Diabetologia (2012) 55:114–122 Introduction The past 4 years have seen a rapid expansion in the number of reproducibly associated loci for type 2 diabetes and related traits. However, progress in translating these genetic associations into biological mechanisms for disease has been slow [1, 2]. Contributing factors have included small effect sizes and the mapping of association signals to poorly annotated, often intergenic, sequences [3]. Signals that include an obvious (e.g. non-synonymous) variant in a biologically plausible gene provide tractable targets for functional follow-up and a framework for exploring approaches to variant characterisation [2]. Glucokinase (GCK) regulates glucose storage and disposal in the liver. Hepatic GCK activity is modulated by glucokinase regulatory protein (GKRP; gene symbol GCKR), a competitive inhibitor of GCK. Fructose 6- phosphate (F6P) and fructose 1-phosphate (F1P) enhance and reduce GKRP-mediated inhibition, respectively [4, 5]. A further layer of regulation exists in the form of cellular localisation: GKRP sequesters GCK to the nucleus at low glucose concentrations, further inhibiting GCK activity and glycolysis [6]. At higher glucose concentrations, glucose- mediated dissociation of GCK from GKRP activates GCK and exposes its nuclear export signal [7, 8]. A common GCKR single nucleotide polymorphism (SNP) (rs1260326 [c.1337C>T; p.P446L]) has been repro- ducibly associated with an inverse relationship between glucose and triacylglycerol levels [9]. This seemingly paradoxical relationship also extends to other glucose- and lipid-related traits. The glucose-lowering leucine allele is also associated with decreased fasting insulin, HOMA- estimated insulin resistance and reduction of type 2 diabetes risk [10] but increased triacylglycerol and total cholesterol levels [11]. This variant is one of only a handful of candidate non-synonymous variants identified to date from type 2 diabetes association studies [2, 12]. We have previously shown that variant P446L-GKRP increases GCK activity as a result of diminished regulation by F6P and have proposed that this results in increased hepatic glycolytic flux and de novo lipogenesis, accounting for the observed associated phenotypes [12]. Interestingly, this kinetic effect was not seen with rat P446L-GKRP [13], likely due to species differences in regulation by F6P [14]. However, kinetic mechanisms alone are not sufficient to explain GKRP regulation of GCK in vivo. For example, Gckr-knockout mice display impaired postprandial glucose handling and paradoxical reductions in GCK protein levels and activity in spite of the removal of GKRP inhibition [15, 16]. This suggests that nuclear interaction with GKRP plays an important role in regulating GCK protein concentration and stability and is essential for the maintenance of glucose homeostasis. In addition, the P446L variant is associated 115 with inverse modulation of fasting and 2 h glucose levels, an observation not readily explained by kinetic data [10]. As the contribution of GKRP and GCK cellular localisation to phenotypes associated with the P446L-GKRP variant protein is currently unexplored, the aims of the current study were to assess the impact of this variant on the ability of GKRP to sequester GCK in the nucleus and to further explore potential differences between rat and human regulatory proteins. Methods Cloning of fluorescent fusion plasmids Human GCK (Mammalian Gene Collection; ATCC, Manassas, VA, USA) was amplified using primers containing BamHI and HindIII restriction sites and subcloned into the pEGFP-c3 vector encoding enhanced green fluorescent protein (EGFP). Generation of enhanced cyan fluorescent protein (ECFP)-tagged GCK (pECFP-GCK) has been described by Baltrusch et al. [17]. PCR primers containing Eco47III and EcoRI sites were used to amplify human GCKR (Mamma- lian Gene Collection) and replace rat Gckr in pEYFP-N1- GCKR [17]. The P446L mutation was introduced into rat and human pEYFP-N1-GCKR plasmids by PCR-based site- directed mutagenesis (Agilent Biotechnologies, Santa Clara, CA, USA). The enhanced yellow fluorescent protein (EYFP) in both rat and human pEYFP-N1-GCKR [17] was replaced by mCherry [18] using AgeI and BsrGI restriction sites. Two independent preparations of all plasmids were generated and verified by sequencing. Primer sequences are available upon request. Cell culture and transient transfection of HeLa cells HeLa cells were grown in DMEM with 5.5 or 25 mmol/l glucose supplemented with 10% (vol./vol.) dialysed fetal bovine serum and penicillin/streptomycin (all Invitrogen, Carlsbad, CA, USA). For quantitative fluorescence microscopy, 4 ×104 cells were seeded into four-chamber slides (BD Biosciences, San Jose, CA, USA) and cultured for 24 h. Co-transfection of mCherry-GCKR and EGFP-GCK was initially carried out with a range of plasmid DNA concentrations at ten separate GCK/GCKR molar ratios between 40:1 and 1:40. Nuclear localisation of GCK was observed for GCK/GCKR ratios between 1:1 and 1:40. A ratio of 1:4 was used for all future experiments as this is in agreement with the published GCK/GCKR mRNA ratio in liver [12]. Optimal transfection efficiency and fluorescence intensity was observed with transfection of 100 ng total DNA and 0.75 μl of Lipofectamine 2000 (Invitrogen). Transfection was carried out according to the manufac- turer’s instructions at 5.5 mmol/l glucose. The medium was replaced 5 h after transfection.