Human Islet Amyloid Polypeptide Overexpression in INS-1E Cells Influences Amylin Oligomerization under ER Stress and Oxidative Stress

Human amylin or islet amyloid polypeptide (hIAPP) is synthesized in the pancreatic β-cells and has been shown to contribute to the pathogenesis of type 2 diabetes (T2D) in vitro and in vivo. This study compared amylin oligomerization/expression and signal transduction under endoplasmic reticulum (ER) stress and oxidative stress. pCMV-hIAPP-overexpressing INS-1E cells presented different patterns of amylin oligomerization/expression under ER stress and oxidative stress. Amylin oligomerization/expression under ER stress showed three amylin oligomers of less than 15 kDa size in pCMV-hIAPP-overexpressing cells, while one band was detected under oxidative stress. Under ER stress conditions, HIF1α, p-ERK, CHOP, Cu/Zn-SOD, and Bax were significantly increased in pCMV-hIAPP-overexpressing cells compared to the pCMV-Entry-expressing cells (control), whereas p-Akt, p-mTOR, Mn-SOD, catalase, and Bcl-2 were significantly decreased. Under oxidative stress conditions, HIF1α, p-ERK, CHOP, Mn-SOD, catalase, and Bcl-2 were significantly reduced in pCMV-hIAPP-overexpressing cells compared to the control, whereas p-mTOR, Cu/Zn-SOD, and Bax were significantly increased. In mitochondrial oxidative phosphorylation (OXPHOS), the mitochondrial complex I and complex IV were significantly decreased under ER stress conditions and significantly increased under oxidative stress conditions in pCMV-hIAPP-overexpressing cells compared to the control. The present study results demonstrate that amylin undergoes oligomerization under ER stress in pCMV-hIAPP-overexpressing cells. In addition, human amylin overexpression under ER stress in the pancreatic β cells may enhance amylin protein aggregation, resulting in β-cell dysfunction.

Cyanidin-3-O-Glucoside improves the viability of human islet cells treated with amylin or Aβ1-42 in vitro

Islet transplantation is being considered as an alternative treatment for type 1 diabetes. Despite recent progress, transplant recipients continue to experience progressive loss of insulin independence. Cyanidin-3-O-Glucoside (C3G) has shown to be protective against damage that may lead to post-transplant islet loss. In this study, human islets cultured with or without C3G were treated with human amylin, Aβ1-42, H2O2, or rapamycin to mimic stresses encountered in the post-transplant environment. Samples of these islets were collected and assayed to determine C3G’s effect on cell viability and function, reactive oxygen species (ROS), oxidative stress, amyloid formation, and the presence of inflammatory as well as autophagic markers. C3G treatment of human islets exposed to either amylin or Aβ1-42 increased cell viability (p<0.01) and inhibited amyloid formation (p<0.01). A reduction in ROS and an increase in HO-1 gene expression as well as in vitro islet function were also observed in C3G-treated islets exposed to amylin or Aβ1-42, although not significantly. Additionally, treatment with C3G resulted in a significant reduction in the protein expression of inflammatory markers IL-1β and NLRP3 (p<0.01) as well as an increase in LC3 autophagic marker (p<0.05) in human islets treated with amylin, Aβ1-42, rapamycin, or H2O2. Thus, C3G appears to have a multi-faceted protective effect on human islets in vitro, possibly through its anti-oxidant property and alteration of inflammatory as well as autophagic pathways.

The Fluorescent Dye 1,6-Diphenyl-1,3,5-Hexatriene Binds to Amyloid Fibrils Formed by Human Amylin and Provides a New Probe of Amylin Amyloid Kinetics

The fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) is widely used as a probe of membrane order. We show that DPH also interacts with amyloid fibrils formed by human amylin (also known as islet amyloid polypeptide) in solution and this results in a 100-fold increase in DPH fluorescence for a sample of microM human amylin and 0.25 microM DPH. No increase in DPH fluorescence is observed with the non-amyloidogenic rat amylin or with freshly dissolved, non-fibrillar human amylin. The time course of amyloid formation by amylin was followed by monitoring the fluorescence of added DPH as a function of time and was similar to that monitored by the standard fluorescent probe thioflavin-T. The inclusion of DPH in the buffer did not perturb the time course of amyloid formation under the conditions examined and the time course was independent of the range of DPH concentrations tested (0.25 to 5 microM). Maximum final fluorescence intensity is observed at substoichiometric ratios of DPH to amylin. No significant increase in fluorescence was observed during the lag phase of amyloid formation, and the implications for the structure of amylin pre-fibril oligomers are discussed. Human amylin contains three aromatic residues. A triple aromatic to leucine mutant forms amyloid and DPH binds to the resulting fibrils, indicating that interactions with aromatic side chains are not required for DPH amylin amyloid interactions. DPH may be especially useful for studies on mutant amylins and other polypeptides in which changes in charged residues might complicate interpretation of thioflavin-T fluorescence.

α-CGRP disrupts amylin fibrillization and regulates insulin secretion: implications on diabetes and migraine

CGRP concentration is elevated in migraine conditions. The protective effect of migraine against type 2 diabetes is attributed to the ability of CGRP to remodel human amylin aggregation and to suppress the secretion of mouse insulin 2 (the orthologue of human insulin).