METABOLISM OF ISLETS OF LANGERHANS AND HORMONE RELEASE

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DESCRIPTION: The proposed work has the objective to explore critical metabolic steps of the signaling pathway(s) in fuel-stimulated beta-cells and to provide mechanistic explanations at the molecular level for selected relevant physiological syndromes in man and animals. The project focuses on the glucokinase glucose sensor governing glycolysis and on islet cell mitochondria as generators of metabolic coupling factors that link intermediary metabolism with biophysical cell membrane events. MODY-2 and hyperinsulinemias linked to glucokinase (HI-GK) or glutamate dehydrogenase (HI-GDH) mutations are the relevant clinical entities in man and the BHEcdb rat is a pertinent animals model of type II diabetes caused by a primary defect of mitochondrial DNA. Recombinant GKs with known missense mutations defined in MODY-2 and HI-GK patients will be characterized by enzyme kinetics. Substrate affinities, sigmoidicity, and enzyme stability will be the primary focus. Isolated mitochondria of betaHC, betaTC, and alphaTC cells will be characterized with novel dynamic tests including 31P- and 13C-NMR, allowing the study of ATP kinetics and of preferred metabolic pathways, respectively. Metabolic coupling will be explored in intact cells of these same cell lines and with isolated mouse or rat islets. Large batches of tumorous beta-or alpha-cells will be embedded in agarose to form "pseudoislets" which will allow the application of non-invasive techniques of respirometry, and 13C- as well as 31P-NMR, providin an opportunity for exact balance studies of fuel flow (including ATP kinetics) that was heretofore not possible. Metabolic coupling in intact islets will be studied physiologically, biochemically, and by imaging techniques. The tempora development of metabolic and functional lesions in intact beta-cells will be explored in the BHEcdb rat model, to search for the earliest functional manifestations of the reported mutation in the Fo component of ATP synthase.