Strategic Reduction of Hybrid Insulin Peptide Formation Significantly Delays Diabetes Onset in NOD Mice

Type 1 diabetes (T1D) is characterized by autoimmune destruction of insulin-producing β-cells. Recent evidence has implicated hybrid insulin peptides (HIPs) as targets of autoreactive CD4 T cells in human T1D patients and as critical autoantigens recognized by diabetogenic T cells in nonobese diabetic (NOD) mice. HIPs form within pancreatic islets through cross-linking reactions between proinsulin fragments and various β-cell peptides. In the NOD mouse model, highly pathogenic CD4 T cells specifically target HIPs generated through transpeptidation mediated by cathepsin D (CatD). These disease-relevant HIPs consistently incorporate a C-peptide fragment terminating in a critical leucine residue that binds to other β-cell peptides. In vitro experiments demonstrated that substituting isoleucine for this leucine residue in human C-peptide inhibited CatD-mediated HIP formation. To investigate the in vivo significance of this finding, we engineered NOD mice carrying a leucine-to-isoleucine mutation in the insulin 2 gene (NOD INS2 I/I ). Mass spectrometric analysis revealed significantly reduced HIP formation in islets from NOD INS2 I/I mice. Significantly decreased activation of HIP-reactive T cells to islets from these mice was also observed. Furthermore, the NOD INS2 I/I mice showed significantly delayed diabetes onset, with 43% remaining disease-free at 1 year compared with only 10% of wild-type NOD controls. These findings implicate HIPs as key mediators in T1D pathogenesis and demonstrate that targeted disruption of HIP formation significantly alters disease progression. Inhibiting CatD-mediated transpeptidation represents a promising therapeutic approach for preventing or delaying T1D onset in genetically susceptible individuals. Article Highlights Hybrid insulin peptides (HIPs) have been identified as targets of autoreactive T cells in type 1 diabetes, although their causal role in disease pathogenesis has remained unclear. We demonstrated that a single leucine-to-isoleucine substitution in insulin C-peptide significantly disrupts cathepsin D–mediated HIP formation in nonobese diabetic (NOD) mouse islets. NOD mice engineered with this precise modification (NOD INS2 I/I ) showed significantly reduced HIP content, decreased T-cell reactivity, and significantly delayed diabetes onset (43% disease-free at 1 year vs. 10% in controls). These findings establish a mechanistic link between HIP formation and disease progression, revealing cathepsin D–mediated transpeptidation as a potential therapeutic target for intervention in at-risk individuals.