Investigation of glutamate receptors in human pancreatic β-cells and their role in insulin secretion

Caygill, Claire

Abstract

Background and Aims: β-cells share many similarities and features with neuronal cells, including the expression of proteins specialised for synaptic transmission. Glutamate receptors (GluRs) are key players in cellular communication throughout the central nervous system (CNS) and are activated by the major excitatory neurotransmitter glutamate. GluRs are also thought to be present in pancreatic β-cells, where they modulate insulin secretion, however there are many conflicting reports over the presence and function of GluRs particularly in human β-cells. Furthermore, autoantibodies to various GluRs have been associated with diseases of the CNS such as autoimmune encephalitis and epilepsy. It is unclear whether these receptors are also targets on β-cells in patients with Type 1 Diabetes (T1D). The aims of this project were (i) to identify GluRs, specifically Kainate receptor (KAR) subunits, expressed in the human β-cell line, EndoC- βh1; (ii) to elucidate their role in insulin secretion and (iii) to identify if GluR subunits on the β-cell surface are targeted by the immune system in T1D.
Materials and Methods: GluR expression was investigated in EndoC-βh1 human β-cells, both at the mRNA and protein levels using RT-PCR and immunoblotting, respectively. To measure the cell surface targeting of KARs in response to glucose stimulation, biotinylation experiments were carried out using EndoC- βh1 cells. Autoantibodies to KAR subunits were detected in sera from T1D patients (n=40) and aged-matched controls (n=40) using immunocytochemistry. The effect of KAR activation on insulin secretion was measured in the presence of kainate and various GluR agonists/antagonists using the Mercodia human insulin ELISA kit. To investigate if the KAR subunit GluK2 is specifically involved in glucose-stimulated insulin secretion (GSIS) a GluK2 knockdown using GluK2 shRNA lentivirus was attempted.
Results and Conclusions: KAR subunits Gluk2 and Gluk5 and the supporting auxiliary subunits Neto1 and Neto2 are expressed in EndoC-βh1 cells. The presence of GluK2 protein in EndoC-βH1 cells was confirmed using immunoblots, suggesting that functional kainate receptors can be formed. However, neither GluK2, GluK5, GluN1 nor GluN1 plus GluN2B are antigenic targets in T1D patients. The subcellular localisation of KAR was observed to be dynamically regulated as glucose stimulation significantly increased the cell surface targeting of GluK2. Lentiviral knock-down of GluK2 was unsuccessful in both INS-1 and EndoC-βH1 cells and it was therefore not possible to assess of GluK2 was specifically involved in GSIS. However, high glucose and kainate significantly increased insulin secretion compared to high glucose alone. Furthermore, high glucose and kainate induced insulin secretion from EndoC-βH1 cells was significantly reduced by the kainate receptor antagonist, NBQX, suggesting a functional role for KARs in insulin secretion and that KAR activation augments insulin secretion in human β-cells.

Citation

Caygill, C. Investigation of glutamate receptors in human pancreatic β-cells and their role in insulin secretion. (Thesis). University of the West of England. Retrieved from https://uwe-repository.worktribe.com/output/5337152

• Thesis Type: Thesis
• Deposit Date: Feb 7, 2020
• Publicly Available Date: Jun 4, 2020
• Public URL: https://uwe-repository.worktribe.com/output/5337152
• Award Date: Jun 4, 2020