Background: Pancreatic β-cells are specialized for the production of insulin and susceptible to endoplasmic reticulum (ER) stress. Irremediable ER stress is coupled with terminal-unfolded protein response (T-UPR) through activating IRE1α, which leads to β-cell dysfunction and apoptosis. Previous reports show the effects of nAChR signaling against ER stress-induced neurotoxicity. However, it is still unknown if and how nAChR signaling affects T-UPR in β-cells. The aim of the study is to determine if and how nAChR signaling reduces T-UPR by regulating IRE1α activity in β-cells.
Methods: We investigate the role of nAChR signaling on T-UPR signaling under ER stress or specific IRE1αactivation using rat insulinoma cell line, INS-1 cells. Cells were treated with Nicotine; an agonist of nAChR, α-Bungarotoxin(α-BTX); an antagonist of nAChR α7 subunit, and Tunicamycin(Tm) and Thapsigargin(Tg); ER stress inducers. Expression levels of spliced XBP1 and TXNIP/Insulin1 (Proinsulin), as T-UPR markers, were analyzed by real time PCR and/or Western blotting.
Results: We first confirmed the expression levels of nAChR α7 subunits in INS1 cells. ER stress inducers (Tm or Tg) increased XBP1 splicing, and Nicotine (>10 μM) reduced it. Nicotine reversed ER stress-induced increase of TXNIP and decrease of Insulin1 (proinsulin) expression in both mRNA and protein levels. Further, Nicotine reversed increase of TXNIP and decrease of Insulin1 mRNA expression levels induced by IRE1α overexpression in Dox-inducible wild type IRE1α-overexpressing INS1 cells. Those effects of Nicotine were canceled by α-BTX.
Conclusion: Our results suggest that nAChR signaling could prevent T-UPR induced by ER stress in INS1 cells.
T. Ishibashi: None. S. Morita: None. A. Doi: None. H. Iwakura: None. H. Ariyasu: None. M. Nishi: None. H. Furuta: None. T. Akamizu: None.