The Glutamate transporter GLT1/EAAT2: a promising target to arrest β-cell dysfunction and death in diabetes mellitus

Background and aims: The clinical course of Type 2 Diabetes mellitus is characterized by a progressive decline in beta-cell mass and function. beta-cell overstimulation, gluco-, lipotoxicity and oxidative stress are recognized causes of beta-cell death, the discovery of new factors that might harm the beta-cell would increase our knowledge of diabetes mellitus pathogenesis and the chance to design and implement effective treatments. We have recently shown that glutamate, an important signalling molecule in pancreatic islets, also controls beta-cell integrity. Indeed, chronic exposure to elevated extracellular glutamate concentrations causes apoptosis in clonal beta-cell lines and in human islet beta-cells, but not in alpha-cells. Of note, increased glutamate levels have been found in sera of T2D subjects, thus suggesting that abnormal glutamate signalling may play a role in diabetes onset or progression. Our previous work demonstrates that the glutamate transporter GLT1 is a key regulator of glutamate clearance in the islet and in the control of beta-cell integrity and a candidate target for pharmacological intervention. Aim of the proposed research is to verify whether GLT1 dysfunction is an early event in the pathogenesis of T2DM Materials and methods: The localization of GLT1 in human pancreases from 5 healthy subjects (age: 69\ub17 yers; M/F:3/2) and 8 T2DM patients (DMT2 age: 67\ub18 years; M/F:5/3) was analysed by immunohistochemistry. The molecular mechanisms responsible for GLT1 alterations were investigated in human islet preparations exposed to metabolic insults (chronic hyperglycaemia 16.7 mM glucose; pro-inflammatory cytokines TNFalpha, ILbeta, INgamma) by means of western blotting, uptake and immunofluorescence experiments. Results: In healthy control pancreases GLT1 staining was detected exclusively in the islet, and confined to the plasma membrane of beta-cells. In contrast, in 5 of 8 T2DM pancreases GLT1 staining was mainly detected in intracellular compartments. No relationship were found with disease duration or pharmacological treatments, interestingly the degree of GLT1 internalization positively correlated with the severity of amyloid deposition in the islets. Under hyperglycaemic conditions, the total GLT1 expression in human islets was unchanged, but the transporter internalized into intracellular vesicular compartments. Because of this relocalization, the GLT1 surface activity measured by means of [3H]D-glutamate uptake was inhibited by 25\ub15% relative to normoglycemic conditions (p<0.05; n=2 in triplicate). Conversely, three days incubation with pro-inflammatory cytokines caused a significant reduction of GLT1 expression (45\ub111% reduction relative to untreated islets, p