Cell type-specific expression and subcellular localization of the human insulin upstream open reading frame (INSU) protein in pancreatic β-cells

Abstract INSU was evolutionarily selected in human and chimp genomes by deletion of the 16 bp in INS 5'UTR regions present in other primate species that do not contain the upstream open-reading-frame (uORF). We now aim to identify the islet cell type in which INSU is produced, its subcellular distribution, and its response to stress in human islets. To do this, we developed an INSU specific rabbit polyclonal antibody, and we employed immunohistochemistry (IHC), immunofluorescence (IF), and immuno-gold labelling with electron microscopy (EM) technologies. We used LC-MS/MS-based Selected Reaction Monitoring (SRM) proteomic assay to quantify INSU in islets, plasma and cerebral spinal fluid (CSF). Unlike mature insulin, INSU levels were unchanged in plasma and CSF two hours after continuous intravenous glucose infusion. The INSU immunohistochemistry signal partially overlapped with that of insulin and was more intensely polarized in β-cells in islets. INSU was not present in α-, δ-, ε-, or PP-cells. Dual IF and immuno-gold EM showed that INSU was present in immature insulin granules and crinosomes, but there was little to none present in mature secretory granules, implying INSU involvement in quality control of β-cells.