Background and aims: Increasing evidence suggests that the excitatory neurotransmitter L-glutamate functions as a paracrine/autocrine signal in human islet of Langerhans. L-glutamate is released by α-cells together with glucagon and modulates hormone secretion by acting on specific glutamate receptors. When present at elevated concentrations, it may induce beta-cell cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its interstitial concentration is regulated by glutamate transporters of the SLC1A family which are expressed on the plasma membrane of endocrine cells. Their functional activity is essential for islet function as shown by the fact that their pharmacological inhibition increases glutamate concentration in the islets and causes beta-cell death. Aim of this study was to verify whether chronic hyperglycaemia may modulate the glutamate signalling system in human islets of Langerhans. Materials and methods: Human islets were incubated under chronic (3 days) hyperglycaemia (16.7 mmol/l glucose) or normoglycemia (5.5 mmoll glucose), and the expression and function of plasma membrane glutamate transporters and intracellular signalling proteins were studied by quantitative PCR analysis, western blotting, immunofluorescence, [3H]-Glutamate uptake and Ca2+-imaging experiments. Results: Quantitative PCR analysis revealed a 40±3% reduction in the total ASCT2/SLC1A5 expression after incubation in chronic hyperglycaemia. No changes in the total GLT1/SLC1A2 mRNA and protein expression in human islets were detected. Immunofluorescence experiments performed on human islets exposed to hyperglycaemia revealed GLT1 relocalization into intracellular vesicular compartments. Because of this relocalization, the GLT1-mediated surface activity measured by [3H]D-glutamate uptake was inhibited by 31±5% relative to normoglycemic conditions (p<0.05; n=4 in triplicate). Chronic hyperglycaemia induced a downregulation of the PI3K/Akt pathway in human beta-cells, suggesting a possible involvement of this pathway in the modulation of GLT-1 trafficking (35±3 % downregulation of P-Akt expression, n=5 islet preparations). In line with this possibility, PI3K inhibition with 100 M LY293 caused the GLT1 relocalization in intracellular compartments, and a 75±8% downregulation of its activity, relative to control conditions (p<0.001; n=2 different islet isolations, in quadruplicate). Chronic treatments with 10 nM ceftriaxone a drug capable to up-regulate GLT1 significantly prevented hyperglycaemia-induced apoptosis in human islets (65±12% reduction of apoptosis. P<0.001; n=2 preparations, in quadruplicate). Conclusions: Our data indicate that glutamate signalling in human islet is altered in hyperglycaemia and this may further contribute to beta-cell death. Targeting glutamate signalling system components may be a promising approach to prevent beta-cell death and to control glucose homeostasis in diabetes.

Modulation of glutamate signalling in human islets of Langerhans under hyperglycaemia / C. Perego, S. Moretti, E.S. Di Cairano, F. Daniele, S. La Rosa, F. Bertuzzi, A.M. Davalli, F. Folli. - In: DIABETOLOGIA. - ISSN 1432-0428. - 57:suppl. 1(2014), pp. 462.194-462.194. ((Intervento presentato al 50. convegno EASD annual meeting tenutosi a Wien nel 2014.

Modulation of glutamate signalling in human islets of Langerhans under hyperglycaemia

C. Perego
Primo
;
S. Moretti
Secondo
;
E.S. Di Cairano;A.M. Davalli;F. Folli
2014

Abstract

Background and aims: Increasing evidence suggests that the excitatory neurotransmitter L-glutamate functions as a paracrine/autocrine signal in human islet of Langerhans. L-glutamate is released by α-cells together with glucagon and modulates hormone secretion by acting on specific glutamate receptors. When present at elevated concentrations, it may induce beta-cell cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its interstitial concentration is regulated by glutamate transporters of the SLC1A family which are expressed on the plasma membrane of endocrine cells. Their functional activity is essential for islet function as shown by the fact that their pharmacological inhibition increases glutamate concentration in the islets and causes beta-cell death. Aim of this study was to verify whether chronic hyperglycaemia may modulate the glutamate signalling system in human islets of Langerhans. Materials and methods: Human islets were incubated under chronic (3 days) hyperglycaemia (16.7 mmol/l glucose) or normoglycemia (5.5 mmoll glucose), and the expression and function of plasma membrane glutamate transporters and intracellular signalling proteins were studied by quantitative PCR analysis, western blotting, immunofluorescence, [3H]-Glutamate uptake and Ca2+-imaging experiments. Results: Quantitative PCR analysis revealed a 40±3% reduction in the total ASCT2/SLC1A5 expression after incubation in chronic hyperglycaemia. No changes in the total GLT1/SLC1A2 mRNA and protein expression in human islets were detected. Immunofluorescence experiments performed on human islets exposed to hyperglycaemia revealed GLT1 relocalization into intracellular vesicular compartments. Because of this relocalization, the GLT1-mediated surface activity measured by [3H]D-glutamate uptake was inhibited by 31±5% relative to normoglycemic conditions (p<0.05; n=4 in triplicate). Chronic hyperglycaemia induced a downregulation of the PI3K/Akt pathway in human beta-cells, suggesting a possible involvement of this pathway in the modulation of GLT-1 trafficking (35±3 % downregulation of P-Akt expression, n=5 islet preparations). In line with this possibility, PI3K inhibition with 100 M LY293 caused the GLT1 relocalization in intracellular compartments, and a 75±8% downregulation of its activity, relative to control conditions (p<0.001; n=2 different islet isolations, in quadruplicate). Chronic treatments with 10 nM ceftriaxone a drug capable to up-regulate GLT1 significantly prevented hyperglycaemia-induced apoptosis in human islets (65±12% reduction of apoptosis. P<0.001; n=2 preparations, in quadruplicate). Conclusions: Our data indicate that glutamate signalling in human islet is altered in hyperglycaemia and this may further contribute to beta-cell death. Targeting glutamate signalling system components may be a promising approach to prevent beta-cell death and to control glucose homeostasis in diabetes.
islet of Langerhans; glutamate; diabetes; GLT1; PI3K/Akt
Settore BIO/09 - Fisiologia
Settore MED/13 - Endocrinologia
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/244578
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