Diabetologia (2012) 55:114–122 hepatocytes (61%) [17]. Translocation of human GCK in the presence of hGKRP has not been previously explored, but our results suggest there are important differences between the rat and human GKRP translocation systems, and that regulation of translocation in humans will be strongly dependent on the genotype at P446L. Extensive assessment in human hepatocytes of appropriate genotype may provide useful future insight into these questions. In conclusion, we have demonstrated that the human GCKR P446L variant alters the ability of GKRP to sequester GCK in the nucleus. This observation, coupled with our previous kinetic studies, provides a mechanistic link between a common genetic variant associated with diabetes risk and metabolic traits and glucose and triacyl- glycerol metabolism. Acknowledgements This study was supported by the NIH Division of Intramural Research and NHGRI project number Z01-HG000024 (F.S. Collins). This study was funded in Oxford by the Medical Research Council (81696) and the Wellcome Trust (095101/Z/10/Z). A.L. Gloyn is a Wellcome Trust Senior Research Fellow in Basic and Biomedical Science. This study was funded in Rostock by the German Diabetes Association (DDG). Contribution statement MGR wrote the manuscript, contributed to experimental design, and researched and analysed data. SW analysed data and contributed to the manuscript. JS researched and analysed data and contributed to the manuscript. RW researched and analysed data and contributed to the manuscript. NLB researched and analysed data and reviewed and edited the manuscript. SB researched and analysed data and reviewed and edited the manuscript. FSC contributed to experimental design and reviewed and edited the manuscript. ALG contributed to experimental design and wrote the manuscript. All authors approved the final version to be published. Duality of interest The authors declare that there is no duality of interest associated with this manuscript. References 1. McCarthy MI (2010) Genomics, type 2 diabetes, and obesity. New Engl J Med 363:2339–2350 2. van de Bunt M, Gloyn AL (2010) From genetic association to molecular mechanism. Curr Diab Rep 10:452–466 3. Hindorff LA, Sethupathy P, Junkins HA et al (2009) Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc Natl Acad Sci USA 106:9362–9367 4. Van Schaftingen E (1989) A protein from rat liver confers to glucokinase the property of being antagonistically regulated by fructose 6-phosphate and fructose 1-phosphate. Eur J Biochem 179:179–184 5. Vandercammen A, Detheux M, van Schaftingen E (1992) Binding of sorbitol 6-phosphate and of fructose 1-phosphate to the regulatory protein of liver glucokinase. Biochem J 286:253–256 6. Toyoda Y, Miwa I, Satake S, Anai M, Oka Y (1995) Nuclear location of the regulatory protein of glucokinase in rat liver and 121 translocation of the regulator to the cytoplasm in response to high glucose. Biochem Biophys Res Commun 215:467–473 7. Shiota C, Coffey J, Grimsby J, Grippo JF, Magnuson MA (1999) Nuclear import of hepatic glucokinase depends upon glucokinase regulatory protein, whereas export is due to a nuclear export signal sequence in glucokinase. J Biol Chem 274:37125–37130 8. Kaminski MT, Lenzen S, Baltrusch S (2010) New aspects of hepatic glucose phosphorylation by intracellular glucokinase localisation. Diabetologia 53:(Suppl 1). Abstract 629 9. Orho-Melander M, Melander O, Guiducci C et al (2008) Common missense variant in the glucokinase regulatory protein gene is associated with increased plasma triglyceride and C-reactive protein but lower fasting glucose concentrations. Diabetes 57:3112–3121 10. Dupuis J, Langenberg C, Prokopenko I et al (2010) New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk. Nat Genet 42:105–116 11. Teslovich TM, Musunuru K, Smith AV et al (2010) Biological, clinical and population relevance of 95 loci for blood lipids. Nature 466:707–713 12. Beer NL, Tribble ND, McCulloch LJ et al (2009) The P446L variant in GCKR associated with fasting plasma glucose and triglyceride levels exerts its effect through increased glucokinase activity in liver. Hum Mol Genet 18:4081–4088 13. Veiga-da-Cunha M, Delplanque J, Gillain A et al (2003) Mutations in the glucokinase regulatory protein gene in 2p23 in obese French Caucasians. Diabetologia 46:704–711 14. Brocklehurst KJ, Davies RA, Agius L (2004) Differences in regulatory properties between human and rat glucokinase regula- tory protein. Biochem J 378:693–697 15. Farrelly D, Brown KS, Tieman A et al (1999) Mice mutant for glucokinase regulatory protein exhibit decreased liver glucoki- nase: a sequestration mechanism in metabolic regulation. Proc Natl Acad Sci USA 96:14511–14516 16. Grimsby J, Coffey JW, Dvorozniak MT et al (2000) Character- ization of glucokinase regulatory protein-deficient mice. J Biol Chem 275:7826–7831 17. Baltrusch S, Francini F, Lenzen S, Tiedge M (2005) Interaction of glucokinase with the liver regulatory protein is conferred by leucine–asparagine motifs of the enzyme. Diabetes 54:2829– 2837 18. Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572 19. Seglen PO (1976) Preparation of isolated rat liver cells. Methods Cell Biol 13:29–83 20. Bader A, Fruhauf N, Tiedge M et al (1999) Enhanced oxygen delivery reverses anaerobic metabolic states in prolonged sand- wich rat hepatocyte culture. Exp Cell Res 246:221–232 21. Langer S, Kaminski MT, Lenzen S, Baltrusch S (2010) Endog- enous activation of glucokinase by 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase is glucose dependent. Mol Endocrinol 24:1988–1997 22. Vanderklish PW, Krushel LA, Holst BH, Gally JA, Crossin KL, Edelman GM (2000) Marking synaptic activity in dendritic spines with a calpain substrate exhibiting fluorescence resonance energy transfer. Proc Natl Acad Sci USA 97:2253–2258 23. Rodriguez-Enriquez S, Marin-Hernandez A, Gallardo-Perez JC, Moreno-Sanchez R (2009) Kinetics of transport and phosphory- lation of glucose in cancer cells. J Cell Physiol 221:552–559 24. Mukhtar M, Stubbs M, Agius L (1999) Evidence for glucose and sorbitol-induced nuclear export of glucokinase regulatory protein in hepatocytes. FEBS Lett 462:453–458 25. Watanabe F, Furuya E (2010) Quantitative image analysis reveals that phosphorylation of liver-type isozyme of fructose-6- phosphate 2-kinase/fructose-2,6-bisphosphatase does not affect