Un dispositif de stimulation électrique in vitro sur des lignées cellulaires a été optimisé afin de nous permettre d'étudier les mécanismes cellulaires et moléculaires de la SHF. Deux lignées cellulaires (GH3 et PC12) ont été analysées au niveau transcriptomique, protéomique et de la sécrétion hormonale et de neurotransmetteurs.Nous avons comparé les niveaux de sécrétion de PRL des GH3 traitées par SHF, SBF ou par la dopamine; ainsi que les niveaux des catécholamines (DA, AD et NA) des PC12 traitées par SHF, SBF ou par la 6-OHDA. La synthèse protéique des cellules stimulées a été analysée par les techniques d'incorporation de 35S méthionine et de SELDI-TOF-MS. Enfin nous avons recherché les modifications d'expression génique des cellules stimulées en utilisant la technique des microarray à base d'oligonucléotides longs sur nylon et détection radioactive.Les premières expériences montrent une diminution significative de la quantité de prolactine à un niveau comparable à celui obtenu avec l'inhibiteur conventionnel, la dopamine. De la même façon, la production de catécholamines dans le milieu est inhibée. Les données transcriptomiques montrent l'existence des profils d'expression caractéristique de la neurostimulation à haute fréquence, avec environ 100 gènes discriminants impliqués dans la synthèse protéique, la signalisation calcique, l'énergétique cellulaire pour les principaux. Nous avons confirmé l'impact de la neurostimulation sur la synthèse protéique en SELDI-TOF comme en incorporation de méthionine. Un mécanisme original de SHF peut donc être proposé, impliquant la neutralisation de la synthèse protéique dans l'inactivation réactionnelle des structures neuronale

<p><b>Background and aims:</b> Type 2 diabetes mellitus is connected with elevated glucose levels, which cause impaired glucose-stimulated insulin secretion (GSIS) and degeneration of β-cells. Mechanisms for such glucotoxic effects were explored in the present study.</p><p><b>Materials and methods:</b> INS-1E cells were cultured for 5 days in 5.5, 11, 20 or 27 mM glucose in the presence or absence of AMPK-agonist AICAR. GSIS was determined from INS-1E cells and islets obtained from type 2 diabetes and control donors. Human islets and INS-1E cells were functionally characterized (GSIS) and protein profiled (SELDI-TOF MS). Glucose-induced <i>de novo</i> synthesis of fatty acyls (HR-MAS NMR spectroscopy), fatty acid composition (GC-MS), triglyceride content and specific proteins (Western blotting) were determined in INS-1E cells.</p><p><b>Results:</b> Impaired GSIS was observed from INS-1E cells exposed to chronic hyperglycaemia and islets isolated from type 2 diabetics compared to INS-1E cells cultured at normal glucose levels and control islets, respectively. Several glucose-regulated proteins were found when type 2 diabetes and control islets or mitochondria from INS-1E cells cultured at different glucose concentrations were protein profiled. Glucose induced lipid <i>de novo</i> synthesis of both saturated and unsaturated fatty acids in specific proportions. Glucose-induced impairment of function and mass was reverted by inclusion of AICAR, which lowered levels of pro-apoptotic protein CHOP but left triglyceride content unaffected.</p><p><b>Conclusions:</b> Impaired GSIS and increased apoptosis observed in β-cells after prolonged exposure to elevated glucose concentrations involved accumulation of lipid species in specific proportions, AMPK-inactivation, ER-stress activation and complex, coordinated changes in expression patterns of mitochondrial and human islet proteins.</p>

<b>Background and aims:</b> Type 2 diabetes mellitus is connected with elevated glucose levels, which cause impaired glucose-stimulated insulin secretion (GSIS) and degeneration of β-cells. Mechanisms for such glucotoxic effects were explored in the present study. <b>Materials and methods:</b> INS-1E cells were cultured for 5 days in 5.5, 11, 20 or 27 mM glucose in the presence or absence of AMPK-agonist AICAR. GSIS was determined from INS-1E cells and islets obtained from type 2 diabetes and control donors. Human islets and INS-1E cells were functionally characterized (GSIS) and protein profiled (SELDI-TOF MS). Glucose-induced de novo synthesis of fatty acyls (HR-MAS NMR spectroscopy), fatty acid composition (GC-MS), triglyceride content and specific proteins (Western blotting) were determined in INS-1E cells. <b>Results:</b> Impaired GSIS was observed from INS-1E cells exposed to chronic hyperglycaemia and islets isolated from type 2 diabetics compared to INS-1E cells cultured at normal glucose levels and control islets, respectively. Several glucose-regulated proteins were found when type 2 diabetes and control islets or mitochondria from INS-1E cells cultured at different glucose concentrations were protein profiled. Glucose induced lipid de novo synthesis of both saturated and unsaturated fatty acids in specific proportions. Glucose-induced impairment of function and mass was reverted by inclusion of AICAR, which lowered levels of pro-apoptotic protein CHOP but left triglyceride content unaffected. <b>Conclusions:</b> Impaired GSIS and increased apoptosis observed in β-cells after prolonged exposure to elevated glucose concentrations involved accumulation of lipid species in specific proportions, AMPK-inactivation, ER-stress activation and complex, coordinated changes in expression patterns of mitochondrial and human islet proteins.