1722-P: RIPK1 Promotes Thapsigargin-Induced ß-Cell Death Independent of Caspase 3/7 Activity In Vitro

Endoplasmic reticulum (ER) stress promotes β-cell dysfunction and death in diabetes. Although studies of ER stress-induced β-cell death have focused on mechanisms of apoptosis, we recently showed that β cells are also susceptible to a form of programmed cell death called necroptosis. Necroptosis occurs downstream of receptor interacting protein kinase 1 (RIPK1), does not require caspase activation, and results in lytic cell death. Studies in non-islet cell types have linked ER stress-induced cell death to necroptosis. Thus, we hypothesized that RIPK1 promotes ER stress-induced β-cell death in vitro. To test this hypothesis, we quantified cell death and caspase 3/7 activity in NIT-1 β cells, human iPSC-derived β-like cells and intact mouse islets following treatment with thapsigargin (a SERCA2b inhibitor and ER stress-inducing agent, 50 nM or 500 nM) and zVAD-FMK (a pan-caspase inhibitor, 40 μM). After quantifying cell death hourly for 48 hrs, we found that thapsigargin significantly increased cell death in NIT-1 cells, β-like human iPSCs and intact mouse islets, and that this occurred in conjunction with increased caspase 3/7 activity. We next asked whether co-treatment with zVAD was sufficient to prevent thapsigargin-induced cell death. In each cell type evaluated, we found that zVAD inhibited caspase 3/7 activity but failed to protect from thapsigargin-induced cell death. To establish the role of RIPK1 in this process, we next evaluated cell death in gene-edited NIT-1 control (non-targeting gRNA) and NIT-1 Ripk1Δ (gRNA targeting Ripk1 exons 2-3) β cells. After 24 hrs, we found that NIT-1 Ripk1Δ cells were strongly protected from thapsigargin-induced cell death both when caspases were active (n=6, p<0.001) and when they were inhibited (n=6, p<0.001). These data indicate that RIPK1 regulates thapsigargin-induced β-cell death independent of caspase 3/7 activation. Future studies will examine the mechanisms by which RIPK1 regulates ER stress-induced β-cell death in vitro and in vivo. Disclosure N.Mukherjee: None. C.J.Contreras: None. L.Lin: None. E.P.Cai: None. A.T.Templin: None. Funding U.S. Department of Veterans Affairs (IK2BX004659 to A.T.T.); National Institutes of Health (T32DK064466 to C.J.C.); Indiana University School of Medicine (to N.M.)