Presence of immunogenic alternatively spliced insulin gene product in human pancreatic delta cells

Abstract

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: 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-1β 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.

Data availability: The full EM dataset is available via www.nanotomy.org (for review: http://www.nanotomy.org/OA/Tienhoven2021SUB/6126-368/ ). Single-cell RNA-seq data was made available by Segerstolpe et al [13] and can be found at https://sandberglab.se/pancreas . The RNA and protein sequence of INS-splice was uploaded to GenBank (BankIt2546444 INS-splice OM489474).

Keywords: Alternative splicing; Alternative translation; Beta cell; Defective ribosomal product; Delta cell; Human islets of Langerhans; Insulin gene; Insulin-degrading enzyme; Type 1 diabetes.

Fig. 1

Alternative INS RNA splicing in human islets. Analysis of INS splicing by PCR on RNA derived from human pancreatic islets of three different donors, visualised on DNA gel. A schematic overview of the human insulin pre-mRNA is shown with the exons annotated by numbers (1–3) and the intronic regions represented by a black solid line. Normal INS mRNA splicing and alternative INS mRNA splicing are indicated by black and red dashed lines, respectively (showing the start codon, AUG). The resulting mRNA products with translation initiation sites are depicted underneath. For each mRNA molecule the potential protein products are displayed. Regular protein translation of the regular spliced transcript produces preproinsulin (PPI) with the signal peptide (orange), B chain (green), C-peptide (blue) and insulin A chain (yellow). Alternative translation of this transcript produces INS-DRiP, with the previously identified CTL epitope (dark red). Translation of the alternatively spliced transcript produces a splice protein variant, referred to as INS-splice. Amino acid sequences are indicated with corresponding colours and letters indicate the presence of the complete chain. The non-stop characteristic of INS-DRiP and INS-splice proteins is visualised by decreasing gradient. A, insulin A chain; B, insulin B chain; C, C-peptide; E, CTL epitope; M, DNA marker; INS, insulin; SP, signal peptide

Fig. 2

SPLICE_81-95 antiserum stained delta cells and INS-splice protein is localised to somatostatin granules. (a, b) Immunohistochemistry of human pancreas sections with pre-immunisation serum and post-immunisation serum (green) in combination with insulin (red). Serum derived from DRiP_1-13 immunised rabbits (a) and SPLICE_81-95 immunised rabbits (b) was used. Scale bar, 30 μm. (c) Human pancreas sections stained for glucagon (white) and somatostatin (red), and SPLICE_81-95 antiserum (green). Enlarged images of the grey enclosure are shown. Nuclei were visualised by DAPI staining (blue). Scale bar, 30 μm. (d) EM images of human pancreas sections labelled for INS-splice (quantum dots, red arrows) and insulin (immunogold, green arrows) visible as black dots. Scale bar, 200 nm. The granules were identified by their morphology. The full dataset is available via www.nanotomy.org (for review, see http://www.nanotomy.org/OA/Tienhoven2021SUB/6126-368/). (e, f) Quantification of the insulin-immunogold⁺ (e) and INS-splice-quantum dot⁺ (f) granules in beta and delta cells. Each granule is represented as a point. The graphs represent the means of 30 beta and 30 delta cell granules

Fig. 3

Splicing events of insulin transcripts in human beta cells and human delta cells. (a) Splicing pattern of PPI (T1, green) and an in silico predicted INS RNA splice variant (T2, red). Exons are shown as boxes and introns as lines. (b, c) Sashimi plots show splicing events of INS RNA for beta cells (b, green) and delta cells (c, blue). Human pancreas donor identity numbers are indicated by HP. The numbers of splicing events are shown. Splicing of PPI mRNA is shown by alignment with the regular splicing pattern from (a) (T1, green boxes). Alternative splicing of insulin transcripts is defined as any sequence that does not align with the regular splicing pattern as displayed in T1

Fig. 4

Insulin B chain expression in delta cells. (a–c) Pancreas section was stained for insulin (green), insulin B chain (white), somatostatin (red) and Hoechst. Z-stack images were made and used for 3D reconstruction and co-localisation analysis (Imaris). Co-localisation of insulin and insulin B chain (a), insulin B chain and somatostatin (b), and insulin and somatostatin (c) was determined and a co-localisation channel (blue) was created for double-positive voxels. The MCC was 0.22, 0.13 and 0.22, respectively. Scale bar, 10 μm. (d) Enlarged images of the grey enclosure show insulin B chain expression in a delta cell, including a 3D reconstructed image (see ESM Video). Scale bar, 5 μm. (e) Single-cell western blot was performed on dispersed pancreatic islet cells from two different donors and stained for DNA (green), insulin B chain (red), C-peptide (white) and somatostatin (blue). Doublets were excluded by measuring a composite of DNA intensity and hormone content of all endocrine cells. Single cells (black circles) were included for analysis on the basis of their single-cell DNA intensity and hormone content. Doublets (red circles) were excluded because of their high DNA intensity and/or high hormone content. Examples of doublets and single cells are shown. The single-cell example shows single delta cells that are positive for somatostatin and insulin B chain (δ^{B chain+}, black open shapes), of which some are negative for C-peptide (δ^{B chain+, C-pep−}, open squares). DNA intensity and examples of cells are shown from one out of six single-cell western blot chips. In total, 554 single delta cells were included for analysis; 54 single delta cells expressed insulin B chain besides somatostatin (9.7%), of which seven were negative for C-peptide (1.3%). Β, beta cell; δ, delta cell; INS, insulin; INS-B, insulin B chain; SST, somatostatin

Fig. 5

INS-splice activates PPI_15-24-specific CTLs. (a) GFP mRNA expression in HEK293T cells transfected with INS-splice-IRES-GFP for 24 h. Gene expression levels are corrected for GAPDH used as housekeeping gene and presented as the induction ratio (mock control set to 1) (n=3). (b) MIP-1β secretion of PPI_15-24-specific CTLs after co-culture with INS-splice-expressing HEK293T (red) or mock control (blue) cells. Effector/target ratios were 1:1, 2:1 and 4:1. The dotted line shows the basal MIP-1β secretion of PPI_15-24-specific CTLs in the absence of target cells. Data are shown as mean ± SD, n=4. E, effector; T, target

Fig. 6

IDE is not expressed in delta cells and cleaves INS-splice. (a) Pancreatic IDE protein expression was determined by immunohistochemistry of human pancreas sections by staining for proinsulin (green), IDE (white) and somatostatin (red). IDE was expressed ubiquitously in the exocrine and endocrine pancreas except in delta cells. Scale bar, 10 μm. Nuclei were visualised by Hoechst staining (blue). (b) Co-localisation of IDE with insulin and IDE with somatostatin was quantified using MCC (QuPath). Thirty-six islets from six pancreas donors were analysed. Data are shown as mean ± SD and statistical analysis was performed using a paired two-tailed Student’s t test (***p<0.001). (c) Coomassie staining of INS-splice after IDE cleavage assay. Absence or presence of IDE is indicated by − or +, respectively. Full-length recombinant INS-splice is 14 kDa. INS, insulin; M, protein marker; SST, somatostatin