The Pathophysiological Mechanisms of Human Amylin Induced Stress in Pancreatic Cells
Open AccessHuman amylin (hA) is a small 37 amino acid peptide hormone secreted by pancreatic β-cells along with insulin in response to glucose stimulus. It misfolds to form insoluble aggregates human islet amyloid polypeptide (hIAPP), which has been shown to induce oxidative stress and toxicity in cells. While human amylin is amyloidogenic, rat amylin lacks this ability due to proline substitutions in IAPP. The pathology of type II diabetes mellitus (T2DM) is characterized by excessive extracellular and intracellular accumulation of toxic amylin species, soluble oligomers and insoluble fibrils in islets, eventually leading to β-cell loss. However, the exact mechanism of clearance of amylin and exactly how amyloid proteins, such as the pancreatic hormone amylin, aggregate and kill cells is still unclear. Hence, the main goal of this study has been to elucidate the molecular mechanisms and pathways involved in hA turnover and toxicity.Elevated serum copper levels are often implicated in diabetics but the significance of this event in conjunction with human amylin toxicity is still unclear. Additionally studies on another amyloid protein, brain β-amyloid have shown that copper exposure exacerbates β-amyloid pathology. Therefore, studies outlined in the first chapter involved exploring the possibility of an interaction between amylin and copper and its subsequent effect on hA induced cytotoxicity. The findings of this study suggested that hA forms a metalo-complex with Cu2+which surprisingly mitigated metalo-catalyzed ROS accumulation by quenching H2O2 and decreasing hydroxyl radical formation. In line with this finding, hA- Cu2+ complex also inhibited hA cytotoxicity by reversing mitochondrial dysfunction and preventing caspase and JNK activation, suggesting a novel and unexpected protective role of hA-Cu2+complex in pancreatic cells.While previous studies showed that hA is internalized by pancreatic cells and causes mitochondrial dysfunction, the precise cellular mechanisms and compartments involved in its turnover and degradation remain unclear. Hence, in the second chapter of this thesis, using confocal microscopy and biochemical approaches, the roles of the main cellular compartments and organelles such as mitochondria, nucleus, cytosol, Golgi apparatus and lysosomes in hA clearance and detoxification were explored. In addition, the role of the main cellular proteolytic complex, the proteasome was also explored. Results suggest that hA, following its internalization, first accumulates in the cytosol of pancreatic β-cells followed by its translocation into nucleus, and to lesser extent lysosomes. An increase in hA accumulation in the nucleus of pancreatic cells is proportional to its cytotoxicity. Cell fractionation, immunoprecipitation and confocal microscopy studies revealed that hA interacts with the catalytic subunits of the proteasome in the nucleus, which results in a decrease in 20S proteolytic activity, ubiquitination and subsequent protein stress. Inhibition of proteasome activity in turn causes a significant increase in hA accumulation and toxicity suggesting a pivotal role of the proteasomes in hA turnover and detoxification in pancreatic cells.While others and we have shown that hA induces cytotoxicity in pancreatic cells by activation of stress kinases such as JNK and p38 leading to apoptosis, the upstream factors and signaling pathways regulating their activation remain undefined. In an effort to elucidate the molecular mechanism of hA toxicity in pancreatic cells, in the third chapter, the role of apoptotic signaling kinase, ASK1 in this process was investigated. ASK1 is known to be a common target of a wide range of stressors such as free radicals, toxic amyloid proteins and possibly hA. Results revealed that in the course of its toxicity, human amylin also activates ASK1 suggesting its participation in hA evoked β-cell death. Indeed, inhibition of ASK1 activity subsequently attenuates hA cytotoxicity. This finding points to a redox-sensitive ASK1-mediated hA toxicity in pancreatic cells.
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