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Alternative Splicing and Immunogenicity of Insulin

Today we have a look at new a study that investigated alternative splicing of INS mRNA in human islets and determined the expression and immunogenicity of alternative insulin protein products in endocrine cells. Let us first look at the concept of endocrine cell plasticity, which refers to the ability of fully differentiated endocrine cells, such as beta cells in the pancreas, to transform into different types of endocrine cells. Polyhormonal endocrine cells have been found in fetal pancreatic islets, individuals with type 2 diabetes and chronic pancreatitis.

Although insulin gene expression is restricted to beta cells, accumulating data suggest that mature human beta cells are more plastic than previously assumed. Specific circumstances, such as metabolic and mechanical stress, can cause spontaneous dedifferentiation and transdifferentiation of human beta cells. The conversion of beta cells into alpha and delta cell-like states observed in individuals with type 2 diabetes has been proposed to contribute to beta cell failure.

Alternative splicing is a process that generates multiple mRNA transcripts from a single gene, increasing proteome diversity. Tissue-specific splicing patterns allow genes to express protein isoforms with different biological compositions and activities. Splicing patterns are highly dynamic and provide a mechanism to adapt to changes in the local microenvironment. Many tumors undergo alternative splicing, potentially generating neoantigens that are prominent targets in cancer immunotherapy. Likewise, splice variants generated in beta cells may contribute to autoimmunity and type 1 diabetes.

Researchers investigate alternative insulin protein products and splicing in human pancreatic tissues

In a new study, the researchers investigated alternative splicing of insulin INS mRNA in human islets and the expression and immunogenicity of alternative insulin protein products in endocrine cells. They obtained pancreatic islets from human cadaveric donor pancreases with consent and isolated them. Moreover, they used HEK293T cells, which were transfected using polyethylenimine and harvested 48 hours post-transfection. They followed standard western blotting protocols using HEK293T cell lysate and antibodies against insulin, C-peptide, actin, GFP, SPLICE81-95, and somatostatin.

The researchers dispersed human islets into single cells, fixed and permeabilized them, stained them with SPLICE81-95 antiserum, and analyzed them using FACS Aria II. They also generated custom polyclonal antisera by immunizing rabbits with the synthetic peptides MLYQHLLPLPAGEC and LLHRERWNKALEPAK. Using electron microscopy and single-cell transcriptome analysis, they investigated the alternative splicing of INS mRNA. Finally, they used immunohistochemistry and microscopy and single-cell western blot to detect protein expression in human pancreatic tissues.

Two major variants of insulin RNA in human islets, with potential implications for diabetes treatment

Researchers have identified alternative splicing of INS RNA in human islets, resulting in two major INS RNA variants. The larger variant represents a full-length proinsulin that generates an insulin-defective ribosomal product, insulin defective ribosomal product (INS-DRiP), under endoplasmic reticulum stress. In contrast, the smaller, less abundant variant resulted from a cryptic splicing site within exon 3 and is predicted to lead to the translation of a polypeptide, INS-splice.

The C-terminal region of INS-splice is identical to INS-DRiP's C-terminus, except for the immunodominant N-terminus unique to INS-DRiP. Immunization of rabbits with short polypeptides unique to INS-DRIP and the C-terminus shared between INS-DRiP and INS-splice resulted in two antiserums that confirmed the specific detection of INS-DRiP polypeptide by cytolytic T cells in individuals with type 1 diabetes. INS-splice was localized to secretory granules of delta cells, as demonstrated by high-resolution EM with quantum dot-labelled SPLICE81-95 antiserum. Furthermore, the researchers concluded that the alternatively spliced INS mRNA is a template for translation in delta cells.

Identification of subset of delta cells expressing immunogenic insulin B chain: Providing insights into type 1 diabetes immunopathology

So, the researchers found a subset of delta cells expressing insulin B chain besides somatostatin, which indicates the presence of INS-splice and excludes the presence of PPI. INS-splice includes the complete signal peptide and B chain sequences of PPI that contain highly immunogenic epitopes targeted in individuals with type 1 diabetes. Moreover, human cells can generate immunogenic epitopes from the INS-splice polypeptide that are processed and presented to patient-derived cytolytic T cells. This suggests that INS-splice-expressing delta cells may be targeted by autoreactive T cells in type 1 diabetes immunopathology.

The study hypothesized that IDE selectively degrades INS-splice in some endocrine cell types but spares it in others. The study found that IDE even was undetectable in delta cells. Consequently, the researchers suggested a role for IDE in the selective expression of INS-splice protein in delta cells. This in turn reconciles the discrepancy between INS splicing RNA and protein expression in beta and delta cells.