PKC-mediated control of glutamate signaling in islets of Langerhans in response to hyperglycaemia

Background and aims: Islets of Langerhans use a sophisticate system of paracrine and autocrine signals to synchronize their activities, among these is glutamate which controls hormone release and β-cell viability, by activating specific glutamate receptors. The extracellular glutamate concentration is shaped by the glutamate transporter GLT1 but the precise mechanisms by which it modulates islet functions are poorly understood. Aim of the proposed research was to verify the impact of acute changes of glucose concentrations on GLT1 localization/function and glutamate signalling in the islet and to verify the molecular mechanisms of this modulation. Materials and methods: mouse βTC3 cells and human islets were incubated under different glucose concentrations (from 3 to 16.7 mmol/l glucose for up to 30 minutes), and the localization and activity of plasma membrane glutamate transporters and intracellular signalling proteins were studied by in vivo imaging, Total internal reflection microscopy (TIRFM) and [3H]-Glutamate uptake. Hormone release was detected by means of ELISA assays. Results: We found that the acute exposure of βTC3 cell lines and human β-cells to high glucose concentrations caused the transporter relocalization in intracellular compartments (assessed by TIRFM). Accordingly, GLT1 transport activity, measured by [3H]D-glutamate uptake, was inhibited by 25±5%. The GLT1 relocalization was prevented by inhibition of PKC activation, a kinase implicated in the control of vesicular trafficking and upregulated by high glucose, indicating that PKC controls the localization and/or activity of this transporter. In line with this possibility, PKC activation by TPA treatment caused a statistically significant reduction in the glutamate uptake. Accordingly, we found that βTC3 pre-treatment with the PKC inhibitor Bisindoleymide abolished the glutamate uptake downregulation induced by 20 mM glucose incubation. Furthermore, no additive effects were observed when βTC3 cells, maintained in 20 mM glucose, were incubated with the PKC activator TPA. Conclusions: In the context of the islet physiology, glutamate is an important paracrine signals that positively modulate the somatostatin secretion. Being GLT1 the main regulator of extracellular glutamate concentration, its relocalization in intracellular compartments would potentiate the activation of glutamate receptors on δ-cells, thus stimulating the somatostatin release. Understanding the molecular mechanisms controlling glutamate release and clearance in islet of Langerhans may be important to control glucose homeostasis in health and disease.