Molecular mechanism of human amylin-mediated cytotoxicity

Abstract

Human amylin (hA) is a small fibrillogenic protein. It is the major constituent of pancreatic islet amyloid, which occurs in most subjects with type 2 diabetes mellitus (T2DM). The toxicity of hA towards islet β-cells is thought to be responsible for the gradual loss of their function in T2DM. Preventing hA-mediated cytotoxicity has been proposed as a route to halt the progression of this disease. However, the mechanism, by which hA evokes β-cell death is not fully understood. Hence, the aim of this project was to investigate the molecular basis of hA-mediated cytotoxicity. These studies were conducted using a cell culture model of hA-mediated cytotoxicity: the rat insulinoma RINm5F β-cells. In order to determine the relationship between specific residues/functional groups within the amylin molecule and the mechanism of β-cell death, the cytotoxic activity of amylin sequence variants towards RINm5F cells was investigated. Here it is shown that the NH2-terminal (1-7) region of hA, which contains the intramolecular disulphide bridge, is not necessary for cytotoxic activity, but that its presence or absence modifies the level of this activity. Studies of rat amylin (rA) variants containing one, two or three of the following substitutions from the hA sequence: R18H, L23F and V26I, revealed that these hA residues have very little influence on cytotoxicity. These results indicate that proline residues in rA at positions 25, 28 and 29 attenuate not only the amyloidogenic potential of this peptide, but are also largely responsible for its lack of cytotoxic activity. In the second part of the studies, the proposed role of oxidative stress in the mechanism of hA-mediated cytotoxicity was evaluated. The observations that antioxidants protect RINm5F cells against hA-evoked death suggest that hA induces oxidative stress. However, protection afforded by free radical scavengers is only partial. Moreover, intracellular accumulation of reactive oxygen species (ROS) is observed in a small number of hA-treated cells, while the intracellular reduced glutathione (GSH) levels remain unchanged. These findings indicate that oxidative stress is not the only mechanism through which hA evokes β-cell death. By contrast, thiol antioxidants, which not only scavenge ROS, but also modulate intracellular GSH levels and act as thiol reducing agents, afford RINm5F cells almost complete protection against hA toxicity. The investigations into the mechanism of this protection suggest that thiol antioxidants may exert their actions through effects on hA aggregation, via reduction of the disulphide bridge and by modulating the redox status of cellular thiol-containing molecules other than GSH, indicating that an important component of the hA-activated cell deathsignalling pathway is regulated via its thiol redox status. Finally, the relationship between the aggregation state of hA and its cytotoxic activity was addressed. These studies showed that hA in the form of mature fibrils, similar to those present in amyloid deposits of subjects with T2DM, is inert towards β-cells. By contrast, highly toxic hA contains an abundance of spheroidal structures. Hence, these results are consistent with the hypothesis that hA toxicity is mediated by non-fibrillar oligomers representing intermediate assemblies in the process of aggregation. The findings of this research suggest some possible sites for intervention in the process of hA-mediated toxicity of β-cells. These suggestions will hopefully lead to the development of effective strategies to prevent β-cell loss in T2DM.