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  • 1
    ISSN: 1432-0428
    Keywords: Alloxan ; cyclic AMP ; isolated islets ; insulin secretion ; glucose metabolism ; 3-0-methylglucose ; glyceraldehyde
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Insulin secretion was stimulated and cyclic adenosine 3′, 5′-monophosphate (cAMP) levels were elevated in isolated rat islets by 27.5 mmol/l glucose. Alloxan caused a dose-dependent decrease in both variables with complete obliteration of insulin release at a concentration of 1.25 mmol/l. D-glucose, in the presence or absence of extracellular calcium, or 3-0-methyl-D-glucose (both at 27.5 mmol/l) protected completely against the effects of alloxan on both glucose-induced insulin release and cAMP levels. 3-0-Methylglucose did not stimulate insulin secretion or elevate cAMP and did not interfere with glucose-stimulated secretion or elevation of cAMP. When glucose-stimulated insulin release was abolished by alloxan, the metabolism of glucose, determined by the rate of3H2O formation from [5-3H] glucose, was depressed by 20%. It is concluded that alloxan altered the adenylate cyclase system such that it could no longer be stimulated by glucose. Glucose-stimulated insulin secretion or elevation of cAMP did not appear essential for glucose to protect against alloxan. Protection by 3-0-methylglucose did not appear to be mediated through an alteration of cAMP metabolism. Alloxan did not inhibit glucose-induced insulin secretion by grossly altering glycolysis.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-0428
    Keywords: Keywords MODY-2 ; glucokinase ; glucose threshold ; insulin secretion ; beta-cell ; mathematical model.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Aims/hypothesis. Mutations of the glucokinase gene cause hyperglycaemia or hypoglycaemia. A quantitative understanding of these defects of glucose homeostasis linked to the glucokinase gene was lacking. Therefore a database of kinetic variables of wild-type and 20 missense mutants of glucokinase was developed and used in mathematical modelling to predict the thresholds for glucose-stimulated insulin release. Methods. Recombinant human glucokinase was generated in E. coli. The kcat, glucose S0.5, ATP Km, and Hill number of glucokinase were determined. Inhibition by Stearoyl CoA and glucokinase regulatory protein and thermal stability were assayed for all mutants kinetically similar to wild-type glucokinase. A mathematical model predicting the threshold for glucose-stimulated insulin release was constructed. This model is based on the two substrate kinetics of glucokinase and the kinetic variables of the database. It is assumed that both glucokinase gene alleles are equally expressed in beta-cells and that induction of glucokinase occurs as a function of basal blood glucose. Results. Large changes, varying greatly between mutants were found in nearly all variables. Glucokinase flux at threshold for glucose-stimulated insulin release was about 25 % of total phosphorylating potential in the normal beta-cell and this was used to predict thresholds for the mutant heterozygotes. Clinical data for maturity onset diabetes of the young type linked to the glucokinase gene and familial hyperinsulinaemic hypoglycaemia linked to the glucokinase gene and the glucokinase kinetic data of this study were used to test the model. The model predicts fasting blood glucose between 3 and 7 mmol/l in these cases. Conclusion/interpretation. A kinetics database of wild-type and 20 mutants of glucokinase was developed. Many kinetic differences were found for the mutants. The mathematical model to calculate the threshold for glucose-stimulated insulin release predicts fasting blood glucose between 3 and 7 mmol/l in subjects with glucokinase gene mutations. [Diabetologia 42: 1175–1186]
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1432-0428
    Keywords: Glucose ; cyclic AMP ; calcium ; insulin ; insulin secretion ; receptor mechanism ; second messenger
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Adenosine 3′,5′-cyclic monophosphate (cAMP) levels of isolated perifused pancreatic islets were elevated by high levels of glucose concomitantly with initiation of enhanced insulin secretion. The rise of cAMP was biphasic and seemed to be related to the temporal biphasic kinetics of insulin release. However, the temporal profiles of cAMP level changes and of insulin release differed; the major rise of the cAMP levels was seen during the initial phase, whereas insulin secretion was more pronounced during the second phase of release. Glucose-induced cAMP elevation required the presence of extracellular Ca++. Mannoheptulose completely blocked cAMP elevation due to high glucose. Exogenous insulin which has been shown by others to inhibit insulin secretion in vitro, blunted the glucose-induced cAMP rise. These observations and data in the literature are compatible with the concept that under physiological conditions glucose governs the intracellular cAMP levels in a Ca++ dependent manner — either directly or indirectly through metabolic effects.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 37 (1994), S. 398-406 
    ISSN: 1040-452X
    Keywords: Preimplantation embryo ; Insulin ; IGFs ; Protein synthesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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. However, seven IGF response proteins were found to be proteins that showed significant changes in isotope incorporation during the eight-cell to blastocyst stage of development (Shi et al., 1993). In all, 54 proteins were affected, and these were unique; thus, protein synthesis in preimplantation mouse embryos is influenced by insulin and the IGFs, and further, each growth factor affects specific proteins. © 1994 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 42 (1995), S. 173-179 
    ISSN: 1040-452X
    Keywords: Embryonic stem cells ; Insulin ; IGF-I ; IGF-II ; Receptor ; RT-PCR ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Insulin and insulin-like growth factors (IGF-I and -II) are members of a family of growth factors which are known to be developmentally regulated during preimplantation mouse embryogenesis. The physiological actions of the insulin family of growth factors are mediated by interactions with specific cell surface receptors that are detectable on the cells of preimplantation mouse embryos. Mouse embryonic stem (ES) cells are totipotent cells derived directly from the inner cell mass of the blastocyst. ES cells have the ability to differentiate into all three germ layers and have unlimited growth potential under certain culture conditions. The great advantage of ES cells is the ability to obtain large amounts of tissue for biochemical studies as compared with preimplantation embryos. To examine in greater detail the biological actions of the insulin family of growth factors, the expression of their cognate receptors on ES cells was examined. ES cells were cultured in DMEM medium supplemented with leukemia inhibitory factor (LIF) to maintain the undifferentiated state. Receptor expression was evaluated at the mRNA level using the reverse transcription polymerase chain reaction (RT-PCR), and at the protein level by radioactive labeled ligand-receptor binding assay. Using RT-PCR, mRNAs of all three growth factor receptors were detected in ES cells. Messenger RNA from ES cells was reverse transcribed into cDNA by AMV reverse transcriptase at 42°C for 1 hr. The reverse transcription reaction was amplified with Taq polymerase and specific primers for insulin, IGF-I, or IGF-II receptors by PCR. RT-PCR and the control plasmid cDNA PCR products were resolved electrophoretically on 3% agarose gels. Each amplified PCR product showed the predicted correct size. The target sequence of RT-PCR amplified fragments were further verified by restriction enzyme digestion. The expression of receptors at the protein level was confirmed by Scatchard analysis, which showed specific binding of the radiolabeled ligands. This study shows that ES cells may provide a useful model to study the biological actions of the insulin family growth factors. © 1995 wiley-Liss, Inc.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
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