A Real Time Quaking Induced Conversion (RT-QuIC) Assay for Detection of Misfolded Insulin Protein

Abstract

Protein misfolding plays a critical role in aging and disease, yet the involvement of specific proteins in metabolic dysfunction is still poorly understood. Here, we report studies on the development of a Real-time Quaking-Induced Conversion (RT-QuIC) assay to detect misfolded insulin, a peptide hormone required for blood glucose regulation. Although RT-QuIC assays were originally designed to amplify misfolded prion proteins implicated in neurodegeneration, we adapted the method to monitor conformational changes in insulin. We first validated the RT-QuIC insulin assay using recombinant insulin and insulin aggregates recovered from clinical infusion devices. Protein characterization by gel electrophoresis, circular dichroism, and particle size analysis suggests differences in insulin recovered from the infusion device. We then applied the RT-QuIC assay to tissue samples from a mouse model of metabolic disease. This work provides proof-of-concept of a novel assay for studying the role of insulin aggregation in disease progression and aging. The RT-QuIC assay for insulin may also provide new avenues to explore early detection, mechanistic insights, and therapeutic targets of metabolic disorders linked to aging and disease.