Cell biology assessment of glucokinase mutations V62M and G72R in pancreatic beta-cells: evidence for cellular instability of catalytic activity.
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 beta-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.
Arden
Catherine
C
Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.
Trainer
Alison
A
de la Iglesia
Nuria
N
Scougall
Kathleen T
KT
Gloyn
Anna L
AL
Lange
Alex J
AJ
Shaw
James A M
JA
Matschinsky
Franz M
FM
Agius
Loranne
L
D000818:Animals; D002384:Catalysis; D002460:Cell Line; D002678:Chimera; D005941:Glucokinase / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation
D005941:Glucokinase