120 Fig. 5 Localisation of human GCK and direct interaction with WT- hGKRP or P446L-hGKRP in primary mouse hepatocytes. Hepato- cytes 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. a Calculation of the ECFP-GCK nuclear:cytoplasmic ratio. Data are expressed as means ± SEM of two individual experiments with a total of 9–12 cells analysed; *p<0.05 (ANOVA/Bonferroni correction). b Calculation of FRETN from the ECFP and EYFP emission intensities in the nucleus and cytoplasm. Data are expressed as means±SEM of two individual experiments with a total of 6–7 cells analysed; **p <0.01; ***p<0.001 compared with control; ††p<0.01 compared with WT (ANOVA/Bonferroni correction). White bars, 5.5 mmol/l glucose; black bars, 25 mmol/l glucose GCK in the nucleus in the fasting state would result in increased hepatic glucose disposal, glycolytic flux and generation of precursors for synthesis of molecules such as triacylglycerols and cholesterol. Recent findings associating GCKR with increased VLDL particle concentrations [27] suggest this enhancement of glycolytic flux leads to increased levels of de novo triacylglycerol and cholesterol synthesis and export. Increased fasting-state hepatic glucose uptake, use and disposal by GCK in the presence of P446L-hGKRP would be predicted to decrease circulating plasma glucose con- centrations. Further support for this molecular mechanism comes from at least one cellular study linking over- Diabetologia (2012) 55:114–122 expression of GCK in liver to reduced glucose levels and raised triacylglycerol levels [28]. We propose that de- creased fasting plasma glucose levels could result in decreased basal insulin output by the pancreas, which could ultimately contribute to higher insulin sensitivity and decreased risk of type 2 diabetes in spite of increased hepatic lipid production. Our results also suggest that, compared with WT- hGKRP, P446L-hGKRP shows reduced glucose depen- dence in its cellular interaction with GCK (Fig. 4). Accordingly, in spite of higher fasting-state GCK activity in the presence of P446L-hGKRP, GCK may not be activated to the same extent by increased glucose in the presence of this variant. This provides a mechanistic explanation for the observation that the P446L variant is associated with increased 2 h glucose levels but decreased fasting glucose levels [10, 29]. Interestingly, the degree of nuclear localisation of rGKRP is significantly reduced compared with WT-hGKRP in both HeLa cells and hepatocytes. Our studies assessing the localisation of transfected rGKRP in mouse hepatocytes (nuclear/cytoplasmic ratio of 3.37 at 5.5 mmol/l glucose) are highly consistent with our observations of endogenous rGKRP in rat hepatocytes (nuclear/cytoplasmic ratio of 3.44 at 5.5 mmol/l glucose; S. Baltrusch, unpublished observations). However, the abnor- mality in nuclear GCK sequestration for P446L-GKRP appears to be specific to the human regulatory protein as we did not observe a similar phenomenon with rGKRP. This is consistent with previous studies that have established important species- specific differences in both the regulation of human and rat GKRP by F6P and the impact of the P446L variant on this regulation [12–14]. Taking into account our previous assess- ment of the interaction of rGKRP with human GCK by FRETN analysis in COS-1 cells [21], direct interaction determined in our experiments in living cells appeared to be strongest between hGKRP and GCK. This is consistent with in vitro studies highlighting hGKRP as a more potent inhibitor of GCK than rGKRP [14]. These studies serve to emphasise the differences between human and rat regulatory proteins and highlight the need for use of appropriate assays to decipher complex mutational mechanisms driving disease associations. It has previously been reported that glucose-dependent GCK localisation is highly heterogeneous in rat hepatocytes [25], thus necessitating careful analysis in living and fixed cells. Our results comparing the glucose dependence of the nuclear/cytoplasmic ratio for human GCK co-transfected with rGKRP in mouse hepatocytes resulted in a ratio at 25 mmol/l that was 63% of the ratio at 5.5 mmol/l glucose. This is consistent with the ratio calculated from the results of Watanabe et al. [25] analysing endogenous rat GCK (59%) and our previous results measuring the nuclear/ cytoplasmic ratio of transfected human GCK in rat