Islet amyloid deposition occurs in the majority of individuals with type 2 diabetes (T2D) and contributes to beta-cell death by mechanisms which are not fully understood. Necroptosis is an immunogenic form of cell death dependent on tumor necrosis factor receptor 1 (TNFR1) signaling, receptor interacting protein kinase 3 (RIPK3) oligomerization, and mixed lineage kinase domain-like (MLKL) phosphorylation. We previously showed mRNA and protein levels of TNFa, a TNFR1 ligand, are increased in islets by amyloid deposition in vitro and in vivo, raising the possibility that amyloid formation induces necroptosis. Thus, we sought to determine whether the components of necroptosis are present in beta cells, and if islet amyloid-induced TNFa expression elicits beta-cell necroptosis. We observed protein expression of TNFR1, RIPK3, and MLKL in the INS-1 beta-cell line, indicating necroptosis components are present in beta cells. In human islet amyloid polypeptide (hIAPP) transgenic mouse islets cultured in 16.7 mM glucose, an in vitro model of islet amyloidosis, we found necroptosis signaling to be increased compared to amyloid-free wild type (WT) islets. This manifested as increased oligomerization of RIPK3 protein (trimers: 2.7 ± 0.2 [hIAPP] vs. 1.0 [WT], n=3, p<0.01) and phosphorylation of the RIPK3 target MLKL (1.8 ± 0.3 vs. 1.0, n=3, p=0.06), without a change in TNFR1 protein expression (1.1 ± 0.1 vs. 1.0, n=2, p=0.74). As further support that amyloid induces immunogenic cell death, immune cell mRNA abundance was increased in vivo in amyloid containing hIAPP transgenic islets vs. WT islets (macrophages: Emr1, 1.6 ± 0.2 [hIAPP] vs. 1.1 ± 0.1 [WT], n=4, p=0.04, T cells: Cd3, 2.7 ± 0.5 vs. 1.0 ± 0.2, n=4, p=0.03, and B cells: Cd20, 2.3 ± 0.2 vs. 1.2 ± 0.5, n=2, p=0.17). These data suggest that necroptosis may be a previously unrecognized mechanism of beta-cell death in response to islet amyloid formation. Further studies are needed to determine whether inhibition of necroptosis signaling may reduce beta-cell death in T2D.
A.T. Templin: None. M.F. Hogan: None. N. Esser: None. S. Zraika: None. R.L. Hull: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company. S.E. Kahn: Advisory Panel; Self; Boehringer Ingelheim GmbH, Elcelyx Therapeutics, Inc., Eli Lilly and Company, Intarcia Therapeutics, Inc., Janssen Research & Development, Merck & Co., Inc., Novo Nordisk A/S.