Expression and function of the NMDA receptor subunits GluN2A and GluN2B in the context of diabetes mellitus

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Question : The NMDA receptor with its various subunits is discussed as a potential target in diabetes research. We investigate whether the expression level for 2 types of the GluN2 subunit (GluN2A and 2B) varies under physiological vs. pathophysiological conditions. We also examine the extent to which NMDA receptors containing GluN2A or GluN2B differ in their functionality in pancreatic islets. Methods : MIN6 cells and islet cells (C57BL/6N mice) were cultivated. Medium supplemented with 500 µM NMDA or 33 mM glucose and 500 µM sodium palmitate plus 0.28% fatty acid-free BSA was applied for 2 or 7 d to achieve cell stress. For electrophysiology, NMDA and glycine were added acutely to the bath solution. A novel and a known GluN2A antagonist (WMS-58-172 and TCN-201), as well as GluN2B antagonists (WMS-1410 and Ro 25-6981) were used. RT-qPCR (2-ΔΔCt method compared to the reference gene β-Actin), TUNEL assays and patch-clamp measurements were performed. Results : Exposure of MIN6 cells to NMDA for 2 d resulted in dynamic changes in the subunit composition of the NMDA receptor. In 75% of the experiments Grin1 and Grin2A mRNA, encoding GluN1 and GluN2A, respectively, was downregulated by NMDA. The gene expression of the GluN2B subunit was much lower vs. GluN2A, but over-stimulation of the NMDA receptors resulted in a consistent upregulation (p ≤ 0.05, n=6). The same results were obtained when MIN6 cells where exposed to glucolipotoxicity for 7 d: Downregulation of the mRNA levels of Grin1 (0.8±0.2, p ≤ 0.05, n=5) and Grin2A (0.7±0.2, p ≤ 0.05, n=5) and upregulation of the mRNA levels of Grin2B. To reveal possible differences between the GluN1/2A and GluN1/2B receptors for regulation of islet cell mass, cell death was monitored. We have already shown that targeting the NMDA receptor by blocking the GluN2B subunit resulted in a strong protective effect on islet cells challenged by NMDA(1). Our new data illustrates that inhibiting NMDA receptors via the GluN2A subunit by TCN-201 or WMS-58-172 (1 µM) also protects against overstimulation (500 µM NMDA, 2 d, p ≤ 0.001, n=5). Finally, patch-clamp experiments show that the influence of NMDA receptors on K+ channels of islet cells is subunit-dependent. The K+ current density in islet cells increased in response to NMDA/glycin (500/10 µM), which was prevented by the GluN2B antagonists WMS-1410 and Ro 25-6981 (1 µM). Importantly, the application of the two GluN2A antagonists (1 µM) did not have any impact on the NMDA-mediated activation of K+ channels. Conclusion : In terms of expression, the GluN2A subunit appears to be more relevant under physiological conditions. In contrast, its relevance decreases under pathophysiological conditions and the importance of GluN2B-subunits increases. Regarding islet cell mass, antagonists of both subunits are protective. However, signalling pathways depending on NMDA receptor-mediated Ca2+ influx, e.g. activation of Ca2+-gated K+ channels, appear to require the GluN2B subunit. Gresch, A., Hurtado, H.N., Wörmeyer, L., de Luca, V., Wiggers, R., Seebohm, G., Wünsch, B., and Düfer, M. (2021). Selective inhibition of N-methyl-d-aspartate receptors with GluN2B subunit protects β cells against stress-induced apoptotic cell death. The Journal of Pharmacology and Experimental Therapeutics 379: 235–244. Publication History Article published online: 18 April 2024 © 2024. Thieme. All rights reserved. Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart, Germany

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