Aims/hypothesis Transcriptome analyses revealed insulin-gene-derived transcripts in non-beta endocrine islet cells. We studied alternative splicing of human INS mRNA in pancreatic islets. Methods:... Show moreAims/hypothesis Transcriptome analyses revealed insulin-gene-derived transcripts in non-beta endocrine islet cells. We studied alternative splicing of human INS mRNA in pancreatic islets. Methods: Alternative splicing of insulin pre-mRNA was determined by PCR analysis performed on human islet RNA and single-cell RNA-seq analysis. Antisera were generated to detect insulin variants in human pancreatic tissue using immunohistochemistry, electron microscopy and single-cell western blot to confirm the expression of insulin variants. Cytotoxic T lymphocyte (CTL) activation was determined by MIP-1 beta release. Results: We identified an alternatively spliced INS product. This variant encodes the complete insulin signal peptide and B chain and an alternative C-terminus that largely overlaps with a previously identified defective ribosomal product of INS. Immunohistochemical analysis revealed that the translation product of this INS-derived splice transcript was detectable in somatostatin-producing delta cells but not in beta cells; this was confirmed by light and electron microscopy. Expression of this alternatively spliced INS product activated preproinsulin-specific CTLs in vitro. The exclusive presence of this alternatively spliced INS product in delta cells may be explained by its clearance from beta cells by insulin-degrading enzyme capturing its insulin B chain fragment and a lack of insulin-degrading enzyme expression in delta cells. Conclusions/interpretation: Our data demonstrate that delta cells can express an INS product derived from alternative splicing, containing both the diabetogenic insulin signal peptide and B chain, in their secretory granules. We propose that this alternative INS product may play a role in islet autoimmunity and pathology, as well as endocrine or paracrine function or islet development and endocrine destiny, and transdifferentiation between endocrine cells. INS promoter activity is not confined to beta cells and should be used with care when assigning beta cell identity and selectivity. Show less
Thomaidou, S.; Kracht, M.J.L.; Slik, A. van der; Laban, S.; Koning, E.J. de; Carlotti, F.; ... ; Zaldumbide, A. 2020
The signal peptide of preproinsulin is a major source for HLA class I autoantigen epitopes implicated in CD8 T cell (CTL)-mediated beta -cell destruction in type 1 diabetes (T1D). Among them, the... Show moreThe signal peptide of preproinsulin is a major source for HLA class I autoantigen epitopes implicated in CD8 T cell (CTL)-mediated beta -cell destruction in type 1 diabetes (T1D). Among them, the 10-mer epitope located at the C-terminal end of the signal peptide was found to be the most prevalent in patients with recent-onset T1D. While the combined action of signal peptide peptidase and endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) is required for processing of the signal peptide, the mechanisms controlling signal peptide trimming and the contribution of the T1D inflammatory milieu on these mechanisms are unknown. Here, we show in human beta -cells that ER stress regulates ERAP1 gene expression at posttranscriptional level via the IRE1 alpha /miR-17-5p axis and demonstrate that inhibition of the IRE1 alpha activity impairs processing of preproinsulin signal peptide antigen and its recognition by specific autoreactive CTLs during inflammation. These results underscore the impact of ER stress in the increased visibility of beta -cells to the immune system and position the IRE1 alpha /miR-17 pathway as a central component in beta -cell destruction processes and as a potential target for the treatment of autoimmune T1D. Show less
Type 1 diabetes (T1D) results from the immune-mediated destruction of the insulin-producing beta cells. Genetic predisposition, impaired immune regulation, and beta cell (dys)function all... Show moreType 1 diabetes (T1D) results from the immune-mediated destruction of the insulin-producing beta cells. Genetic predisposition, impaired immune regulation, and beta cell (dys)function all contribute to disease initiation and progression. A critical gap in our knowledge is what causes the break in peripheral tolerance that eventually leads to beta cell destruction. We propose that neoepitopes generated by dysfunctional beta cells activate immune surveillance, causing beta cell autoimmunity. ER stress imposed both by intrinsic beta cell physiology and by external secondary triggers seems to be a crucial component in this process. Understanding the molecular mechanisms underlying beta cell dysfunction and neoantigen generation is critical to identify clinically relevant neoepitopes. This subsequently provides more insight in the disease dynamics as well as contribute to translational research in the development of biomarker assays and development of therapeutic strategies targeting autoreactive T-cells and beta cell function. Our task will be to restore the balance between immune reactivity and beta cell function, in order to prevent, treat, or cure type 1 diabetes. Show less
