Cellular characterisation of the GCKR P446L variant associated with type 2 diabetes risk

Abstract

Aims/hypothesis: Translation of genetic association signals into molecular mechanisms for diabetes has been slow. The glucokinase regulatory protein (GKRP; gene symbol GCKR) P446L variant, associated with inverse modulation of glucose- and lipid-related traits, has been shown to alter the kinetics of glucokinase (GCK) inhibition. As GCK inhibition is associated with nuclear sequestration, we aimed to determine whether this variant also alters the direct interaction between GKRP and GCK and their intracellular localisation.

Methods: Fluorescently tagged rat and human wild-type (WT)- or P446L-GCKR and GCK were transiently transfected into HeLa cells and mouse primary hepatocytes. Whole-cell and nuclear fluorescence was quantified in individual cells exposed to low- or high-glucose conditions (5.5 or 25 mmol/l glucose, respectively). Interaction between GCK and GKRP was measured by sensitised emission-based fluorescence resonance energy transfer (FRET) efficiency.

Results: P446L-GKRP had a decreased degree of nuclear localisation, ability to sequester GCK and direct interaction with GCK as measured by FRET compared with WT-GKRP. Decreased interaction was observed between WT-GKRP and GCK at high compared with low glucose, but not between P446L-GKRP and GCK. Rat WT-GKRP and P446L-GKRP behaved quite differently: both variants responded to high glucose by diminished sequestration of GCK but showed no effect of the P446L variant on nuclear localisation or GCK sequestration.

Conclusions/interpretation: Our study suggests the common human P446L-GKRP variant protein results in elevated hepatic glucose uptake and disposal by increasing active cytosolic GCK. This would increase hepatic lipid biosynthesis but decrease fasting plasma glucose concentrations and provides a potential mechanism for the protective effect of this allele on type 2 diabetes risk.

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)

Fig. 2

Localisation of co-transfected human GCK and GKRP in HeLa cells at 5.5 mmol/l glucose. a Visualisation 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

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

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

Fig. 5

Localisation of human GCK and direct interaction with WT-hGKRP or P446L-hGKRP in primary mouse hepatocytes. Hepatocytes 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