Cell-biological assessment of human glucokinase mutants causing maturity-onset diabetes of the young type 2 (MODY-2) or glucokinase-linked hyperinsulinaemia (GK-HI).

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.

Insulin family growth factors have specific effects on protein synthesis in preimplantation mouse embryos.

Previously constructed protein databases for two stages of preimplantation mouse embryogenesis, the compacted eight-cell stage and the fully expanded blastocyst stage, have been used to analyze the effects of insulin, IGF-I, and IGF-II on protein synthesis in these developmental stages. Proteins were labeled by placing, for 2 hr, synchronous cohorts of 35-50 embryos into human tubal fluid (HTF) medium containing L-[35S]-methionine (1 mCi/ml) in the presence or absence of one of the growth factors. The embryos were then washed with medium and lysed. Samples were processed for 2-D gel analysis. For each embryonic stage and each growth factor, four or five experimental replicates were done and the gel images were compared using the PDQUEST system. Using the computer-assisted analysis, we were able to identify proteins that showed a statistically significant (P < 0.05) change in synthesis. At the eight-cell stage of development insulin caused increased synthesis of two proteins and decreased synthesis in three proteins. Insulin-treated blastocyst stage embryos exhibited an increased synthesis in eight proteins and decreased synthesis for one protein. The effect of IGF-I at the eight-cell stage of development was mostly inhibitory; the synthesis of only one protein increased and the synthesis of five proteins showed a decrease. Similar results were obtained with blastocyst stage embryos; four proteins demonstrated an increase in synthesis while 14 proteins showed a decrease. Eight-cell stage embryos incubated with IGF-II had seven proteins with a decreased synthesis, although in blastocyst stage embryos, nine proteins showed increased synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein databases for compacted eight-cell and blastocyst-stage mouse embryos.

High-resolution two-dimensional sodium dodecyl sulfate-polyacrylamide (2D-SDS) gel electrophoresis combined with computerized analysis of gel images was used to construct and analyze protein data-bases for two stages of preimplantation mouse embryogenesis, the compacted eight-cell stage and the fully expanded blastocyst stage. These stages were chosen for their ease in identification of multiple synchronous embryos. Synchronous cohorts of 30-50 embryos were labelled with L-[35S]methionine for 2 hr. The embryos were then lysed in 30 microliters hot SDS sample buffer, and the lysates were stored at -80 degrees C until the gels were run. Five replicates were run for eight-cell embryos, and four for blastocyst-stage embryos. The samples were processed for 2D gel electrophoresis and fluorography; multiple exposures were made. Gel images were analyzed using the PDQUEST system, and databases were constructed. Analysis of the databases for both developmental stages showed high reproducibility of protein spots in multiple gel images. Of 1,674 total spots in eight-cell embryo standards, > 79% of spots had a percentage error (S.E.M./average) < 50%, and > 45% had a percentage error < 30%. Similarly, of 1,653 total spots in blastocyst-stage embryo standards, 74% of spots had a percentage error < 50%, and approximately 47% of spots had a percentage error < 30%. Forty-three spots (approximately 3% of the total spots) were found to be detected only in the eight-cell stage, while 75 spots were detected solely in the blastocyst stage. Sixty-nine proteins showed a greater than threefold increase in isotope incorporation from the eight-cell to the blastocyst stage, with a percentage error < 50% in both the eight-cell and the blastocyst stages. In contrast, 41 of the proteins showed a decrease during this period. Analysis of the protein databases described in this study has allowed us to document the overall quantitative changes in proteins from the compacted eight-cell stage to the blastocyst stage of mouse preimplantation development. These data-bases provide a valuable tool for further detailed quantitative analysis of specific proteins associated with developmental events. In addition they will permit analysis of the effects of environmental factors, such as growth factors, on early embryo development.