The aggregation potential of human amylin determines its cytotoxicity towards islet beta-cells.
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 beta-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 beta-cells. Highly toxic amylin contained few preformed fibrils and initially showed little beta-sheet content, but underwent marked time-dependent aggregation and beta-conformer formation following dissolution. By contrast, low-toxicity amylin contained abundant preformed fibrils, and demonstrated high initial beta-sheet content but little propensity to aggregate further once dissolved. Thus, mature amylin fibrils are not toxic to beta-cells, and aggregates of fibrils such as occur in pancreatic islet amyloid in vivo are unlikely to contribute to beta-cell loss. Rather, the toxic molecular species is likely to comprise soluble oligomers with significant beta-sheet content. Attempts to find ways of protecting beta-cells from amylin-mediated death might profitably focus on preventing the conformational change from random coil to beta-sheet.
Konarkowska
Barbara
B
School of Biological Sciences, Faculty of Science, University of Auckland, New Zealand.
Aitken
Jacqueline F
JF
Kistler
Joerg
J
Zhang
Shaoping
S
Cooper
Garth J S
GJ
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry / Q000494:pharmacology* / Q000633:toxicity; D002460:Cell Line; D002942:Circular Dichroism; D006801:Humans; D058228:Islet Amyloid Polypeptide; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000682:Amyloid; D058228:Islet Amyloid Polypeptide