In vitro and in vivo consequences of inflammatory regulation of Nonsense-Mediated RNA decay (NMD) pathway for pancreatic beta-cell function and viability

Ghiasi SM, et al., 2023: Aims/Hypothesis. Proinflammatory cytokines are implicated in pancreatic beta-cell failure in type 1 and type 2 diabetes and are known to stimulate alternative RNA splicing and the expression of Nonsense-Mediated RNA Decay (NMD) components. Here, we investigate whether cytokines regulate NMD activity and identify transcript isoforms targeted in beta-cells. Methods. A luciferase-based NMD reporter transiently expressed in INS1(832/13), EndoC-betaH3 or dispersed human islet cells was used to examine the effect of proinflammatory cytokines (Cyt) and/or glucolipotoxicity (GLT) on NMD activity. Gain- or loss-of function of two key NMD components UPF3B and UPF2 was used to reveal the effect of cytokines on cell viability and function. RNA-sequencing and siRNA-mediated silencing were deployed using standard techniques. Results. Cyt, but not GLT, attenuated NMD activity in insulin-producing cell lines and primary human beta-cells. These effects involved ER stress and were associated with downregulation of UPF3B. Increases or decreases in NMD activity achieved by UPF3B overexpression (OE) or UPF2 silencing, raised or lowered Cyt-induced cell death, respectively, in EndoC-betaH3 cells, and was associated with decreased or increased insulin content, respectively. No effects of these manoeuvres were observed on glucose-stimulated insulin secretion. Transcriptomic analysis revealed that, in contrast to GLT, Cyt increased alternative splicing (AS)-caused exon skipping in the transcript isoforms, and this was potentiated by UPF2 silencing. Gene enrichment analysis identified transcripts regulated by UPF2 silencing whose proteins are localized and/or functional in extracellular matrix (ECM) including the serine protease inhibitor SERPINA1/alpha-1-antitrypsin, whose silencing sensitised beta-cells to Cyt cytotoxicity. Conclusions/interpretation. Cyt suppress NMD activity via UPR signalling, potentially serving as a protective response against Cyt-induced NMD component expression. Our findings highlight the central importance of RNA turnover in beta-cell responses to inflammatory stress.