title: Generation of N-ethyl-N-nitrosourea (ENU) diabetes models in mice demonstrates genotype-specific action of glucokinase activators.
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.
authors.affiliation: Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.
authors.forename: Deborah
authors.initials: D
authors.lastname: Fenner
authors.forename: Stella
authors.initials: S
authors.lastname: Odili
authors.forename: Hee-Kyung
authors.initials: HK
authors.lastname: Hong
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Akira
authors.initials: A
authors.lastname: Kohsaka
authors.forename: Sandra M
authors.initials: SM
authors.lastname: Siepka
authors.forename: Martha H
authors.initials: MH
authors.lastname: Vitaterna
authors.forename: Pan
authors.initials: P
authors.lastname: Chen
authors.forename: Bogumil
authors.initials: B
authors.lastname: Zelent
authors.forename: Joseph
authors.initials: J
authors.lastname: Grimsby
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Takahashi
authors.forename: Franz M
authors.initials: FM
authors.lastname: Matschinsky
authors.forename: Joseph
authors.initials: J
authors.lastname: Bass
mesh_terms: D000477:Alkylating Agents / Q000009:adverse effects* / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001786:Blood Glucose / Q000235:genetics / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D020536:Enzyme Activators / Q000378:metabolism*; D005038:Ethylnitrosourea / Q000009:adverse effects* / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics; D005941:Glucokinase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D006943:Hyperglycemia / Q000139:chemically induced / Q000201:enzymology / Q000235:genetics; D008099:Liver / Q000201:enzymology* / Q000473:pathology; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D020125:Mutation, Missense; D009928:Organ Specificity; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000477:Alkylating Agents; D001786:Blood Glucose; D020536:Enzyme Activators; D011994:Recombinant Proteins; D005941:Glucokinase; D005038:Ethylnitrosourea