MODULATION OF GLUTAMATE TRANSPORTER 1 (GLT1) FUNCTION AND EXPRESSION IN ISLETS OF LANGERHANS: PHYSIOLOGICAL ROLE AND PATHOLOGICAL IMPLICATIONS

Abstract: Glutamate is the main excitatory neurotransmitter of the mammalian nervous system and is involved in neuronal plasticity, memory and learning. Emerging evidences suggest that glutamate is also present in peripheral tissues, where it plays a role in both cellular homeostasis and in autocrine/paracrine communication as an extracellular signalling molecule (Hedigerand Welbourne, 1999; Nedergaard et al., 2002). Particular interesting is the role played by this amino acid molecule in the endocrine pancreas, the pancreas portion responsible for hormones secretion and glucose homeostasis. In this context, glutamate works as intracellular signalling molecules in beta cells to couple changes in metabolic activity with insulin secretion. More recently, an additional role of glutamate as paracrine and autocrine signal involved in the control of hormone release and islet cells viability has been proposed. The glutamate signalling machinery and the precise mechanisms by which glutamate controls islet function are poorly understood. The extracellular glutamate concentration is tightly controlled by high affinity glutamate transporters, whose expression and modulation in the peripheral tissues have been poorly investigated. The main isoform expressed in the islet is the Na+-dependent high affinity glutamate transporter GLT1/EAAT2 (glutamate transporter 1/ excitatory amino acid transporter 2) which is prevalently expressed on the insulin-secreting cell plasma membrane (\u3b2-pancreatic cells). We previously demonstrated that an excess of extracellular glutamate affects the islet viability, and induces selectively \u3b2-cell death. The GLT1 transporter, by transporting glutamate within the cells, exerts a key role in the control of extracellular glutamate concentration and in the preservation of the \u3b2 cell excito-toxicity induced by glutamate (Di Cairano et al., 2011). Given the key role of GLT1 in islet physiology, in this study, we focus on GLT1 and we examine the molecular mechanisms which control its expression and function, using murine clonal \u3b2-cell lines and isolated human islets of Langerhans as experimental models. In chapter I we specifically investigate the intracellular pathways responsible for the acute regulation of the GLT1 localization and function at the \u3b2-cell plasma membrane, where it physiologically works. In chapter II we analyse the modulation of GLT1 expression and activity in condition of chronic hyperglycaemia (a condition typical of diabetes mellitus) and we investigate the consequences of such as modulation on \u3b2-cell viability. Given the anatomical, morphological and structural differences between human and rodent islets, we directly used human isolated islets, the most appropriate model to study the islets pathophysiology. The localization of GLT1 was also assessed on pancreas sections from control and diabetic subjects. In chapter III we highlight the possible pathological role of GLT1 in type 1 of diabetes mellitus (T1DM). We investigate the potential intervention of this transporter in the development of T1DM autoimmunity, as a new membrane antigen of diabetes mellitus. Chapter IV reports two side projects of these three-years of doctoral activity. The first project is related to the characterization of islet remodelling during diabetes, in baboons. This is collaboration with Prof. Folli Franco of Texas University and my role in the project was to characterize the apoptotic cells in islets of Langerhans. The second project is a technical project; the lab is interested in developing imaging techniques and protocols for studying vesicle dynamics. Using the genetically encoded synapto-pHluorin and Total Internal Reflection Fluorescence Microscopy we developed a new macro for the analysis of endocytic and exocytic events at the plasma membrane. This macro could be very useful for future studies related to the effect of glutamate on hormone secretion