose. Thereafter, fluorescence images were taken in living cells and the FRETN was calculated from the ECFP and EYFP emission intensities in the nucleus. Data are expressed as means±SEM of two individual experiments with a total of 7–10 cells analysed; *p<0.05; ***p<0.001 (ANOVA/Bonferroni correction). White bars, 5.5 mmol/l glucose; black bars, 25 mmol/l glucose 119 ECFP-GCK. This may be due to an abundance of EYFP- P446L-hGKRP protein in the cytoplasm preventing GCK from localising to the nucleus even at low glucose concen- trations, as suggested by our assessment of GCK localisation in HeLa cells (Fig. 3). As in HeLa cells, we observed significantly decreased interaction between GCK and P446L- hGKRP compared with WT-hGKRP at 5.5 mmol/l glucose in hepatocytes (Fig. 5b). At 25 mmol/l glucose in hepatocytes, accelerated metabolism results in generation of high levels of NADH and NADPH, resulting in autofluorescence. Addition- ally, there is a reduction in the absolute value of FRETN compared with HeLa cells due to quenching by endogenous mouse GCK and GKRP. Accordingly, the reduced interaction between GCK and GKRP at 25 mmol/l glucose was not significantly detectable above this increased background (Fig. 5b). Discussion To capitalise on recent advances in our understanding of the genetic basis of diabetes risk, it is necessary to translate association signals into detailed molecular mechanisms that may underlie disease predisposition. We have previously shown that P446L-hGKRP, inversely associated with triacylglycerol levels and glucose levels, affects GCK activity in liver through diminished regulation by F6P [12]. However, the impact of this variant on the cellular localisation of GCK and GKRP has not previously been explored. Recent studies using rat hepatocytes have suggested reliable estimation of GCK localisation can be accomplished by quantification of localisation in multiple fields of cells [25, 26]. We therefore sought to image a large number of cells using a quantitative method to assess nuclear and cytoplasmic fluorescence, and to combine this with assessment of direct interaction of GKRP and GCK by measuring FRETN. We have demonstrated for the first time differences in the degree of nuclear sequestration of GCK between WT-hGKRP and P446L-hGKRP and in the direct cellular interaction of P446L-hGKRP with GCK compared with WT-hGKRP. In spite of the presence of endogenous mouse GCK and GKRP, we were also able to demonstrate these differences in localisation, sequestration and direct interaction in primary mouse hepatocytes. In combination with our previously described kinetic studies, the P446L-hGKRP variant protein would be expected to result in an increased pool of active cytoplas- mic GCK, particularly in the presence of low (i.e. fasting state) glucose concentrations. Our findings suggest a mechanism of action for P446L-hGKRP consistent with the associated clinical phenotypes [9–11]. The reduced ability of P446L-hGKRP to sequester and interact with