The glutamate transporter GLT1/EAAT2 in islets of Langerhans : a key player in the control of β-cell function and integrity in health and disease

Background and aim: The clinical course of Diabetes Mellitus is characterized by a progressive decline in β-cell function and mass. Understanding the causes of β-cell failure and death is of capital importance to develop new and more effective therapeutic strategies. We have shown that glutamate represents a new insult for β-cells. Indeed, chronic exposure to elevated extracellular glutamate concentrations causes apoptosis in clonal β-cell lines and in human islet β-cells, but not in α-cells. In the islet, L-glutamate is released by α-cells with glucagon and it modulates hormone secretion and β-cell viability by acting on ionotropic and metabotropic glutamate receptors. Its extracellular concentration is locally controlled 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 is prevalently localized on β-cell membrane and its normal function is essential to prevent glutamate-induced β-cell death. Given the particular role of GLT1 in β-cell protection, aim of the proposed research was to verify whether chronic hyperglycemia might modulate GLT1 expression and/or its activity in human islets of Langerhans. Material and methods: Human isolated islets were exposed for three days to 5.5 mM (normoglycemia) or 16.7 mM (hyperglycemia) glucose. Transporters expression, localization and function were assessed by RT-PCR, western blotting, indirect immunofluorescence and [3H]-D glutamate uptake. Results: Quantitative PCR analysis showed a 40±3% reduction in the total ASCT2/SLC1A5 expression, after incubation in chronic hyperglycemia. No changes in the total GLT1/SLC1A2 mRNA and protein expression in human islets were found. Immunofluorescence experiments performed on human islets exposed to hyperglycemia revealed GLT1 relocalization into intracellular vesicular compartments of β-cells. 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 hyperglycemia induced a downregulation of the PI3K/Akt pathway in human β-cells (35±3% downregulation of P-Akt expression, n=5 islet preparations), suggesting a possible involvement of this pathway in the modulation of GLT1 trafficking. According to this possibility, PI3K inhibition with 100 μM LY294002 in human islets caused the GLT1 relocalization in intracellular compartments and a 75±8% downregulation of its activity (p<0.001; n=3 different islet isolations, in quadruplicate). Chronic treatment with 10 nM ceftriaxone, a drug able to upregulate GLT1 expression, increases the glutamate transport activity and significantly prevented hyperglycemia-induced apoptosis in human islets (65±12% reduction of apoptosis. P<0.001; n=2 preparations, in quadruplicate). Conclusion: Our data indicate that hyperglycemia alters glutamate signaling in human islets and this may further contribute to β-cell dysfunction and death. Glutamate signalling system components may be promising therapeutic targets to prevent β-cell dysfunction and to regulate glucose homeostasis in diabetes

Mechanotransduction in human islets of Langerhans : implications for β cell fate

Background and aim. The interaction between cells and the extracellular environment plays a pivotal role in tissue differentiation and fate, both in physiological and pathological conditions. Like other cells, β-cell behaviour is strongly influenced by extracellular matrix interactions which are organized at the nanoscale level, but the contribution of nanotopography on β-cell fate has never been explored. Aim of the proposed research was to investigate whether human β-cells can regulate their behaviour in response to nanoscale features and characterize the molecular mechanisms involved. Material and methods. Transition metal oxide nanostructured surfaces were fabricated as substrates to study the effects of nanoscale topography on β-cell behaviour. Human islets were grown on these substrates for 15 days and β-cell function and viability were assessed by measuring insulin secretion and apoptosis. The mechanotransductive signalling complexes were characterized by proteomic analysis and super-resolution imaging techniques. Results. β-cell survival and function were improved on nanostructured substrates as revealed by insulin secretion experiments and TUNEL assays. Proteomic analysis demonstrates that β-cells respond to the substrate nanotopography through the up-regulation of proteins involved in the integrin signalling and actin polymerization. Super-resolution imaging techniques and quantitative immunofluorescence analyses confirmed modifications in cell-substrate adhesion complexes and reorganization of the actin cytoskeleton. Conclusions. Our data reveal that β-cells respond to the microenvironment morphology by activating a mechanotransductive signalling pathway which greatly promotes their survival and function. Characterizing the mechanotransductive signalling involved at the molecular level may offer a unique opportunity for identifying potential therapeutic targets of intervention in diabetes mellitus

Mechanotransduction in human and mouse beta cell lines: reliable models to characterise novel signalling pathways controlling beta cell fate

Background and aim: in recent years, there are evidences suggesting that the interaction between cells and extracellular environment has a pivotal role in tissue differentiation and fate, both in physiological and pathological conditions. Moreover, it has become clear that not only chemical composition, but also physical properties of the extracellular matrix (ECM) affect cell behaviour. The cell ability to respond to changes in their physical environment is known as mechanotransduction and it is prevalently mediated by heterodimeric transmembrane receptors of the family of integrins. Like other tissues, β-cells behaviour is influenced by cell-ECM interaction and there is significant evidence that β-cells depend on extracellular cues to replicate, survive and differentiate. However, the architecture and physical interactions within and surrounding the islets are not completely understood. Thus, aim of the proposed research was to evaluate the mechanotrasductive signalling components in the islets of Langerhans. Material and methods: we used metal oxide layers with tailored nanoscale roughness to fabricate scaffolds that mimic ECM morphology. Isolated human islets grew on these different substrates for 20 days and mechanotrasductive signalling components were evaluated by proteomic analysis; nuclear architecture and focal adhesion were assessed by indirect immunofluorescence. Results: we found that nanostructured substrates cause a relocalization of focal adhesion which may influence protein expression. Proteomic analysis confirmed this hypothesis and showed an up-regulation of focal adhesion molecular components (GO:0005925) and proteins important for actin polymerization (GO:0005856) like TNS1, ARPC2, ARPC1B, DYNLL1, DNAH14, KANK2 protein complexes. The modifications in actin cytoskeleton cause a tension on the nuclear envelope which in turn modulates the program of gene transcription and cellular modelling. Accordingly, we found an up-regulation of the proteins involved in the control of nuclear architecture (GO:0031891) and also a modification of nuclear shape. Conclusion: understanding the mechanotrasductive signalling system may offer a unique possibility to identify molecular or pharmacological targets to prevent and treat diabetes mellitus

Cluster-assembled zirconia substrates promote long-term differentiation and functioning of human islets of Langerhans

Ex vivo expansion and differentiation of human pancreatic ß-cell are enabling steps of paramount importance for accelerating the development of therapies for diabetes. The success of regenerative strategies depends on their ability to reproduce the chemical and biophysical properties of the microenvironment in which ß-cells develop, proliferate and function. In this paper we focus on the biophysical properties of the extracellular environment and exploit the cluster-assembled zirconia substrates with tailored roughness to mimic the nanotopography of the extracellular matrix. We demonstrate that ß-cells can perceive nanoscale features of the substrate and can convert these stimuli into mechanotransductive processes which promote long-term in vitro human islet culture, thus preserving ß-cell differentiation and function. Proteomic and quantitative immunofluorescence analyses demonstrate that the process is driven by nanoscale topography, via remodelling of the actin cytoskeleton and nuclear architecture. These modifications activate a transcriptional program which stimulates an adaptive metabolic glucose response. Engineered cluster-assembled substrates coupled with proteomic approaches may provide a useful strategy for identifying novel molecular targets for treating diabetes mellitus and for enhancing tissue engineering in order to improve the efficacy of islet cell transplantation therapies