Glutamate (GLUT) is the major excitatory neurotransmitter of the central nervous system and may induce cytotoxicity through activation of glutamate receptors and oxidative stress. Its extracellular concentration is regulated by high affinity glutamate transporters including the glial glutamate transporter GLT-1. GLUT is co-secreted with glucagon by the alpha-cells in islets , acting as a signalling molecule and hormone secretion modulator. We investigated whether GLUT plays a role on islet cells viability and the role of GLT-1 in this process. We used mouse betaTC3, alphaTC1 cell lines and human islets of Langerhans to test whether the effects of an acute and chronic exposure to GLUT exerts a cytotoxic effect in alpha and beta cells. Cells were grown for 24 h and then exposed to either GLUT, dihydrokainate (DHK) a selective GLT-1 inhibitor, Ceftriaxone (CEF) or GLUT receptor inhibitors (APV and CNQX) for 5 days. Viability was measured by the MTT and apoptosis by TUNEL. GLT1 expression and content in betaTC3 cells and human islets was tested by RT-PCR, Western Blotting and confocal microscopy. 5 days incubation with 0.05, 0.5 and 5 mM GLUT increased betaTC3 apoptosis by in a dose-dependent fashion up to 53% (p<0.01) as compared to only 20% at 5mM in alphaTC1 cells (p<0.05). GLT1 was expressed in betaTC3 cells while no expression was found in alphaTC1. These data were confirmed by Na-dependent [3H]-D-aspartate uptake in betaTC3 cells, that was inhibited by DHK. GLT-1-shRNA induced down-regulation of 35%, also caused a 2-4 fold increase in apoptosis in betaTC3 incubated in 0.5mM GLUT (p<0.001). In contrast, CEF induced 2 fold increased of GLT1 expression and provided dose dependent protection from glutamate-induced toxicity (p<0.05). GLT1 was expressed exclusively in the cell membrane in the beta cells in human pancreas, as demonstrated by double-confocal immunocytochemistry with GLT-1and insulin antibodies. GLT-1 inhibition with DHK inhibited Na-dependent [3H]-D-aspartate uptake by 60% in human islets. 5 mM GLUT caused a 80% increase and 0.1 mM DHK caused a 60% in human islets' apoptosis and 50% reduction in 16.7mM glucose-stimulated insulin release and 70% increase in the proinsulin release (all p<0.05). Quantitation by electron-, immunoelectron and double confocal microscopy of apoptotic/degenerated confirmed that apoptosis occured exclusively in beta-cells. GLT1 is a new player in glutamate homeostasis in the islet of Langerhans, controlling extracellular glutamate levels and beta-cells integrity.
The Glial Glutamate Transporter 1 (GLT1) is Expressed by Pancreatic beta-Cells and Prevents Glutamate-Induced beta-Cell Death / E.S. Di Cairano, A.M. Davalli, L. Perego, S. Sala, V.F. Sacchi, S. La Rosa, C. Placidi, C. Cappella, P. Conti, V.E. Centonze, F. Casiraghi, F. Bertuzzi, F. Folli, C. Perego. - In: ENDOCRINE REVIEWS. - ISSN 0163-769X. - 32:Supplemnt 1(2011 Jun). ((Intervento presentato al 93. convegno ENDO tenutosi a Boston nel 2011.
|Titolo:||The Glial Glutamate Transporter 1 (GLT1) is Expressed by Pancreatic beta-Cells and Prevents Glutamate-Induced beta-Cell Death|
|Parole Chiave:||glutamate transporter ; glutamate ; islet of Langerhans ; beta-cells ; diabetes|
|Settore Scientifico Disciplinare:||Settore BIO/09 - Fisiologia|
|Data di pubblicazione:||giu-2011|
|Enti collegati al convegno:||Endocrine Society|
|Appare nelle tipologie:||01 - Articolo su periodico|