Abstract
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.
Highlights
Human amylin or islet amyloid polypeptide and rat IAPP have 83% sequence identity, human amylin or islet amyloid polypeptide (hIAPP) rapidly aggregates into amyloid fibrils in vitro while rIAPP does not form fibrils under the same experimental conditions [1,2,3]
endoplasmic reticulum (ER) stress is induced by thapsigargin (1 μM) or tunicamycin (2 μg/mL) treatments with/without 4-phenylbutyric acid (4-PBA, 20 μM). pCMV6-Entry and pCMV-hIAPP vectors were stably transfected in the INS-1E cells and selected with G418
The introduced hIAPP gene was confirmed by hIAPP overexpression determined using Western blot
Summary
Human amylin or islet amyloid polypeptide (hIAPP) and rat IAPP (rIAPP) have 83% sequence identity, hIAPP rapidly aggregates into amyloid fibrils in vitro while rIAPP does not form fibrils under the same experimental conditions [1,2,3]. When comparing these two peptides sequences, the variation is evident in the region spanning residues 20–29. They suggested that N21Q fibrillar structure enables conformational self-assembly, and the resulting IAPP assembly can be cytotoxic
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