Expression of S561F CDKAL1 variant modifies the constitutive trafficking and affects insulin release in INS1E cells

Background and aims: Congenital hyperinsulinism (CHI) is a rare disorder (OMIM#256450), characterized by hypoglycaemia due to inappropriate insulin secretion. A Whole Exome Sequencing (WES) analysis performed on CHI patients lacking mutations in ABCC8/KCNJ11 identified a polimorfism in the CDKAL transcript (S561F-CDKAL1 variant). CDKAL is a methylthiotransferase that modifies tRNA(Lys) to enhance translational fidelity of transcripts, including the one encoding proinsulin. Interestingly, CDKAL is a susceptibility genes for type 2 diabetes and CDKAL knock-out (cdkal1 -/-) mice showed impaired glucose homeostasis, thus indicating the protein involvement in beta-cell function. Aim of this work was to understand the impact of the CDKAL1 variant S561F on the insulin content, trafficking and release in pancreatic beta cells. Materials and methods: Clonal INS1-E cells expressing Wild Type (WT) or S561F CDKAL1 were generated and used as a model to characterize S561F-CDKAL1 impact on beta cell function. The localization of the variant protein was monitored by immunofluorescence and insulin content and release were measured with ELISA test. An acridine orange assay was performed to evaluate the constitutive and regulated trafficking and possible alterations in vesicle protein expression were evaluated by western blotting. Results: Wild type CDKAL1 overexpressed in INS1-E cells localized in the reticular compartment. The S561F variant was similarly confined to the reticular compartment, although its localization was enriched in spot-like structures distributed in the perinuclear region. Insulin content was increased by overexpression of WT CDKAL1 (2 fold over INS1E, p<0.05) while it was decreased by S561F-CDKAL1 variant expression. Conversely, insulin release measured in overnight culture medium or in 30 minutes static incubation in normal glucose concentrations was 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/secretion in the mutant CDKAL1. An acridina orange assay performed to measure the constitutive and regulated trafficking in INS1E cells revealed more basal exocytosis in S561F-CDKAL1 then WT clones. Interestingly, the basal release was not further increased by potassium chloride or high glucose. Western blotting experiments revealed up-regulation of proteins involved in the secretion machinery in mutant clones compared to WT. Conclusion: The S561F-CDKAL1 variant expression leads to an increased basal insulin release in INS1E cell line. Such an increase is associated to a defect in the vesicle trafficking and correlates with altered expression of proteins involved in the secretory machinery , further studies are needed to clarify molecular mechanisms linking CDKAL to insulin processing and membrane trafficking. Our findings confirm the importance of CDKAL1 in insulin release and suggest a possible mechanism by which this variant can participate to the development of congenital hyperinsulinism.