Receptor-mediated toxicity of human amylin fragment aggregated by short- and long-term incubations with copper ions
Human amylin (hA1–37) is a polypeptide hormone secreted in conjunction with insulin from the pancreatic β-cells involved in the pathogenesis of type 2 diabetes mellitus (T2DM). The shorter fragment hA17–29 than full-length peptide is capable to form amyloids "in vitro". Here, we monitored the time course of hA17–29 β-amyloid fibril and oligomer formation [without and with copper(II)], cellular toxicity of different amyloid aggregates, and involvement of specific receptors (receptor for advanced glycation end-products, RAGE; low-affinity nerve growth factor receptor, p75-NGFR) in aggregate toxicity. Fibril and oligomer formation of hA17–29 incubated at 37 °C for 0, 48, and 120 h, without or with copper(II), were measured by the thioflavin T fluorescence assay and ELISA, respectively. Toxicity of hA17–29 aggregates and effects of anti-RAGE and anti-p75-NGFR antibodies were evaluated on neuroblastoma SH-SY5Y viability. Fluorescence assay of hA17–29 indicates an initial slow rate of soluble fibril formation (48 h), followed by a slower rate of insoluble aggregate formation (120 h). The highest quantity of oligomers was recorded when hA17–29 was pre-aggregated for 48 h in the presence of copper(II) showing also the maximal cell toxicity (−44% of cell viability, p < 0.01 compared to controls). Anti-RAGE or anti-p75-NGFR antibodies almost abolished cell toxicity of hA17–29 aggregates. These results indicate that copper(II) influences the aggregation process and hA17–29 toxicities are especially attributable to oligomeric aggregates. hA17–29 aggregate toxicity seems to be mediated by RAGE and p75-NGFR receptors which might be potential targets for new drugs in T2DM treatment.
KeywordsAggregation Amylin Oligomers Cell viability Copper Receptor-mediated cell toxicity
The International Internship Programme, University of Catania, Italy, supported G.C. during his Internship to the University of Kansas, School of Pharmacy. The present work was financially supported by the University of Catania (Italy) “Athenaeum Research Projects”: Glycation.
Compliance with ethical standards
Conflict of interests
The authors declare that they have no conflict of interests.
Not applicable. This is an in vitro study.
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