BACKGROUND and AIMS. Glutamate is the major excitatory neurotrasmitter in the Central Nervous System but it also present in non-neuronal tissues, as in endocrine cells of the pancreas. Our previous work demonstrated that glutamate is secreted by α-cells and acts as an important signalling molecule to modulate hormone secretion and control β-cell integrity and function (1, 2). Indeed, chronic exposure to glutamate exerts a cytotoxic effect on clonal and human β-cells, but not on α-cells. The extra-cellular glutamate level is maintained under physiological concentrations by high affinity glutamate transporters of the solute carrier 1 family (SLC1), in particular by GLT1/EAAT2 (glutamate transporter 1/excitatory amino acid transporters 2). GLT1 prevalently localized on β-cell’s membrane and its normal function is essential to prevent glutamate-induced β–cell death. Given the particular GLT1 localization and its role in β-cell protection, aim of the proposed research was to verify whether the GLT1 activity and localization may change in diabetic conditions, in particular in the presence of high levels of glucose (hyperglicemia). MATERIALS and METHODS: Western blotting techniques, uptake of radiolabelled glutamate ([3H]D-glutamate), total internal reflection fluorescence microscopy (TIRFM), and immunofluorescence assays were used to analyzed the expression, activity and localization of GLT1 in βTC3 cells, a mouse β-cell line, and in human islet of Langerhans. RESULT: We found that exposure of human and clonal β-cells to elevated glucose concentrations (16.7 mM glucose) did not significantly affect the total GLT1 expression (95±4% of 5.5 mM glucose) but caused the transporter relocalization in intracellular compartments. Accordingly, GLT1 transport activity measured by [3H]D-glutamate uptake was inhibited by 25±5% as compared to 5.5 mM glucose (P<0.01). These results were confirmed by total internal reflection microscopy performed on β-TC3 cells transfected with the GFP-tagged GLT1 transporter. Exposure to elevated glucose concentrations resulted in the progressive e rapid disappearance of GLT1 from the cell surface and its concomitant internalization in intracellular vesicular compartments. A similar relocalization was observed when the cells was treated with LY294, an inhibitor of PI3K, a kinase implicated in the control of vesicular trafficking, and severely downregulated in diabetes mellitus (3) CONCLUSION: These results indicate that abnormal function of the glutamate transporter GLT1 is an early event in diabetes mellitus pathogenesis and it may contribute significantly to β-cell loss. Understanding the factors and the molecular mechanism that control GLT1 in islet of Langerhans may be important to control β-cell function and integrity in health and disease.

The glutamate transporter GLT1 in Islets of Langerhans: localization and function / S. Moretti, E.S. Di Cairano, D. Mengacci, A. Davalli, F. Bertuzzi, F. Folli, C. Perego. ((Intervento presentato al 7. convegno Annual Meeting of Young Researchers tenutosi a Anacapri nel 2013.

The glutamate transporter GLT1 in Islets of Langerhans: localization and function

S. Moretti
Primo
;
E.S. Di Cairano
Secondo
;
A. Davalli
Penultimo
;
F. Folli;C. Perego
2013-05

Abstract

BACKGROUND and AIMS. Glutamate is the major excitatory neurotrasmitter in the Central Nervous System but it also present in non-neuronal tissues, as in endocrine cells of the pancreas. Our previous work demonstrated that glutamate is secreted by α-cells and acts as an important signalling molecule to modulate hormone secretion and control β-cell integrity and function (1, 2). Indeed, chronic exposure to glutamate exerts a cytotoxic effect on clonal and human β-cells, but not on α-cells. The extra-cellular glutamate level is maintained under physiological concentrations by high affinity glutamate transporters of the solute carrier 1 family (SLC1), in particular by GLT1/EAAT2 (glutamate transporter 1/excitatory amino acid transporters 2). GLT1 prevalently localized on β-cell’s membrane and its normal function is essential to prevent glutamate-induced β–cell death. Given the particular GLT1 localization and its role in β-cell protection, aim of the proposed research was to verify whether the GLT1 activity and localization may change in diabetic conditions, in particular in the presence of high levels of glucose (hyperglicemia). MATERIALS and METHODS: Western blotting techniques, uptake of radiolabelled glutamate ([3H]D-glutamate), total internal reflection fluorescence microscopy (TIRFM), and immunofluorescence assays were used to analyzed the expression, activity and localization of GLT1 in βTC3 cells, a mouse β-cell line, and in human islet of Langerhans. RESULT: We found that exposure of human and clonal β-cells to elevated glucose concentrations (16.7 mM glucose) did not significantly affect the total GLT1 expression (95±4% of 5.5 mM glucose) but caused the transporter relocalization in intracellular compartments. Accordingly, GLT1 transport activity measured by [3H]D-glutamate uptake was inhibited by 25±5% as compared to 5.5 mM glucose (P<0.01). These results were confirmed by total internal reflection microscopy performed on β-TC3 cells transfected with the GFP-tagged GLT1 transporter. Exposure to elevated glucose concentrations resulted in the progressive e rapid disappearance of GLT1 from the cell surface and its concomitant internalization in intracellular vesicular compartments. A similar relocalization was observed when the cells was treated with LY294, an inhibitor of PI3K, a kinase implicated in the control of vesicular trafficking, and severely downregulated in diabetes mellitus (3) CONCLUSION: These results indicate that abnormal function of the glutamate transporter GLT1 is an early event in diabetes mellitus pathogenesis and it may contribute significantly to β-cell loss. Understanding the factors and the molecular mechanism that control GLT1 in islet of Langerhans may be important to control β-cell function and integrity in health and disease.
Glutamate ; GLT1 transporter ; islet of Langerhans ; diabetes
Settore BIO/09 - Fisiologia
Settore MED/13 - Endocrinologia
Società italiana di fisiologia
The glutamate transporter GLT1 in Islets of Langerhans: localization and function / S. Moretti, E.S. Di Cairano, D. Mengacci, A. Davalli, F. Bertuzzi, F. Folli, C. Perego. ((Intervento presentato al 7. convegno Annual Meeting of Young Researchers tenutosi a Anacapri nel 2013.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/233710
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