Generation of N-ethyl-N-nitrosourea (ENU) diabetes models in mice demonstrates genotype-specific action of glucokinase activators

Deborah Fenner, Stella Odili, Hee Kyung Hong, Yumiko Kobayashi, Akira Kohsaka, Sandra M. Siepka, Martha H. Vitaterna, Pan Chen, Bogumil Zelent, Joseph Grimsby, Joseph S. Takahashi, Franz M. Matschinsky, Joseph Bass

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

We performed genome-wide mutagenesis in C57BL/6J mice using N-ethyl-N-nitrosourea to identify mutations causing high blood glucose early in life and to produce new animal models of diabetes. Of a total of 13 new lines confirmed by heritability testing, we identified two semi-dominant pedigrees with novel missense mutations (Gck K140E and Gck P417R) in the gene encoding glucokinase (Gck), the mammalian glucose sensor that is mutated in human maturity onset diabetes of the young type 2 and the target of emerging anti-hyperglycemic agents that function as glucokinase activators (GKAs). Diabetes phenotype corresponded with genotype (mild-to-severe: Gck +/+ < Gck P417R/+, Gck K140E/+ < Gck P417R/P417R, Gck P417R/K140E, and Gck K140E/K140E) and with the level of expression of GCK in liver. Each mutant was produced as the recombinant enzyme in Escherichia coli, and analysis of k cat and tryptophan fluorescence (I 320/360) during thermal shift unfolding revealed a correlation between thermostability and the severity of hyperglycemia in the whole animal. Disruption of the glucokinase regulatory protein-binding site (GCK K140E), but not the ATP binding cassette (GCK P417R), prevented inhibition of enzyme activity by glucokinase regulatory protein and corresponded with reduced responsiveness to the GKA drug. Surprisingly, extracts from liver of diabetic GCK mutants inhibited activity of the recombinant enzyme, a property that was also observed in liver extracts from mice with streptozotocin-induced diabetes. These results indicate a relationship between genotype, phenotype, and GKA efficacy. The integration of forward genetic screening and biochemical profiling opens a pathway for preclinical development of mechanism-based diabetes therapies.

Original languageEnglish (US)
Pages (from-to)39560-39572
Number of pages13
JournalJournal of Biological Chemistry
Volume286
Issue number45
DOIs
StatePublished - Nov 11 2011

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Fenner, D., Odili, S., Hong, H. K., Kobayashi, Y., Kohsaka, A., Siepka, S. M., Vitaterna, M. H., Chen, P., Zelent, B., Grimsby, J., Takahashi, J. S., Matschinsky, F. M., & Bass, J. (2011). Generation of N-ethyl-N-nitrosourea (ENU) diabetes models in mice demonstrates genotype-specific action of glucokinase activators. Journal of Biological Chemistry, 286(45), 39560-39572. https://doi.org/10.1074/jbc.M111.269100