Gangliosides GM3 and GD3 modulate insulin aggregation pathways and reduce cytotoxicity through structural remodeling.

Insulin amyloid aggregation is a key pathological and pharmaceutical concern, particularly in the context of Type-2 Diabetes (T2D), where amyloid deposition can impair therapeutic efficacy and contribute to local tissue damage. While gangliosides are known to modulate amyloid formation in neurodegenerative systems, their influence on insulin aggregation remains largely unexplored. In this study, we investigate the effects of gangliosides GM3 and GD3 on insulin amyloid aggregation using Thioflavin-T (ThT) based fluorescence kinetics, Fourier Transform Infrared (FTIR) spectroscopy, Circular Dichroism (CD) spectroscopy, Small Angle X-ray Scattering (SAXS), Nuclear Magnetic Resonance (NMR) spectroscopy, and Transmission Electron Microscopy (TEM) to examine the aggregation pathway, changes in the secondary structure and morphology of insulin aggregates. Our results show that both GM3 and GD3 lipids accelerated insulin aggregation in a concentration-dependent manner while steering the pathway away from classical fibril formation, producing short, beaded structures distinct from the extended fibrils observed under lipid-free conditions. Structural analyses revealed distinct non-fibrillar intermediates with β-sheet-rich globular clusters in presence of GD3 and α-helical intermediates in GM3-treated samples. Notably, these ganglioside-induced aggregates exhibit significantly reduced cytotoxicity when compared to insulin-only aggregates. Furthermore, ganglioside-bound insulin species retain seeding capacity, suggesting that they can nucleate further aggregation despite their non-fibrillar morphology. These findings underscore the role of gangliosides in modulating insulin amyloid polymorphism and toxicity, offering new insights into their potential impact on the pathology of T2D and treatment strategies.