The mechanism of death evoked by human amylin in pancreatic islet B cells

Abstract

Amylin is a 37-amino acid peptide usually cosecreted with insulin from pancreatic islet β-cells. It is implicated in the regulation of normal glucose metabolism and thought to induce pathological features of non-insulin-dependent diabetes mellitus (NIDDM). In particular, human amylin (hA) deposits as islet amyloid, and is associated with the loss of insulin-producing islet β-cells in NIDDM. The biochemical mechanism of hA-evoked death in cultured RINm5F pancreatic islet β-cells has been investigated in this thesis. Synthetic hA but not rat amylin (rA) aggregated in aqueous solution, formed fibrils, and evoked β-cell death in a time- and concentration-dependent manner. The cell death exhibited apoptotic features, including inter-nucleosomal DNA fragmentation, mitochondrial dysfunction, delayed membrane lysis, aurintricarboxylic acid suppression and cell membrane blebbling. Cytotoxicity of hA was inhibited by Congo red (an amyloid-binding dye), 8-37hA fragment (fibril-forming but non-toxic), 1-40βA or 25-35βA (Alzheimer-associated peptide), but neither by sorbitol (inhibitory to hA fibril formation), rA nor its 8-37rA peptide (non-fibril-forming and non-toxic). Preformed large amyloid deposits of hA were less potent in causing β-cell death than small aggregates. These data suggest that hA induces β-cell apoptosis via small aggregates through a possible membrane receptor pathway. Inhibitors of protein and mRNA synthesis did not inhibit hA-evoked apoptosis, but rather enhanced or directly triggered β-cell death during prolonged exposure. Likewise, Ca2+ modulators, which alter intracellular free Ca2+ concentration ([Ca2+]i), failed to prevent hA cytotoxicity and were ultimately cytotoxic themselves. Fura-2 loading and 45Ca2+ uptake studies indicated that hA did not mobilise intracellular Ca2+ during its toxicity. These results indicate a protein synthesis- and Ca2+-independent process of hA toxicity RINm5F islet β-cells. The studies reported in this thesis have established a new in vitro model of hA-evoked apoptosis using cultured RINm5F pancreatic islet β-cells. A new model of NIDDM pathogenesis is presented and discussed.