Exendin-4 protects INS-1 cells against palmitate-induced apoptosis through the IRE1α-Xbp1 signaling pathway

Abstract

The anti-apoptotic effect of the incretin analog, exendin-4 (EX-4) on pancreatic β cells is mediated via the activation of protein kinase B (Akt) signaling, and its effect is partly produced through the inhibition of endoplasmic reticulum (ER) stress. However, the molecular mechanisms that underlie the effect of EX-4 on the suppression of ER stress and the upregulation of Akt signaling are poorly understood. Inositol-requiring enzyme 1 (IRE1), a member of the ER-localized transmembrane protein family, activates its downstream transcription factor X-box binding protein 1 (XBP1) to mediate a key part of the cellular unfolded protein response in order to cope with ER stress. Using the clonal rat pancreatic β cell line INS-1, the present study produced an in vitro model of ER stress using palmitate (PA) in order to determine whether the beneficial effect of EX-4 under ER stress was regulated by the IRE1α-Xbp1 signaling pathway. The results demonstrated that the reduction in ER stress and the activation Akt by EX-4 may be associated with the upregulation of IRE1α phosphorylation and the splicing of Xbp1 mRNA, which improved PA-reduced cell viability. This effect was partially abrogated by the knockdown of IRE1α with small interfering RNA. Additionally, cellular IRE1α was phosphorylated by the protein kinase A (PKA) associated with EX-4 and the activation of IRE1α, as IRE1α phosphorylation was attenuated by the inhibition of PKA with its inhibitor. In conclusion, the data identified the IRE1α-Xbp1 signaling pathway as an essential mediator that associates EX-4 with the intracellular mechanism that inhibits ER stress and activates Akt in order to regulate β cell survival. This may provide important evidence for the use of EX-4 in treatments for type 2 diabetes.

Keywords: X-box binding protein 1; apoptosis; endoplasmic reticulum stress; exendine-4; inositol-requiring enzyme 1α.

Figure 1.

EX-4 exerts a protective role on palmitate-induced apoptosis and stimulates the IRE1α-Xbp1 signaling pathway in INS-1 cells. INS-1 cells were treated with PA (0.5 mM) and to which EX-4 (50 nM) was then added for 24 h. (A) Western blot analysis of p-eIF2α, eIF2α, p-Akt, Akt, p-FoxO1, FoxO1, p-IRE1α, IRE1α, XBP1s and Tubulin. (B) Cell viability. (C) Reverse transcription-quantitative polymerase chain reaction for Bim mRNA expression. (D) Cell apoptosis rate was determined by flow cytometry using Annexin V and propidium iodide. Tubulin was used as the internal control. Data are presented as the mean ± standard error mean of three independent experiments. *P<0.05, as indicated. IRE1α, inositol-requiring enzyme 1α; Xbp1, X-box binding protein 1; EX-4, exendin-4; p-, phosphorylated-; eIF2α, eukaryotic initiation factor 2α; Akt, protein kinase B; FoxO1, forkhead box protein O1; Bim, B-cell lymphoma-2-like protein 11; BSA, bovine serum albumin; PA, sodium palmitate.

Figure 2.

IRE1α-Xbp1 signaling pathway mediates the protective role of EX-4 on the palmitate-induced apoptosis in INS-1 cells. INS-1 cells with RNAi-mediated knockdown of IRE1α, coupled with or without the overexpression of XBP1s by transfection with a plasmid containing human XBP1s, were treated with PA (0.5 mM), with or without EX-4 (50 nM), for 24 h. (A) Western blot analysis of IRE1α, XBP1s, p-Akt, Akt and Tubulin. (B) Cell viability. (C) Reverse transcription-quantitative polymerase chain reaction for Bim mRNA expression. (D) Cell apoptosis rate was determined by flow cytometry using Annexin V and propidium iodide. Tubulin was used as the internal control. Data are presented as the mean ± standard error mean of three independent experiments. *P<0.05, as indicated. IRE1α, inositol-requiring enzyme 1α; Xbp1, X-box binding protein 1; EX-4, exendin-4; p-, phosphorylated-; si-, small interfering; Akt, protein kinase B; Bim, B-cell lymphoma-2-like protein 11; BSA, bovine serum albumin; PA, sodium palmitate.

Figure 3.

Protein kinase A is required for EX-4-induced phosphorylation of IRE1α. INS-1 cells were treated with Sodium palmitate (0.5 mM), to which FSK (10 µM), EX-4 (50 nM) or EX-4 with H89 (10 µM), was then added for 24 h. Western blot analysis was then performed for p-CREB, CREB, p-IRE1α, IRE1α and Tubulin. Tubulin was used as the internal control. Tubulin was used as the internal control. IRE1α, inositol-requiring enzyme 1α; EX-4, exendin-4; FSK, Forskolin; p-, phosphorylated-; CREB, cAMP response element-binding protein.

Figure 4.

RACK1 is essential for PKA-dependent IRE1α phosphorylation in response to EX-4 treatment. (A) INS-1 cells were treated with Sodium palmitate (0.5 mM) and then with or without EX-4 (50 nM) for 24 h. Immunoprecipitation was performed with anti-IRE1α, followed by immunoblotting with RACK1, PKA and IRE1α antibodies. Immunoprecipitation reactions were replicated three times. (B) INS-1 cells with RNAi-mediated knockdown of RACK1, were then treated with Sodium palmitate (0.5 mM), with or without EX-4 (50 nM) for 24 h. Western blot analysis of RACK1, p-CREB, CREB, p-IRE1α, IRE1α and Tubulin was then performed. Tubulin was used as the internal control. RACK1, receptor for activated C kinase 1; PKA, protein kinase A; IRE1α, inositol-requiring enzyme 1α; EX-4, exendin-4; BSA, bovine serum albumin; p-, phosphorylated-; CREB, cAMP response element-binding protein.