Expression of the CHI-linked S561F CDKAL1 variant affects the insulin processing and release in INS1E cells

Background and aims: Congenital Hyperinsulinism (CHI) is a rare disorder, characterized by hypoglycemia due to inappropriate insulin release from pancreatic \u3b2-cells. Despite the advances in understanding the molecular pathogenesis of CHI, specific genetic determinants in about 50 % of the CHI patients remain unknown. A whole-exome sequencing analysis performed on 17 CHI patients lacking mutations in ABCC8/KCNJ11 identified a polymorphism in the CDKAL transcript (S561F-CDKAL1 variant). CDKAL1 is a methylthiotransferase that modifies cytoplasmic tRNALys to enhance translational fidelity of transcripts. Interestingly, CDKAL1 is a susceptibility gene for type 2 diabetes and CDKA1 knock-out mice showed impaired glucose homeostasis, thus indicating the protein involvement in \u3b2-cell function. Since a lysine residue is located at the cleavage site for proinsulin to insulin processing, aim of this work was to investigate the impact of the CDKAL1 variant S561F on the insulin/proinsulin content and the formation of mature hormone granules. Materials and methods: INS1-E clones expressing Wild Type (WT) or S561F CDKAL1 were generated and used as a model to characterize the S561F CDKAL1 impact on beta-cell function. Western Blot experiments and ELISA assays were performed in order to evaluate the expression, processing and release of insulin/proinsulin in the different INS-1E clones. The distribution of insulin granules in clones was monitored by indirect immunofluorescence and total internal fluorescence reflection microscopy (TIRFM). Results: The insulin content in clones overexpressing the S561F-CDKAL1 variant was decreased as compared to WT clones (2 folds increase over WT, p<0.05). Conversely, insulin release measured in overnight culture medium or in 30 minutes static incubation, in normal glucose concentrations, was significantly increased in the S561F-CDKAL1 as compared to WT clones (2 to 4 folds increase over WT; p<0.05), thus suggesting a different insulin processing and/or secretion in the mutant CDKAL1. In line with these results, we found a higher proinsulin content in INS-1E-S561F clones than in CDKAL1 WT. Data were confirmed by western blotting analysis. Differences between the clones were highlighted by the proinsulin/insulin ratio which was significantly higher (; p<0.05) in mutant than in WT clones. Immunofluorescence experiments showed an abnormal enrichment of insulin-positive granules in the perinuclear region, positive for Golgi markers, and a decreased granules density in the TIRF zone in mutant clones compared to WT CDKAL1, thus suggesting that alterations in proinsulin-insulin processing probably impact on insulin granules formation and secretio