Abstract
Protein aggregation is associated with various diseases caused by protein misfolding. Among them, amylin deposition is a prominent feature of type 2 diabetes. At present, the mechanism of amylin aggregation remains unclear, and this has hindered the treatment of type 2 diabetes. In this study, we analyzed the aggregation process of amylin using the quantum dot (QD) imaging method. QD fluorescence imaging revealed that in the presence of 100 μM amylin, aggregates appeared after 12 h of incubation, while a large number of aggregates formed after 24 h of incubation, with a standard deviation (SD) value of 5.435. In contrast, 50 μM amylin did not induce the formation of aggregates after 12 h of incubation, although a large number of aggregates were observed after 24 h of incubation, with an SD value of 2.883. Confocal laser microscopy observations revealed that these aggregates were deposited in three dimensions. Transmission electron microscopy revealed that amylin existed as misfolded fibrils in vitro and that QDs were uniformly bound to the amylin fibrils. In addition, using a microliter-scale high-throughput screening (MSHTS) system, we found that rosmarinic acid, a polyphenol, inhibited amylin aggregation at a half-maximal effective concentration of 852.8 μM. These results demonstrate that the MSHTS system is a powerful tool for evaluating the inhibitory activity of amylin aggregation. Our findings will contribute to the understanding of the pathogenesis of amylin-related diseases and the discovery of compounds that may be useful in the treatment and prevention of these diseases.