Copper(ii)–human amylin complex protects pancreatic cells from amylin toxicity

Abstract

Human amylin-derived oligomers and aggregates are believed to play an important role in the pathogenesis of type II diabetes mellitus (T2DM). In addition to amylin-evoked cell attrition, T2DM is often accompanied by elevated serum copper levels. Although previous studies have shown that human amylin, in the course of its aggregation, produces hydrogen peroxide (H₂O₂) in solution, and that this process is exacerbated in the presence of copper(II) ions (Cu²⁺), very little is known about the mechanism of interaction between Cu²⁺ and amylin in pancreatic β-cells, including its pathological significance. Hence, in this study we investigated the mechanism by which Cu²⁺ and human amylin catalyze formation of reactive oxygen species (ROS) in cells and in vitro, and examined the modulatory effect of Cu²⁺ on amylin aggregation and toxicity in pancreatic rat insulinoma (RIN-m5F) β-cells. Our results indicate that Cu²⁺ interacts with human and rat amylin to form metalo-peptide complexes with low aggregative and oxidative properties. Human and non-amyloidogenic rat amylin produced minute (nM) amounts of H₂O₂, the accumulation of which was slightly enhanced in the presence of Cu²⁺. In a marked contrast to human and rat amylin, and in the presence of the reducing agents glutathione and ascorbate, Cu²⁺ produced μM concentrations of H₂O₂ surpassing the amylin effect by several fold. The current study shows that human and rat amylin not only produce but also quench H₂O₂, and that human but not rat amylin significantly decreases the amount of H₂O₂ in solution produced by Cu²⁺ and glutathione. Similarly, human amylin was found to also decrease hydroxyl radical formation elicited by Cu²⁺ and glutathione. Furthermore, Cu²⁺ mitigated the toxic effect of human amylin by inhibiting activation of pro-apoptotic caspase-3 and stress-kinase signaling pathways in rat pancreatic insulinoma cells in part by stabilizing human amylin in its native conformational state. This sacrificial quenching of metal-catalyzed ROS by human amylin and copper's anti-aggregative and anti-apoptotic properties suggest a novel and protective role for the copper–amylin complex.