Susceptibility of Glucokinase-MODY Mutants to Inactivation by Oxidative Stress in Pancreatic β-Cells
- 1Institute of Cellular Medicine, Newcastle University, Newcastle Upon Tyne, U.K.
- 2Department of Biochemistry and Biophysics and Institute for Diabetes, Obesity and Metabolism, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
- Corresponding author: Catherine Arden, .
L.A. and C.A. contributed equally to this work.
OBJECTIVE The posttranslational regulation of glucokinase (GK) differs in hepatocytes and pancreatic β-cells. We tested the hypothesis that GK mutants that cause maturity onset diabetes of the young (GK-MODY) show compromised activity and posttranslational regulation in β-cells.
RESEARCH DESIGN AND METHODS Activity and protein expression of GK-MODY and persistent hyperinsulinemic hypoglycemia of infancy (PHHI) mutants were studied in β-cell (MIN6) and non–β-cell (H4IIE) models. Binding of GK to phosphofructo-2-kinase, fructose-2,6 bisphosphatase (PFK2/FBPase2) was studied by bimolecular fluorescence complementation in cell-based models.
RESULTS Nine of 11 GK-MODY mutants that have minimal effect on enzyme kinetics in vitro showed decreased specific activity relative to wild type when expressed in β-cells. A subset of these were stable in non-β-cells but showed increased inactivation in conditions of oxidative stress and partial reversal of inactivation by dithiothreitol. Unlike the GK-MODY mutants, four of five GK-PHHI mutants had similar specific activity to wild-type and Y214C had higher activity than wild-type. The GK-binding protein PFK2/FBPase2 protected wild-type GK from oxidative inactivation and the decreased stability of GK-MODY mutants correlated with decreased interaction with PFK2/FBPase2.
CONCLUSIONS Several GK-MODY mutants show posttranslational defects in β-cells characterized by increased susceptibility to oxidative stress and/or protein instability. Regulation of GK activity through modulation of thiol status may be a physiological regulatory mechanism for the control of GK activity in β-cells.
- Received March 29, 2011.
- Accepted August 31, 2011.
- © 2011 by the American Diabetes Association.
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