Abstract
Amylin is a pancreatic peptide hormone that regulates glucose homeostasis but also aggregates to form islet amyloid in type-2 diabetes. Given its role in both health and disease, there is renewed interest in proteolytic degradation of amylin by insulin-degrading enzyme (IDE) and other proteases. Here, we describe the development and detailed characterization of three novel assays for amylin degradation, two based on a fluoresceinated and biotinylated form of rodent amylin (fluorescein-rodent amylin-biotin, FrAB), which can be used for any amylin protease, and another based on an internally quenched fluorogenic substrate (FRET-based amylin, FRAM), which is more specific for IDE. The FrAB-based substrate can be used in a readily implemented fluorescence-based protocol or in a fluorescence polarization (FP)-based protocol that is more amenable to high-throughput screening (HTS), whereas the FRAM substrate has the advantage of permitting continuous monitoring of proteolytic activity. All three assays yield highly quantitative data and are resistant to DMSO, and the FRAM and FP-based FrAB assay are ideally suited to HTS applications.
Highlights
Amylin, known as islet amyloid polypeptide, is a peptide hormone that is stored in pancreatic beta-cells and co-secreted with insulin in response to many factors, including glucose, arginine, and fatty acids [1]
The second FrAB-based assay utilizes an fluorescence polarization (FP)-based protocol; this one is better suited for high-throughput screening (HTS) applications because it is a pure “mix-and-measure” assay [22], but it requires specialized equipment capable of reading FP or fluorescence anisotropy
The development of successful assays for amylin degradation based on the agarose precipitation (AP) and FP protocols was more complicated than expected
Summary
Known as islet amyloid polypeptide, is a peptide hormone that is stored in pancreatic beta-cells and co-secreted with insulin in response to many factors, including glucose, arginine, and fatty acids [1]. IDE degrades several other peptide hormones relevant to glucose homeostasis, including insulin and glucagon [7,8]; there is growing interest in disentangling the different ways that IDE activity can regulate glucose homeostasis, in particular by developing substrate-selective inhibitors such as the insulin-selective IDE inhibitor recently developed by Maianti and colleagues [16]. Despite growing interest in amylin degradation, only expensive and cumbersome assays based on ELISA and HPLC are currently available. To address this need, we describe here the development and characterization of three novel assays for amylin degradation that are inexpensive, easy to use, and highly quantitative
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