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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 (Cu(2+)), very little is known about the mechanism of interaction between Cu(2+) and amylin in pancreatic ?-cells, including its pathological significance. Hence, in this study we investigated the mechanism by which Cu(2+) and human amylin catalyze formation of reactive oxygen species (ROS) in cells and in vitro, and examined the modulatory effect of Cu(2+) on amylin aggregation and toxicity in pancreatic rat insulinoma (RIN-m5F) ?-cells. Our results indicate that Cu(2+) 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 Cu(2+). In a marked contrast to human and rat amylin, and in the presence of the reducing agents glutathione and ascorbate, Cu(2+) produced ?M 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 Cu(2+) and glutathione. Similarly, human amylin was found to also decrease hydroxyl radical formation elicited by Cu(2+) and glutathione. Furthermore, Cu(2+) 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.

SUBMITTER: Lee EC

PROVIDER: S-EPMC4041291 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Copper(II)-human amylin complex protects pancreatic cells from amylin toxicity.

Lee Elizabeth C EC Ha Emmeline E Singh Sanghamitra S Legesse Linda L Ahmad Sana S Karnaukhova Elena E Donaldson Robert P RP Jeremic Aleksandar M AM

Physical chemistry chemical physics : PCCP 20130801 30

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 (Cu(2+)), very little is known about the mech ...[more]

PMID: 23793354

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Project description:The objective of these studies was to clarify the role of Ca(2+) in the mechanism of death evoked by human amylin (hA) in islet beta-cells. hA forms fibrils in vitro and islet amyloid in vivo. Here we show that pure synthetic hA aggregated in solution, formed fibrils and evoked death in cultured RINm5F islet beta-cells in a time-dependent (0-24 h) and concentration-dependent (0-20 microM) manner. Dying cells underwent shrinkage of the nucleus, with clumping and segregation of chromatin into masses that lay against the nuclear envelope, and internucleosomal DNA fragmentation. These cells therefore show many features of apoptosis, although aspects of the morphology might be characteristic of this particular cell type rather than of a general apoptotic nature. Aurintricarboxylic acid, an inhibitor of both Ca(2+)-dependent and Ca(2+)-independent nucleases, suppressed this DNA fragmentation and inhibited apoptosis at concentrations between 25 and 200 microM. Direct measurements of the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) in fura-2 acetoxymethyl ester (AM)-loaded beta-cells showed that neither hA nor its non-cytotoxic homologue, rat amylin were effective in raising [Ca(2+)](i). Modulators of Ca(2+) regulation were tested for their effects on hA-induced beta-cell apoptosis. Ca(2+) ionophore (A23187) and thapsigargin (an inhibitor of endoplasmic reticular Ca(2+)-ATPase activity) by themselves evoked apoptosis accompanied by increased [Ca(2+)](i). Neither the Ca(2+) channel blocker verapamil, the extracellular Ca(2+) chelator EGTA nor the cytosolic Ca(2+) buffer bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid ('BAPTA')/AM protected beta-cells from hA-evoked apoptosis. Prolonged incubation of beta-cells with a lethal dose of hA altered neither the basal [Ca(2+)](i) nor the thapsigargin-induced release of Ca(2+) from intracellular stores. Furthermore, (45)CaCl(2) uptake by RINm5F cells did not differ in the presence or absence of hA. These results suggest that, whereas alterations in cytosolic Ca(2+) homoeostasis do have a significant role in certain forms of beta-cell death, they do not contribute to the pathway of apoptosis evoked by hA in islet beta-cells.

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Copper(II)-human amylin complex protects pancreatic cells from amylin toxicity.

Publications

Copper(II)-human amylin complex protects pancreatic cells from amylin toxicity.

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