Cell Biology Assessment of Glucokinase Mutations V62M and G72R in Pancreatic β-Cells

Evidence for Cellular Instability of Catalytic Activity

  1. Catherine Arden1,
  2. Alison Trainer1,
  3. Nuria de la Iglesia1,
  4. Kathleen T. Scougall1,
  5. Anna L. Gloyn2,
  6. Alex J. Lange3,
  7. James A.M. Shaw1,
  8. Franz M. Matschinsky4 and
  9. Loranne Agius1
  1. 1Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, U.K
  2. 2Diabetes Research Laboratories, Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, U.K
  3. 3Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota
  4. 4Department of Biochemistry and Biophysics, Diabetes Research Center, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
  1. Address correspondence and reprint requests to Loranne Agius, School of Clinical Medical Sciences, The Medical School, Newcastle upon Tyne, NE2 4HH, U.K. E-mail: loranne.agius{at}ncl.ac.uk

Abstract

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 green fluorescent protein (GFP) GK chimaeras in MIN6 β-cells to study the phenotype defect of V62M and G72R. GK activity in lysates of MIN6 cell lines stably expressing wild-type or mutant GFP GK showed the expected affinity for glucose and response to pharmacological activators, indicating the expression of catalytically active enzymes. MIN6 cells stably expressing GFP V62M or GFP G72R had a lower GK activity–to–GK immunoreactivity ratio and GK activity–to–GK mRNA ratio but not GK immunoreactivity–to–GK mRNA ratio than wild-type GFP GK. Heterologous expression of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2/FDP2) in cell lines increased GK activity for wild-type GK and V62M but not for G72R, whereas expression of liver GK regulatory protein (GKRP) 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 hyperglycemia in subjects with V62M and G72R mutations.

Footnotes

  • Published ahead of print at http://diabetes.diabetesjournals.org on 27 March 2007. DOI: 10.2337/db06-1151.

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    • Accepted March 21, 2007.
    • Received August 16, 2006.
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  1. Diabetes vol. 56 no. 7 1773-1782
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