Assessment of New Compound Inhibitors of 12-Lipoxygenase in Beta-Cell Protection

Abstract

The contribution of 12-lipoxygenase (12-LO) to the development of diabetes may involve beta cell inflammation and immune activation. Recent studies with targeted deletion of 12-LO in islet beta cells confirm a beta cell component to pathology mediated by 12-LO. Our identification and characterization of first-in-class selective inhibitors of 12-LO have enabled investigation of 12-LO activity in pro-inflammatory cytokine (PIC)-induced beta cell dysfunction. Lead 8-hydroxyquinoline-based compounds (NCTT- 956, NCTT-685) were studied. These show >20 fold selectivity for 12-LO inhibition over other LO isotypes. In human donor islets, NCTT-956/685 blocked 12-HETE (product of 12-LO activity) and inhibited gene expression of autoimmune-promoting cytokines (IL-12, IFNγ) induced by PICs (p<0.05). In primary mouse islets, NCTT-956/685 inhibited 80% (p<0.05) IFNγ gene expression induced by PICs and blocked PIC-induced islet apoptosis measured by YO-PRO-1 fluorescent microscopy. In INS-1 beta cell line, NCTT-956 inhibited production of reactive oxygen species and induction of caspase-3 activity triggered by PIC-stimulation (90±5% and 85±14%, p<0.01, resp.). Functionally, NCTT-956/685 prevented uncoupling of the glucose-stimulated insulin secretion by PIC-stimulation in INS-1 cells. Protection was not observed using structurally related molecules that were not active for 12-LO inhibition. In vivo, mice dosed with NCTT-685 at 50mg/kg for three days resulted in a 60% reduction in basal 12-HETE levels in plasma (p<0.05). Collectively these data support a key role of 12-LO in PIC-induced beta cell dysfunction. NCTT- 956 and NCTT-685 are efficacious inhibitors and validate selective inhibition of 12-LO as a promising new strategy to protect beta cells exposed to elevated PICs.