title: Cell-biological assessment of human glucokinase mutants causing maturity-onset diabetes of the young type 2 (MODY-2) or glucokinase-linked hyperinsulinaemia (GK-HI).
abstract: Mutations in the glucokinase (GK) gene cause type-2 maturity-onset diabetes of the young type 2 (MODY-2) and GK-linked hyperinsulinaemia (GK-HI). Recombinant adenoviruses expressing the human wild-type islet GK or one of four mutant forms of GK, (the MODY-2 mutants E70K, E300K and V203A and the GK-HI mutant V455M) were transduced into glucose-responsive insulin-secreting beta-HC9 cells and tested functionally in order to initiate the first analysis in vivo of recombinant wild-type and mutant human islet GK. Kinetic analysis of wild-type human GK showed that the glucose S(0. 5) and Hill coefficient were similar to previously published data in vitro (S(0.5) is the glucose level at the half-maximal rate). E70K had half the glucose affinity of wild-type, but similar enzyme activity. V203A demonstrated decreased catalytic activity and an 8-fold increase in glucose S(0.5) when compared with wild-type human islet GK. E300K had a glucose S(0.5) similar to wild-type but a 10-fold reduction in enzyme activity. E300K mRNA levels were comparable with wild-type GK mRNA levels, but Western-blot analyses demonstrated markedly reduced levels of immunologically detectable protein, consistent with an instability mutation. V455M was just as active as wild-type GK, but with a markedly reduced S(0.5). The effects of the different GK mutants on glucose-stimulated insulin release support the kinetic and expression data. These experiments show the utility of a combined genetic, biochemical and cell-biological approach to the quantification of functional and structural changes of human GK that result from MODY-2 and GK-HI mutations.
authors.affiliation: Harrison Department of Surgical Research, University of Pennsylvania Medical Center, 313 Stemmler Hall, 36th and Hamilton Walk, Philadelphia, PA 19104, USA.
authors.forename: C V
authors.initials: CV
authors.lastname: Burke
authors.forename: C W
authors.initials: CW
authors.lastname: Buettger
authors.forename: E A
authors.initials: EA
authors.lastname: Davis
authors.forename: S J
authors.initials: SJ
authors.lastname: McClane
authors.forename: F M
authors.initials: FM
authors.lastname: Matschinsky
authors.forename: S E
authors.initials: SE
authors.lastname: Raper
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002460:Cell Line; D003920:Diabetes Mellitus / Q000201:enzymology* / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D005941:Glucokinase / Q000235:genetics* / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000097:blood* / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D007700:Kinetics; D017354:Point Mutation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D007328:Insulin; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005941:Glucokinase; D005947:Glucose