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Diabetes Publish Ahead of Print published online ahead of print March 27, 2007
DOI: 10.2337/db06-1151
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Original Research

Cell biology assessment of glucokinase mutations V62M and G72R in pancreatic beta-cells: evidence for cellular instability of catalytic activity

Catherine Arden1, Alison Trainer1, Nuria de la Iglesia1, Kathleen T. Scougall1, Anna L. Gloyn2, Alex J. Lange3, James AM Shaw1, Franz M. Matschinsky4, and Loranne Agius1

1Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne NE2 4HH, UK
2Diabetes Research Laboratories, Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, Oxford OX3 7LJ
3Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455
4Department of Biochemistry & Biophysics and Diabetes Research Center, University of Pennsylvania School of Medicine, Philadelphia PA 19104

Correspondence: Loranne.Agius{at}ncl.ac.uk

Mutations in the glucokinase (GK) gene cause defects in blood glucose homeostasis. In some cases (V62M and G72R) the phenotype cannot be explained by altered enzyme kinetics or protein instability. We used transient and stable expression of GFP-GK chimaeras in MIN6 beta-cells to study the phenotype defect of V62M and G72R. GK activity in lysates of MIN6 cell lines stably expressing GFP-GK wild-type or mutants showed the expected affinity for glucose and response to pharmacological activators indicating expression of catalytically active enzyme. MIN6 cells stably expressing GFP-V62M or GFP-G72R had a lower GK-activity/GK-immunoreactivity ratio and GK activity/GK-mRNA ratio but not GK-immunoreactivity/GK-mRNA ratio than GFP-GK-wildtype. Heterologous expression of liver PFK2-FDP2 in the cell lines increased GK activity for GK-wild-type and V62M but not for G72R whereas expression of GKRP (liver GK regulatory protein) increased GK activity for wild-type but not V62M or G72R. Lack of interaction of these mutants with GKRP was also evident in hepatocyte transfections from the lack of nuclear accumulation. These results suggest that cellular loss of GK catalytic activity rather than impaired translation or enhanced protein degradation may account for the hyperglycaemia in subjects with V62M and G72R mutations.


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