Exploratory investigation of insulin–glucagon interactions using fluorescence-based LigandTracer assays

Sammanfattning

Diabetes mellitus is characterized by dysregulated glucose homeostasis involving the pancreatic hormones insulin and glucagon. While these hormones are traditionally regarded as physiological antagonists, previous studies have suggested that direct molecular interactions between them may also occur under certain conditions. The aim of this study was to investigate whether insulin–glucagon complex formation could be detected in surface-based fluorescence assays and whether such interactions influence molecular stability or antibody epitope accessibility. Experiments were performed using LigandTracer technology together with antibody-based sandwich assays, disturbance assays and serial dilution experiments. Multiple assay configurations involving anti-glucagon and anti-insulin antibodies were evaluated using fluorescently labelled ligands and nonspecific control proteins. Several sandwich assays demonstrated altered fluorescence following insulin addition, suggesting possible modulation of glucagon epitope accessibility. In addition, FITC-insulin serial dilution experiments showed reproducible concentration associated signal progression that differed from nonspecific control conditions. Control experiments further indicated that the observed signal depended on the presence of glucagon. Due to the complexity of the experimental system and technical limitations associated with surface-based fluorescence measurements, the findings should be interpreted cautiously. Nevertheless, the collective results are consistent with possible insulin associated modulation of glucagon and support further investigation of insulin–glucagon interactions using complementary biophysical approaches.