We recently observed that Schwann cells, the myelin-forming cells in the peripheral nervous syste... more We recently observed that Schwann cells, the myelin-forming cells in the peripheral nervous system, express the high-affinity glutamate transporter EAAC1/EAAT3 in the plasma membrane and in intracellular vesicular compartments in vitro and in vivo [1]. The ...
Background and aims: Increasing evidence suggests that the excitatory neurotransmitter L-glutamat... more 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 \u3b1-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\ub13% 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\ub15% 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\ub13 % downregulation of P-Akt expression, n=5 islet preparations). In line with this possibility, PI3K inhibition with 100 \uf06dM LY293 caused the GLT1 relocalization in intracellular compartments, and a 75\ub18% 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\ub112% 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
Pancreatic β-cells, by secreting insulin, play a key role in the control of glucose homeostasis, ... more Pancreatic β-cells, by secreting insulin, play a key role in the control of glucose homeostasis, and their dysfunction is the basis of diabetes development. The metabolic milieu created by high blood glucose and lipids is known to play a role in this process. In the last decades, cholesterol has attracted significant attention, not only because it critically controls β-cell function but also because it is the target of lipid-lowering therapies proposed for preventing the cardiovascular complications in diabetes. Despite the remarkable progress, understanding the molecular mechanisms responsible for cholesterol-mediated β-cell function remains an open and attractive area of investigation. Studies indicate that β-cells not only regulate the total cholesterol level but also its redistribution within organelles, a process mediated by vesicular and non-vesicular transport. The aim of this review is to summarize the most current view of how cholesterol homeostasis is maintained in pancrea...
Background and aims: Studies in the last years indicate that the adult pancreas has an inherent r... more Background and aims: Studies in the last years indicate that the adult pancreas has an inherent regenerative capacity to produce new \u3b2-cells in response to increased metabolic demand or after injury. Different mechanisms of beta cell regeneration have been proposed to occur in the adult pancreas: i) replication from endogenous \u3b2-cell, ii) neogenesis from non-\u3b2-cells precursors and iii) \u3b2-cell formation by a transdifferentiation program. This last mechanism consists in the conversion of one endocrine cell type into another (non-beta cells into beta) by lineage reprogramming. It has been clearly identified in animal models; its existence and relevance in humans has never been demonstrated. Aim of this study was to examine the islet composition and architecture in control and diabetic subjects and to verify the existence of the transdifferentiation process in diabetic islets. Materials and methods: we collected human pancreatic sections from 12 normal (7M/5F, mean age 6...
Background and aims: Congenital hyperinsulinism (CHI) is a rare disorder (OMIM#256450), character... more Background and aims: Congenital hyperinsulinism (CHI) is a rare disorder (OMIM#256450), characterized by hypoglycaemia due to inappropriate insulin secretion. A Whole Exome Sequencing (WES) analysis performed on CHI patients lacking mutations in ABCC8/KCNJ11 identified a polimorfism in the CDKAL transcript (S561F-CDKAL1 variant). CDKAL is a methylthiotransferase that modifies tRNA(Lys) to enhance translational fidelity of transcripts, including the one encoding proinsulin. Interestingly, CDKAL is a susceptibility genes for type 2 diabetes and CDKAL knock-out (cdkal1 -/-) mice showed impaired glucose homeostasis, thus indicating the protein involvement in beta-cell function. Aim of this work was to understand the impact of the CDKAL1 variant S561F on the insulin content, trafficking and release in pancreatic beta cells. Materials and methods: Clonal INS1-E cells expressing Wild Type (WT) or S561F CDKAL1 were generated and used as a model to characterize S561F-CDKAL1 impact on beta cell function. The localization of the variant protein was monitored by immunofluorescence and insulin content and release were measured with ELISA test. An acridine orange assay was performed to evaluate the constitutive and regulated trafficking and possible alterations in vesicle protein expression were evaluated by western blotting. Results: Wild type CDKAL1 overexpressed in INS1-E cells localized in the reticular compartment. The S561F variant was similarly confined to the reticular compartment, although its localization was enriched in spot-like structures distributed in the perinuclear region. Insulin content was increased by overexpression of WT CDKAL1 (2 fold over INS1E, p&lt;0.05) while it was decreased by S561F-CDKAL1 variant expression. Conversely, insulin release measured in overnight culture medium or in 30 minutes static incubation in normal glucose concentrations was increased in the S561F-CDKAL1 as compared to WT clones (2 to 4 folds increase over WT; p&lt;0.05), thus suggesting a different insulin processing/secretion in the mutant CDKAL1. An acridina orange assay performed to measure the constitutive and regulated trafficking in INS1E cells revealed more basal exocytosis in S561F-CDKAL1 then WT clones. Interestingly, the basal release was not further increased by potassium chloride or high glucose. Western blotting experiments revealed up-regulation of proteins involved in the secretion machinery in mutant clones compared to WT. Conclusion: The S561F-CDKAL1 variant expression leads to an increased basal insulin release in INS1E cell line. Such an increase is associated to a defect in the vesicle trafficking and correlates with altered expression of proteins involved in the secretory machinery , further studies are needed to clarify molecular mechanisms linking CDKAL to insulin processing and membrane trafficking. Our findings confirm the importance of CDKAL1 in insulin release and suggest a possible mechanism by which this variant can participate to the development of congenital hyperinsulinism
Background and aims: Congenital Hyperinsulinism (CHI) is a rare disorder, characterized by hypogl... more Background and aims: Congenital Hyperinsulinism (CHI) is a rare disorder, characterized by hypoglycemia due to inappropriate insulin release from pancreatic \u3b2-cells. Despite the advances in understanding the molecular pathogenesis of CHI, specific genetic determinants in about 50 % of the CHI patients remain unknown. A whole-exome sequencing analysis performed on 17 CHI patients lacking mutations in ABCC8/KCNJ11 identified a polymorphism in the CDKAL transcript (S561F-CDKAL1 variant). CDKAL1 is a methylthiotransferase that modifies cytoplasmic tRNALys to enhance translational fidelity of transcripts. Interestingly, CDKAL1 is a susceptibility gene for type 2 diabetes and CDKA1 knock-out mice showed impaired glucose homeostasis, thus indicating the protein involvement in \u3b2-cell function. Since a lysine residue is located at the cleavage site for proinsulin to insulin processing, aim of this work was to investigate the impact of the CDKAL1 variant S561F on the insulin/proinsulin content and the formation of mature hormone granules. Materials and methods: INS1-E clones expressing Wild Type (WT) or S561F CDKAL1 were generated and used as a model to characterize the S561F CDKAL1 impact on beta-cell function. Western Blot experiments and ELISA assays were performed in order to evaluate the expression, processing and release of insulin/proinsulin in the different INS-1E clones. The distribution of insulin granules in clones was monitored by indirect immunofluorescence and total internal fluorescence reflection microscopy (TIRFM). Results: The insulin content in clones overexpressing the S561F-CDKAL1 variant was decreased as compared to WT clones (2 folds increase over WT, p&lt;0.05). Conversely, insulin release measured in overnight culture medium or in 30 minutes static incubation, in normal glucose concentrations, was significantly increased in the S561F-CDKAL1 as compared to WT clones (2 to 4 folds increase over WT; p&lt;0.05), thus suggesting a different insulin processing and/or secretion in the mutant CDKAL1. In line with these results, we found a higher proinsulin content in INS-1E-S561F clones than in CDKAL1 WT. Data were confirmed by western blotting analysis. Differences between the clones were highlighted by the proinsulin/insulin ratio which was significantly higher (; p&lt;0.05) in mutant than in WT clones. Immunofluorescence experiments showed an abnormal enrichment of insulin-positive granules in the perinuclear region, positive for Golgi markers, and a decreased granules density in the TIRF zone in mutant clones compared to WT CDKAL1, thus suggesting that alterations in proinsulin-insulin processing probably impact on insulin granules formation and secretio
BACKGROUND and AIMS. Glutamate is the major excitatory neurotrasmitter in the Central Nervous Sys... more 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 \u3b1-cells and acts as an important signalling molecule to modulate hormone secretion and control \u3b2-cell integrity and function (1, 2). Indeed, chronic exposure to glutamate exerts a cytotoxic effect on clonal and human \u3b2-cells, but not on \u3b1-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 \u3b2-cell\u2019s membrane and its normal function is essential to prevent glutamate-induced \u3b2\u2013cell death. Given the particular GLT1 localization and its role in \u3b2-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 \u3b2TC3 cells, a mouse \u3b2-cell line, and in human islet of Langerhans. RESULT: We found that exposure of human and clonal \u3b2-cells to elevated glucose concentrations (16.7 mM glucose) did not significantly affect the total GLT1 expression (95\ub14% 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\ub15% as compared to 5.5 mM glucose (P<0.01). These results were confirmed by total internal reflection microscopy performed on \u3b2-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 \u3b2-cell loss. Understanding the factors and the molecular mechanism that control GLT1 in islet of Langerhans may be important to control \u3b2-cell function and integrity in health and disease
We recently observed that Schwann cells, the myelin-forming cells in the peripheral nervous syste... more We recently observed that Schwann cells, the myelin-forming cells in the peripheral nervous system, express the high-affinity glutamate transporter EAAC1/EAAT3 in the plasma membrane and in intracellular vesicular compartments in vitro and in vivo [1]. The ...
Background and aims: Increasing evidence suggests that the excitatory neurotransmitter L-glutamat... more 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 \u3b1-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\ub13% 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\ub15% 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\ub13 % downregulation of P-Akt expression, n=5 islet preparations). In line with this possibility, PI3K inhibition with 100 \uf06dM LY293 caused the GLT1 relocalization in intracellular compartments, and a 75\ub18% 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\ub112% 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
Pancreatic β-cells, by secreting insulin, play a key role in the control of glucose homeostasis, ... more Pancreatic β-cells, by secreting insulin, play a key role in the control of glucose homeostasis, and their dysfunction is the basis of diabetes development. The metabolic milieu created by high blood glucose and lipids is known to play a role in this process. In the last decades, cholesterol has attracted significant attention, not only because it critically controls β-cell function but also because it is the target of lipid-lowering therapies proposed for preventing the cardiovascular complications in diabetes. Despite the remarkable progress, understanding the molecular mechanisms responsible for cholesterol-mediated β-cell function remains an open and attractive area of investigation. Studies indicate that β-cells not only regulate the total cholesterol level but also its redistribution within organelles, a process mediated by vesicular and non-vesicular transport. The aim of this review is to summarize the most current view of how cholesterol homeostasis is maintained in pancrea...
Background and aims: Studies in the last years indicate that the adult pancreas has an inherent r... more Background and aims: Studies in the last years indicate that the adult pancreas has an inherent regenerative capacity to produce new \u3b2-cells in response to increased metabolic demand or after injury. Different mechanisms of beta cell regeneration have been proposed to occur in the adult pancreas: i) replication from endogenous \u3b2-cell, ii) neogenesis from non-\u3b2-cells precursors and iii) \u3b2-cell formation by a transdifferentiation program. This last mechanism consists in the conversion of one endocrine cell type into another (non-beta cells into beta) by lineage reprogramming. It has been clearly identified in animal models; its existence and relevance in humans has never been demonstrated. Aim of this study was to examine the islet composition and architecture in control and diabetic subjects and to verify the existence of the transdifferentiation process in diabetic islets. Materials and methods: we collected human pancreatic sections from 12 normal (7M/5F, mean age 6...
Background and aims: Congenital hyperinsulinism (CHI) is a rare disorder (OMIM#256450), character... more Background and aims: Congenital hyperinsulinism (CHI) is a rare disorder (OMIM#256450), characterized by hypoglycaemia due to inappropriate insulin secretion. A Whole Exome Sequencing (WES) analysis performed on CHI patients lacking mutations in ABCC8/KCNJ11 identified a polimorfism in the CDKAL transcript (S561F-CDKAL1 variant). CDKAL is a methylthiotransferase that modifies tRNA(Lys) to enhance translational fidelity of transcripts, including the one encoding proinsulin. Interestingly, CDKAL is a susceptibility genes for type 2 diabetes and CDKAL knock-out (cdkal1 -/-) mice showed impaired glucose homeostasis, thus indicating the protein involvement in beta-cell function. Aim of this work was to understand the impact of the CDKAL1 variant S561F on the insulin content, trafficking and release in pancreatic beta cells. Materials and methods: Clonal INS1-E cells expressing Wild Type (WT) or S561F CDKAL1 were generated and used as a model to characterize S561F-CDKAL1 impact on beta cell function. The localization of the variant protein was monitored by immunofluorescence and insulin content and release were measured with ELISA test. An acridine orange assay was performed to evaluate the constitutive and regulated trafficking and possible alterations in vesicle protein expression were evaluated by western blotting. Results: Wild type CDKAL1 overexpressed in INS1-E cells localized in the reticular compartment. The S561F variant was similarly confined to the reticular compartment, although its localization was enriched in spot-like structures distributed in the perinuclear region. Insulin content was increased by overexpression of WT CDKAL1 (2 fold over INS1E, p&lt;0.05) while it was decreased by S561F-CDKAL1 variant expression. Conversely, insulin release measured in overnight culture medium or in 30 minutes static incubation in normal glucose concentrations was increased in the S561F-CDKAL1 as compared to WT clones (2 to 4 folds increase over WT; p&lt;0.05), thus suggesting a different insulin processing/secretion in the mutant CDKAL1. An acridina orange assay performed to measure the constitutive and regulated trafficking in INS1E cells revealed more basal exocytosis in S561F-CDKAL1 then WT clones. Interestingly, the basal release was not further increased by potassium chloride or high glucose. Western blotting experiments revealed up-regulation of proteins involved in the secretion machinery in mutant clones compared to WT. Conclusion: The S561F-CDKAL1 variant expression leads to an increased basal insulin release in INS1E cell line. Such an increase is associated to a defect in the vesicle trafficking and correlates with altered expression of proteins involved in the secretory machinery , further studies are needed to clarify molecular mechanisms linking CDKAL to insulin processing and membrane trafficking. Our findings confirm the importance of CDKAL1 in insulin release and suggest a possible mechanism by which this variant can participate to the development of congenital hyperinsulinism
Background and aims: Congenital Hyperinsulinism (CHI) is a rare disorder, characterized by hypogl... more Background and aims: Congenital Hyperinsulinism (CHI) is a rare disorder, characterized by hypoglycemia due to inappropriate insulin release from pancreatic \u3b2-cells. Despite the advances in understanding the molecular pathogenesis of CHI, specific genetic determinants in about 50 % of the CHI patients remain unknown. A whole-exome sequencing analysis performed on 17 CHI patients lacking mutations in ABCC8/KCNJ11 identified a polymorphism in the CDKAL transcript (S561F-CDKAL1 variant). CDKAL1 is a methylthiotransferase that modifies cytoplasmic tRNALys to enhance translational fidelity of transcripts. Interestingly, CDKAL1 is a susceptibility gene for type 2 diabetes and CDKA1 knock-out mice showed impaired glucose homeostasis, thus indicating the protein involvement in \u3b2-cell function. Since a lysine residue is located at the cleavage site for proinsulin to insulin processing, aim of this work was to investigate the impact of the CDKAL1 variant S561F on the insulin/proinsulin content and the formation of mature hormone granules. Materials and methods: INS1-E clones expressing Wild Type (WT) or S561F CDKAL1 were generated and used as a model to characterize the S561F CDKAL1 impact on beta-cell function. Western Blot experiments and ELISA assays were performed in order to evaluate the expression, processing and release of insulin/proinsulin in the different INS-1E clones. The distribution of insulin granules in clones was monitored by indirect immunofluorescence and total internal fluorescence reflection microscopy (TIRFM). Results: The insulin content in clones overexpressing the S561F-CDKAL1 variant was decreased as compared to WT clones (2 folds increase over WT, p&lt;0.05). Conversely, insulin release measured in overnight culture medium or in 30 minutes static incubation, in normal glucose concentrations, was significantly increased in the S561F-CDKAL1 as compared to WT clones (2 to 4 folds increase over WT; p&lt;0.05), thus suggesting a different insulin processing and/or secretion in the mutant CDKAL1. In line with these results, we found a higher proinsulin content in INS-1E-S561F clones than in CDKAL1 WT. Data were confirmed by western blotting analysis. Differences between the clones were highlighted by the proinsulin/insulin ratio which was significantly higher (; p&lt;0.05) in mutant than in WT clones. Immunofluorescence experiments showed an abnormal enrichment of insulin-positive granules in the perinuclear region, positive for Golgi markers, and a decreased granules density in the TIRF zone in mutant clones compared to WT CDKAL1, thus suggesting that alterations in proinsulin-insulin processing probably impact on insulin granules formation and secretio
BACKGROUND and AIMS. Glutamate is the major excitatory neurotrasmitter in the Central Nervous Sys... more 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 \u3b1-cells and acts as an important signalling molecule to modulate hormone secretion and control \u3b2-cell integrity and function (1, 2). Indeed, chronic exposure to glutamate exerts a cytotoxic effect on clonal and human \u3b2-cells, but not on \u3b1-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 \u3b2-cell\u2019s membrane and its normal function is essential to prevent glutamate-induced \u3b2\u2013cell death. Given the particular GLT1 localization and its role in \u3b2-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 \u3b2TC3 cells, a mouse \u3b2-cell line, and in human islet of Langerhans. RESULT: We found that exposure of human and clonal \u3b2-cells to elevated glucose concentrations (16.7 mM glucose) did not significantly affect the total GLT1 expression (95\ub14% 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\ub15% as compared to 5.5 mM glucose (P<0.01). These results were confirmed by total internal reflection microscopy performed on \u3b2-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 \u3b2-cell loss. Understanding the factors and the molecular mechanism that control GLT1 in islet of Langerhans may be important to control \u3b2-cell function and integrity in health and disease
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