Susceptibility of glucokinase-MODY mutants to inactivation by oxidative stress in pancreatic β-cells.
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.


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.
Institute of Cellular Medicine, Newcastle University, Newcastle Upon Tyne, U.K.
Kirsty S
KS
Cullen
Franz M
FM
Matschinsky
Loranne
L
Agius
Catherine
C
Arden
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics* / Q000378:metabolism*; D005941:Glucokinase / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D008297:Male; D051379:Mice; D009154:Mutation; D009569:Nitric Oxide / Q000378:metabolism; D018384:Oxidative Stress; D025481:Phosphofructokinase-2 / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar
D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D025481:Phosphofructokinase-2; D005941:Glucokinase; D005978:Glutathione