The aggregation potential of human amylin determines its cytotoxicity towards islet β-cells

Abstract

Human amylin is a small fibrillogenic protein that is the major constituent of pancreatic islet amyloid, which occurs in most subjects with type 2 diabetes. There is evidence that it can elicit in vitro apoptosis in islet β-cells, but the physical properties that underpin its cytotoxicity have not been clearly elucidated. Here we employed electron microscopy, thioflavin T fluorescence and CD spectroscopy to analyze amylin preparations whose cytotoxic potential was established by live-dead assay in cultured β-cells. Highly toxic amylin contained few preformed fibrils and initially showed little β-sheet content, but underwent marked time-dependent aggregation and β-conformer formation following dissolution. By contrast, low-toxicity amylin contained abundant preformed fibrils, and demonstrated high initial β-sheet content but little propensity to aggregate further once dissolved. Thus, mature amylin fibrils are not toxic to β-cells, and aggregates of fibrils such as occur in pancreatic islet amyloid in vivo are unlikely to contribute to β-cell loss. Rather, the toxic molecular species is likely to comprise soluble oligomers with significant β-sheet content. Attempts to find ways of protecting β-cells from amylin-mediated death might profitably focus on preventing the conformational change from random coil to β-sheet. © 2006 The Authors