Glucokinase Thermolability and Hepatic Regulatory Protein Binding Are Essential Factors for Predicting the Blood Glucose Phenotype of Missense Mutations

Maria F Pino,Kyoung-Ah Kim,Kathy D Shelton,Jill Lindner,Stella Odili,Changhong Li,Heather W Collins,Masakazu Shiota,Franz M Matschinsky,Mark A Magnuson

doi:10.1074/jbc.m610094200

Abstract

To better understand how glucokinase (GK) missense mutations associated with human glycemic diseases perturb glucose homeostasis, we generated and characterized mice with either an activating (A456V) or inactivating (K414E) mutation in the gk gene. Animals with these mutations exhibited alterations in their blood glucose concentration that were inversely related to the relative activity index of GK. Moreover, the threshold for glucose-stimulated insulin secretion from islets with either the activating or inactivating mutation were left- or right-shifted, respectively. However, we were surprised to find that mice with the activating mutation had markedly reduced amounts of hepatic GK activity. Further studies of bacterially expressed mutant enzymes revealed that GK(A456V) is as stable as the wild type enzyme, whereas GK(K414E) is thermolabile. However, the ability of GK regulatory protein to inhibit GK(A456V) was found to be less than that of the wild type enzyme, a finding consistent with impaired hepatic nuclear localization. Taken together, this study indicates that it is necessary to have knowledge of both thermolability and the interactions of mutant GK enzymes with GK regulatory protein when attempting to predict in vivo glycemic phenotypes based on the measurement of enzyme kinetics.

Highlights

Studies over the past 2 decades have firmly established that glucokinase (GK)3 plays a key role in determining the blood glucose concentration in mammals
Chimeric mice generated by blastocyst microinjection were either crossed with 129S6 to directly place both alleles into an inbred background or mated with EIIa-Cre transgenic mice to remove the neomycin resistance cassette and back-crossing the mutant gk alleles for at least 10 generations to create C57Bl/6J congenic lines
By introducing two human disease-associated GK mutations into the mouse, we have gained a new appreciation of the key role that both GK stability and GK regulatory protein (GKRP) binding play in modulating GK activity and how this affects the blood glucose concentration

Summary

Introduction

Studies over the past 2 decades have firmly established that glucokinase (GK) plays a key role in determining the blood glucose concentration in mammals. Heterozygous gene mutations that diminish enzyme expression or otherwise lower catalytic flux cause maturity onset diabetes of the young type. 3 The abbreviations used are: GK, glucokinase; MODY, maturity onset diabetes of the young type; PHHI, persistent hyperinsulinemic hypoglycemia of infancy; GKRP, GK regulatory protein; GST, glutathione S-transferase; RIA, radioimmunoassay. Heterozygous gene mutations that increase the activity of GK cause persistent hyperinsulinemic hypoglycemia of infancy (PHHI)-GK, a disease that is characterized by clinically significant hypoglycemia (6 – 8). The analysis of genetically engineered rodent models has unequivocally established that GK gene expression in both pancreatic ␤-cells and hepatocytes independently contributes to the maintenance of blood glucose homeostasis (9 –14). In addition to sequestering the enzyme away from other glycolytic enzymes, the binding of GKRP to GK may protect it from degradation [35, 36].

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