SAT069 Targeting Human β-Cell G Protein-coupled Receptors To Alter Cytokine-mediated Caspase Activation

Abstract

Abstract Disclosure: K. Rodrigues dos Santos: None. N. Mukherjee: None. L.F. Barella: None. F. Armoo: None. D.L. Eizirik: None. A.T. Templin: None. M.A. Kalwat: None. In type 1 diabetes (T1D) autoimmune destruction of pancreatic islet β-cells causes a lack of insulin, leading to hyperglycemia. The development of therapies that can prevent β-cell death is critical. β-cell proliferation and survival pathways are affected by signaling through G protein-coupled receptors (GPCRs). Using high-depth RNA-seq, we found altered expression of 123 different GPCRs in human islets exposed to cytokines (IL1β 50U/ml + IFNγ 1000U/ml, 48 h) to model the T1D milieu. We hypothesized these candidates may be exploited for β-cell protection. Among the candidates, adenosine receptors (ADORA1 and ADORA2A), calcium-sensing receptor (CASR), and metabotropic glutamate receptor 4 (GRM4) stood out. Cytokines decreased the expression of ADORA1 and CASR, but increased ADORA2A and GRM4. To gauge the impact of agonizing/antagonizing these receptors, we pre-treated human EndoC-β H1 β-cells or islets with receptor ligands and co-treated with cytokines (IL1β 50U/ml + IFNγ or IL1β + 1000U/ml IFNγ + 1000U/ml TNFɑ, 48 h) and measured glucose-stimulated insulin secretion, cell viability, and gene expression. Each cytokine combination induced target gene expression (e.g. CXCL10), but only the combination of IL1β + IFNγ + TNFɑ reduced glucose-stimulated insulin secretion. Preliminary experiments indicated that adenosine receptor ligands had little impact in our assays. However, ligands for CASR and GRM4 exhibited significant suppression of cytokine-induced caspase 3/7 activation in EndoC-βH1 cells. Interestingly, suppressed caspase activation did not result in observable impacts on live/dead cell ratios or live-cell analysis using Incucyte in EndoC-βH1 cells. Consistently, we also observed cytokine-mediated cell death in the presence of the pan-caspase inhibitor ZVAD-fmk. We conclude that a variety of β-cell GPCR-mediated signaling pathways may converge to prevent cytokine-induced caspase activation, but additional measures are needed to enable protection from eventual cell death. Our results and this line of investigation is significant because understanding mechanisms of GPCR-cytokine crosstalk in β-cells will inform strategies to protect islets in other models of T1D. Funding: JDRF 1-INO-2022-1113-A-N and Diabetes Research Connection #33. Presentation: Saturday, June 17, 2023