A FRET-Based Assay Identifies a Zinc–Albumin–Fatty Acid Axis Regulating Insulin Disassembly

Insulin is stored and secreted as Zn2+-stabilised hexamers that must dissociate into monomers for receptor activation; yet tools to monitor this process in real time under physiological conditions remain limited. Here we report a Förster resonance energy transfer (FRET)-based assay that enables direct tracking of insulin oligomerisation in solution. Labelling at LysB29 with a donor-acceptor dye pair converts changes in oligomeric state into a quantitative fluorescence readout, allowing resolution of Zn2+-dependent hexamerisation, pH effects across the physiological range of insulin secretory granules, and the kinetics of albumin-mediated decomplexation. Single molecule total internal reflection microscopy was used to confirm Zn2+-dependent oligomerisation of fluorescent insulin monomers. Using the FRET-based approach, we show that human serum albumin promotes Zn2+-dependent hexamer dissociation, while long-chain free fatty acids, including palmitate, inhibit this process by impairing albumin’s Zn2+ binding. Application to clinical plasma samples reveals reduced insulin decomplexation in those from type 2 diabetes individuals compared to healthy controls and identifies correlations between oligomer stability and specific fatty acid species. These findings establish a Zn2+-albumin-lipid axis governing insulin speciation in circulation and introduce a general platform for quantifying insulin oligomer dynamics, with applications in analogue design, formulation assessment, and the investigation of mechanisms underpinning insulin resistance.