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.

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Human amylin is a 37-residue peptide hormone (hA1-37) secreted by β-cells of the pancreas and, along with insulin, is directly associated with type 2 diabetes mellitus (T2DM). Amyloid deposits within the islets of the pancreas represent a hallmark of T2DM. Additionally, amylin aggregates have been found in blood vessels and/or brain of patients with Alzheimer’s disease, alone or co-deposited with β-amyloid. The purpose of this study was to investigate the neuroprotective potential of human amylin in the context of endothelial-neuronal “cross-talk”. We initially performed dose-response experiments to examine cellular toxicity (quantified by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT assay) of different hA17–29 concentrations in endothelial cells (RBE4). In the culture medium of these cells, we also measured heat shock protein B5 (HspB5) levels by ELISA, finding that even a sub-toxic concentration of hA17–29 (3 µM) produced an increase of HspB5. Using a cell medium of untreated and RBE4 challenged for 48 h with a sub-toxic concentration of hA17–29, we determined the potential beneficial effect of their addition to the medium of neuroblastoma SH-SY5Y cells. These cells were subsequently incubated for 48 h with a toxic concentration of hA17–29 (20 µM). We found a complete inhibition of hA17–29 toxicity, potentially related to the presence in the conditioned medium not only of HspB5, but also of vascular endothelial growth factor (VEGF). Pre-treating SH-SY5Y cells with the anti-Flk1 antibody, blocking the VEGF receptor 2 (VEGFR2), significantly decreased the protective effects of the conditioned RBE4 medium. These data, obtained by indirectly measuring VEGF activity, were strongly corroborated by the direct measurement of VEGF levels in conditioned RBE4 media as detected by ELISA. Altogether, these findings highlighted a novel role of sub-toxic concentrations of human amylin in promoting the secretion of proteic factors by endothelial cells (HspB5 and VEGF) that support the survival and proliferation of neuron-like cells.

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