GLUTAMATE TRANSPORTERS IN PANCREAS AND EPITHELIA: PHYSIOLOGICAL ROLES AND DYNAMIC REGULATION

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 [Hediger and Welbourne, 1999; Nedergaard et al., 2002]. The extracellular glutamate concentration is tightly controlled by high affinity glutamate transporters, whose expression and modulation in the peripheral tissues have been poorly investigated. In this work we analyse the high affinity glutamate transporters EAAC1 and GLT1 in epithelia and endocrine pancreas, respectively. EAAC1 was cloned from rabbit intestine [Kanai and Hediger, 1992] and its expression and function have been well characterised in absorptive epithelia, such as intestine and kidney, where it represents the major transporter for the dicarboxylic amino acids [Peghini et al., 1997]. Less is known about the molecular mechanisms that regulate its surface expression and activity. During the past few years, it has become clear that the activity of these transporters can be rapidly regulated by redistribution of proteins to and from the plasma membrane: a process that can be controlled by dynamic protein-protein interactions. Therefore, the research presented in the chapter II focuses on the molecular mechanisms which regulate EAAC1 trafficking in epithelial cells. EAAC1 has a conserved sequence present in the C-terminal domain of EAAC1, that mediates interactions with class I PDZ proteins. In the past years, we demonstrated that the PDZ-target sequence and PDZ proteins are responsible for the retention and stability of EAAC1 at the plasma membrane [D\u2019Amico et al., 2010]. The aim of the present work is to verify whether this PDZ-target sequence is also important for the transporter\u2019s biosynthetic delivery. Our data indicate that PDZ interactions occur early in the biosynthetic pathways and are involved in the ER-to-Golgi trafficking, as well as in Post-Golgi trafficking of EAAC1. Removal of the PDZ motif delays rather than prevents the ER export and the plasma membrane delivery of the transporter, thus indicating that PDZ interactions facilitate the ER-Golgi trafficking. Possibly, PDZ-interactions favour the transporters homo-oligomerization, a process required for the efficient ER export of EAAC1. Alternatively, PDZ domain-proteins may couple EAAC1 with protein complexes required for the efficient fusion of carrier vesicles to the appropriate target membranes. Further studies will be needed to identify the PDZ protein/s involved in the EAAC1 biosynthetic delivery. On the other hand, the presence of glutamate as an intercellular signal mediator in endocrine pancreas is well established [Moriyama and Hayashi, 2003]. In the Central Nervous System (CNS), glutamate may cause cell death by excitotoxicity, that is physiologically prevented by glutamate clearance systems [Choi et al., 1988]. The effect of glutamate on islet viability, the expression of glutamate transporters and their physiological roles in the endocrine pancreas are still unclear. Thus, in the III chapter we examine the effects of glutamate and the function of sodium dependent glutamate transporters in clonal beta-cells and in human isolated islets of Langerhans. We demonstrate that exposure to elevated glutamate concentrations induces a significant cytotoxic effect in pancreatic beta-cells, due to the prolonged activation of ionotropic glutamate receptors. We provide evidence that the key regulator of the extracellular glutamate clearance in the islet is the glial glutamate transporter 1 (GLT1/EAAT2). GLT1 is the only high affinity glutamate transporter expressed in the islets and localizes to the beta-cell plasma membrane. Finally, as diabetes is characterized by selective beta-cell death, and our data indicate that GLT1 controls beta-cell survival, we investigate the expression of GLT1 in type 2 diabetes mellitus (T2DM) patients. We show an altered GLT1 localisation in pancreases from T2DM patients, suggesting a decreased glutamate clearance ability in these subjects. In chapter IV, is reported the experience at University of Texas Health Science Center at San Antonio (UTHSCSA), USA. The aim of this project is to find interactors of IAPP, a protein involved in diabetes, by means of Yeast Two Hybrid Screening, a technique which allows the identification of direct protein-protein interactions. In prospect, this technique will be useful to find proteins that are associated with glutamate transporters and that potentially regulate their expression and function

Pancreas endocrino e metabolismo del colesterolo

Il diabete di tipo 2 comprende un complesso gruppo di disordini metabolici eterogenei caratterizzati da un deficit di secrezione di insulina accompagnato da insulino-resistenza. E\u2019 spesso associato a dislipidemia che contribuisce sia alla progressiva disfunzione beta-pancreatica sia all\u2019aumentato rischio di complicanze cardiovascolari. Queste ultime rappresentano la principale causa di mortalit\ue0 nei soggetti diabetici. Studi condotti in questi anni supportano un ruolo chiave del colesterolo non solo nello sviluppo di complicanze cardiovascolari ma anche direttamente nel controllo della funzionalit\ue0 beta-pancreatica, fondamentale per garantire la secrezione di insulina e l\u2019omeostasi glicemica. Le cellule beta-pancreatiche sono sensibili alle variazioni di colesterolo ed hanno sviluppato un complesso sistema per controllarne omostaticamente la concentrazione intracellulare. Dopo una breve introduzione sul pancreas endocrino e sui meccanismi molecolari che sono alla base del controllo della secrezione di insulina, la rassegna prende in esame il metabolismo del colesterolo nella cellula beta-pancreatica ed esamina l\u2019impatto del lipide sulla funzione e disfunzione di questa cellula. Infine, nell\u2019ultima parte, vengono analizzate le evidenze che legano il metabolismo del colesterolo, alla funzionalit\ue0 beta-pancreatica e al metabolismo glucidico nell\u2019uomo. La conoscenza degli specifici meccanismi molecolari responsabili del metabolismo e della tossicit\ue0 del colesterolo nella cellula beta-pancreatica rappresenta un fondamentale obiettivo della ricerca sperimentale per identificare nuovi target di intervento farmacologico per il trattamento del diabete e delle sue complicanze, possibilmente ancora pi\uf9 efficaci e sicuri di quelle ora disponibili.Type 2 diabetes includes a complex of heterogeneous metabolic disorders characterized by a lack of insulin secretion accompanied by insulin resistance. Dyslipidemia is often associated with both progressive beta-cell dysfunction and increased risk of cardiovascular complications. The latter are the main cause of mortality in diabetic subjects. Studies carried out in recent years support a key role of cholesterol not only in the development of cardiovascular complications, but also directly in the control of beta-pancreatic function, which is fundamental for ensuring insulin secretion and glycemic homeostasis. Beta-pancreatic cells are sensitive to changes in cholesterol and have developed a complex system to control their homeostatic intracellular concentration. After a brief introduction on the endocrine pancreas and the molecular mechanisms that underlie the control of insulin secretion, the review examines the metabolism of cholesterol in the beta-pancreatic cell and examines the impact of lipid on the function and dysfunction of this cell. Finally, in the last part, the evidence linking cholesterol metabolism, beta-pancreatic function and glucose metabolism in humans are analyzed. Knowledge of the specific molecular mechanisms of metabolism and cholesterol toxicity in the beta-pancreatic cell is a fundamental objective of experimental research to identify new targets for pharmacological intervention for the treatment of diabetes and its complications, possibly even more effective and safe than those now available

Plasticità delle cellule endocrine dell’isola del Langerhans di pazienti con diabete di tipo 2

L\u2019isola pancreatica mantiene, anche nell\u2019adulto, una incredibile plasticit\ue0 e capacit\ue0 di modificarsi in risposta ad una crescente richiesta metabolica o dopo un severo danneggiamento delle popolazioni endocrine, come avviene nel diabete di tipo 1 e di tipo 2 (DT2). Particolarmente interessante \ue8 il processo di transdifferenziamento che si osserva tra cellule endocrine pancreatiche. Consiste nella conversione da un tipo cellulare endocrino non-beta verso quello beta, attraverso un processo di riprogrammazione. E\u2019 stato chiaramente identificato in modelli animali di diabete ma la sua presenza nell\u2019uomo e i meccanismi alla base di questo processo devono ancora essere dimostratati. Obiettivo dello studio \ue8 stato quello di valutare modificazione nella composizione e nell\u2019architettura dell\u2019isola in pazienti con diabete di DT2 e di verificare l\u2019esistenza di un possibile processo di transdifferenziamento tra cellule endocrine pancreatiche umane. Lo studio \ue8 stato condotto su sezioni di pancreas di 12 soggetti controllo (7M/5F, et\ue0 69\ub17 anni) e 14 soggetti con DT2 (8M/ 6F; et\ue0 66.4\ub110.34 anni). Le sezioni sono state colorate con anticorpi diretti contro i diversi ormoni e analizzate mediante microscopia confocale e analisi morfometrica. I nostri dati mostrano che l\u2019isola del Langerhans, in pazienti con DT2, va incontro a un rimodellamento caratterizzato da un progressiva riduzione dell\u2019area e della densit\ue0 cellulare dell\u2019isola (riduzione del 27\ub15% e del 15\ub17.9%, rispettivamente. P<0.05) per aumentata apoptosi di cellule beta e delta e amiloidosi. Per quanto riguarda le diverse popolazioni dell\u2019isola abbiamo evidenziato una diminuzione significativa delle cellule beta e delta e un aumento delle cellule co-esprimenti insulina e glucagone (valutata mediante indice di colocalizzazione) suggerendo l\u2019esistenza di un processo di transdifferenziamento tra cellule endocrine dell\u2019isola. L\u2019indice di colocalizzazione correla in modo negativo con l\u2019area delle cellule beta e risulta essere particolarmente elevato in pazienti sotto terapia insulinica, suggerendo una severa disfunzione dell\u2019isola. La comprensione dei meccanismi molecolari alla base di questo processo di transdifferenziamento potrebbe essere di estrema importanza per lo sviluppo di terapie mirate al controllo della progressione del DT2

Exenatide regulates pancreatic islet integrity and insulin sensitivity in the nonhuman primate baboon Papio hamadryas.

The glucagon-like peptide-1 receptor agonist exenatide improves glycemic control by several and not completely understood mechanisms. Herein, we examined the effects of chronic intravenous exenatide infusion on insulin sensitivity, β cell and α cell function and relative volumes, and islet cell apoptosis and replication in nondiabetic nonhuman primates (baboons). At baseline, baboons received a 2-step hyperglycemic clamp followed by an l-arginine bolus (HC/A). After HC/A, baboons underwent a partial pancreatectomy (tail removal) and received a continuous exenatide (n = 12) or saline (n = 12) infusion for 13 weeks. At the end of treatment, HC/A was repeated, and the remnant pancreas (head-body) was harvested. Insulin sensitivity increased dramatically after exenatide treatment and was accompanied by a decrease in insulin and C-peptide secretion, while the insulin secretion/insulin resistance (disposition) index increased by about 2-fold. β, α, and δ cell relative volumes in exenatide-treated baboons were significantly increased compared with saline-treated controls, primarily as the result of increased islet cell replication. Features of cellular stress and secretory dysfunction were present in islets of saline-treated baboons and absent in islets of exenatide-treated baboons. In conclusion, chronic administration of exenatide exerts proliferative and cytoprotective effects on β, α, and δ cells and produces a robust increase in insulin sensitivity in nonhuman primates

Neurosteroid allopregnanolone regulates EAAC1-mediated glutamate uptake and triggers actin changes in Schwann cells

Recent evidence shows that neurotransmitters (e.g. GABA, Ach, adenosine, glutamate) are active on Schwann cells, which form myelin sheaths in the peripheral nervous system under different pathophysiologic conditions. Glutamate, the most important excitatory neurotransmitter, has been recently involved in peripheral neuropathies, thus prevention of its toxic effect is desirable to preserve the integrity of peripheral nervous system and Schwann cells physiology. Removal of glutamate from the extracellular space is accomplished by the high affinity glutamate transporters, so we address our studies to analyze their functional presence in Schwann cells. We first demonstrate that Schwann cells express the EAAC1 transporter in the plasma membrane and in intracellular vesicular compartments of the endocytic recycling pathways. Uptake experiments confirm its presence and functional activity in Schwann cells. Secondly, we demonstrate that the EAAC1 activity can be modulated by exposure to the neurosteroid allopregnanolone 10 nM (a progesterone metabolite proved to support Schwann cells). Transporter up-regulation by allopregnanolone is rapid, does not involve protein neo-synthesis and is prevented by actin depolymerization. Allopregnanolone modulation involves GABA-A receptor and PKC activation, promotes the exocytosis of the EAAC1 transporter from intracellular stores to the Schwann cell membrane, in actin-rich cell tips, and modifies the morphology of cell processes. Finally, we provide evidence that glutamate transporters control the ALLO-mediated effects on cell proliferation. Our findings are the first to demonstrate the presence of a functional glutamate uptake system, which can be dynamically modulated by ALLO in Schwann cells. Glutamate transporters may represent a potential therapeutic target to control Schwann cell physiolog

The neurosteroid allopregnanolone upregulates glutamate transport in Schwann cells by changing the surface density of the EAAC1/EAAT3 glutamate transporter

Glutamate has been recently involved in the proliferation and differentiation of Schwann cells, the myelinating cells of the Peripheral Nervous System. Thus, the control of extracellular glutamate level may be crucial for Schwann cell physiology. We recently observed that functional glutamate transporters were expressed by Schwann cells and modulated by exposure to 10 nM allopregnanolone (ALLO), a progesterone's metabolite. Aim of this study was to identify the molecular mechanisms by which ALLO modulated glutamate uptake. We found that transport up-regulation did not involve protein neo-synthesis and was prevented by actin depolimerization, suggesting a change in the transporters' trafficking. To verify this hypothesis, Schwann cells were transfected with the GFP-tagged EAAC1 transporter and its surface density was measured by Total Internal Reflection Microscopy (TIRFM). ALLO induced the actin cytoskeleton reorganization and caused the EAAC1 redistribution from intracellular compartments to the plasma membrane, at the tips of filopodia structures. Taken together, these data demonstrate that ALLO up-regulates glutamate transport by increasing the delivery of EAAC1 to the cell surface

High affinity glutamate transporters are expressed in Schwann cells and regulated by allopregnanolone

Aim: L-Glutamate, the major excitatory neurotransmitter in the central nervous system (CNS), is implicated in several neurological disorders including peripheral neuropathies (Watkins, 2000). Among therapeutic approaches for treating these diseases, it is considered the block of glutamate receptors with small antagonist molecules. Alternative therapeutic targets are the Na+-dependent high affinity glutamate transporters, the main regulators of extracellular glutamate. Recently, glutamate transporters were identified in vivo in the peripheral nervous system (PNS), whereas their cellular location has not been investigated. Our results give a complete characterization of the glutamate transporters system in the Schwann cell cultures, the myelin forming cells in the PNS. Methods: We observed that Schwann cells expressed EAAC1 and GLAST in the plasma membrane as well as in intracellular vesicular compartments. Results: Uptake experiments confirmed that these transporters were present and functional in Schwann cells and that their activity was modulated by exposure to allopregnanolone 10 nM (progesterone's metabolite). Up-regulation by allopregnanolone did not require protein's neosynthesis and was prevented by actin depolimerization. Conclusion: Studies are in progress in order to verify whether the transport up-regulation relies on allosteric modulation or changes in the surface density of glutamate transporters, via redistribution from intracellular compartments to the plasma membrane

TIRFM and pH-sensitive GFP-probes to evaluate neurotransmitter vesicle dynamics in SH-SY5Y neuroblastoma cells: cell imaging and data analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease. Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (<150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events. In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented. To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling. With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion

The neuroactive steroid allopregnanolone up-regulates EAAC1-mediated glutamate uptake in Schwann cells: a neuroprotective mechanism?

The identification of signals regulating Schwann cell proliferation and differentiation under physiological or pathological conditions is of great importance for the development and regeneration of the peripheral nervous system. A role for the neuroactive steroid allopregnanolone (ALLO), which is synthesized de novo in Schwann cells, and classical neurotransmitters in such a control has been recently proposed. Among these must be considered glutamate which is the major excitatory neurotransmitter in the nervous system. Schwann cells express a variety of glutamate receptors and enzymes involved in the glutamate metabolism. We recently observe that Schwann cells also express the high-affinity glutamate transporter EAAC1 in the plasma membrane and in intracellular vesicular compartments. The transporter is functional and its activity is increased by exposure to 10 nM ALLO. The ALLO effect is rapid, does not involve protein neo-synthesis and is prevented by actin depolymerisation. ALLO modulation involves GABA-A receptors and PKC activation, modifies the morphology of cell processes and causes the relocation of the EAAC1 transporter from intracellular stores to the Schwann cell membrane, in actin-rich cell tips. Finally, we provide evidence that the EAAC1 activity mediates the ALLO effect on Schwann cell proliferation. In conclusion, we propose a mechanism of ALLO action on Schwann cells differentiation and proliferation which involves the GABA-A receptor and the glutamate transporter EAAC1. ALLO induces a dynamic remodeling of the actin cytoskeleton and leads to increased surface activity of the glutamate transporter EAAC1 which represents the principal glutamate clearance system in Schwann cells. Understanding the molecular mechanisms through which ALLO and glutamate control Schwann cell differentiation and proliferation may be of interest to develop new therapeutic approaches for treating peripheral neuropathies and promoting regenerative processe