Nicotinic acetylcholine receptor signaling regulates inositol-requiring enzyme 1α activation to protect β-cells against terminal unfolded protein response under irremediable endoplasmic reticulum stress

Abstract

Aims/introduction: Under irremediable endoplasmic reticulum (ER) stress, hyperactivated inositol-requiring enzyme 1α (IRE1α) triggers the terminal unfolded protein response (T-UPR), causing crucial cell dysfunction and apoptosis. We hypothesized that nicotinic acetylcholine receptor (nAChR) signaling regulates IRE1α activation to protect β-cells from the T-UPR under ER stress.

Materials and methods: The effects of nicotine on IRE1α activation and key T-UPR markers, thioredoxin-interacting protein and insulin/proinsulin, were analyzed by real-time polymerase chain reaction and western blotting in rat INS-1 and human EndoC-βH1 β-cell lines. Doxycycline-inducible IRE1α overexpression or ER stress agents were used to induce IRE1α activation. An α7 subunit-specific nAChR agonist (PNU-282987) and small interfering ribonucleic acid for α7 subunit-specific nAChR were used to modulate nAChR signaling.

Results: Nicotine inhibits the increase in thioredoxin-interacting protein and the decrease in insulin 1/proinsulin expression levels induced by either forced IRE1α hyperactivation or ER stress agents. Nicotine attenuated X-box-binding protein-1 messenger ribonucleic acid site-specific splicing and IRE1α autophosphorylation induced by ER stress. Furthermore, PNU-282987 attenuated T-UPR induction by either forced IRE1α activation or ER stress agents. The effects of nicotine on attenuating thioredoxin-interacting protein and preserving insulin 1 expression levels were attenuated by pharmacological and genetic inhibition of α7 nAChR. Finally, nicotine suppressed apoptosis induced by either forced IRE1α activation or ER stress agents.

Conclusions: Our findings suggest that nAChR signaling regulates IRE1α activation to protect β-cells from the T-UPR and apoptosis under ER stress partly through α7 nAChR. Targeting nAChR signaling to inhibit the T-UPR cascade may therefore hold therapeutic promise by thwarting β-cell death in diabetes.

Keywords: Inositol-requiring enzyme 1α; Nicotinic acetylcholine receptor; Pancreatic β cell.

Figure 1

Nicotine reverses terminal unfolded protein response induced by overexpression of Flag‐tagged full‐length inositol‐requiring enzyme 1α in INS‐1 cells. (a,b) Relative messenger ribonucleic acid (mRNA) expression levels of (a) TXNIP and (b) insulin 1 (Ins1) in IRE1α overexpressing INS‐1 cells (n = 3‐8). The cells were treated with doxycycline hyclate (Dox; 1 μg/mL) with or without nicotine at indicated concentrations for indicated times. Relative mRNA levels of those in the cells treated with only nicotine for 24 h are shown in the second lane as a control. (c) Western blots of TXNIP and proinsulin in IRE1α overexpressing INS‐1 cells treated with Dox with or without 1 mmol/L nicotine for 24 h. All data are expressed as the mean ± standard error of the mean. ***P < 0.001 versus control.

Figure 2

Nicotine regulates inositol‐requiring enzyme 1α (IRE1α) activation induced by endoplasmic reticulum stress in INS‐1 and EndoC‐βH1 cells. (a) EtBr‐stained agarose gel electrophoresis of Pst1‐digested X‐box‐binding protein‐1 (XBP1) amplicons obtained by polymerase chain reaction in INS‐1 cells treated with Tg (500 nmol/L, 6 h) with or without nicotine (1 mmol/L) for 3 h. (b,c) The numbers above are calculated from the ratio metric quantitation of the level of spliced XBP1 complementary deoxyribonucleic acid to that of total XBP1 complementary deoxyribonucleic acids. sXBP1 expression measured by quantitative reverse transcription polymerase chain reaction treated with (b) thapsigargin (Tg; 500 nmol/L, 6 h; n = 10) or (c) tunicamycin (Tm; 100 μg/mL, 3 h; n = 3) with or without nicotine (1 mmol/L) for 6 h in INS‐1 cells. (d) Immunoblots with anti‐IRE1α antibody for proteins separated using Phos‐tag gel for INS‐1 cells treated with Tg (500 nmol/L) with or without nicotine (1 mmol/L) for 6 h. (e) Expression of CHRNA7 messenger ribonucleic acid (mRNA) by polymerase chain reaction in human EndoC‐βH1 cells. The expression of the in pcDNA3.1‐human CHRNA7 vector is shown as the positive control. (f) Relative sXBP1 mRNA expression in EndoC‐βH1 cells treated with Tg (10 μmol/L) with or without nicotine (1 mmol/L) for 6 h (n = 3). All data are expressed as the mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001 versus control.

Figure 3

Nicotine attenuates terminal unfolded protein response induced by endoplasmic reticulum stress in INS‐1 and EndoC‐βH1 cells. (a–d) Relative messenger ribonucleic acid (mRNA) levels of (a,c) TXNIP and (b,d) Ins1 by quantitative polymerase chain reaction in INS‐1 cells treated with thapsigargin (Tg; 500 nmol/L, 6 h) or tunicamycin (Tm; 200 nmol/L, 24 h) with or without nicotine (1 mmol/L; n = 6‐9). (e,h) Western blots of TXNIP and proinsulin in INS‐1 cells incubated with (e) Tg (500 nmol/L, 6 h; n = 3) or with (h) high medium glucose (28 mmol/L, 24 h) with or without nicotine (1 mmol/L). (f,g) Relative mRNA levels of (f) TXNIP and (g) insulin in EndoC‐βH1 cells treated with Tg (10 μmol/L) with or without nicotine (100 μmol/L) for 6 h. All data are expressed as the mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001 versus control.

Figure 4

The nicotinic acetylcholine receptor (nAChR) α7 subunit‐specific agonist, PNU‐282987, regulates terminal unfolded protein response induced by inositol‐requiring enzyme 1α (IRE1α) overexpression and endoplasmic reticulum stress in INS‐1 and EndoC‐βH1 cells. (a,b) Relative messenger ribonucleic acid (mRNA) expression levels of (a) TXNIP and (b) Ins1 in IRE1α‐overexpressing INS‐1 cells treated with doxycycline hyclate (Dox; 1 μg/mL) with or without PNU‐282987 (PNU), the agonist of nAChR α7 at indicated doses for 24 h (n = 4). (c) Western blots of TXNIP and proinsulin in IRE1α‐overexpressing INS‐1 cells treated with Dox (1 μg/mL) with or without PNU at indicated doses for 24 h. (d,e) Relative mRNA expression levels of splice X‐box‐binding protein‐1 (sXBP1) in (d) INS‐1 cells treated with Tm (200 nmol/L, 24 h) or in (e) EndoC‐βH1 cells with thapsigargin (Tg; 10 μmol/L, 6 h) with or without PNU (10 μmol/L) (n = 3–4). (f,g) Relative mRNA levels of (f) TXNIP (n = 3) and (g) Ins1 (n = 6) in INS‐1 cells treated with Tg (500 nmol/L) with or without PNU (10 μmol/L) for 6 h. (h) Western blots of TXNIP and proinsulin in INS‐1 cells treated with Tg (500 nmol/L) with or without PNU (10 μmol/L) for 6 h. All data are expressed as the mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001 versus control.

Figure 5

Nicotinic acetylcholine receptor (nAChR) signaling regulates inositol‐requiring enzyme 1α (IRE1α) signaling through the α7 subunits in INS‐1 cells. (a) Relative messenger ribonucleic acid (mRNA) levels of TXNIP in INS‐1 cells co‐treated with tunicamycin (Tm; 200 nmol/L) and nicotine (1 mmol/L) for 24 h, pretreated with or without α‐bungarotoxin (αBTX, 1 μmol/L), an α7‐specific nAChR inhibitor, for 3 h (n = 6). (b,c) IRE1α‐overexpressing INS‐1 cells were transfected with a non‐specific control small interfering ribonucleic acid (siCTL) or two independent small interfering ribonucleic acids targeting CHRNA7 (siCHRNA7 #1 and #2). After 24 h, the cells were treated with doxycycline hyclate (Dox; 1 μg/mL) with or without nicotine (1 mmol/L) or PNU (20z0 μmol/L) for 24 h. Relative (b) TXNIP and(c) Ins1 mRNA levels were determined by quantitative polymerase chain reaction (n = 3‐5). All data are expressed as the mean ± standard error of the mean. **P < 0.01, ***P < 0.001 versus control.

Figure 6

Nicotinic acetylcholine receptor (nAChR) signaling inhibits apoptosis induced by inositol‐requiring enzyme 1α (IRE1α) activation and endoplasmic reticulum stress in INS‐1 cells. (a,b) Caspase‐3/7 fluorescence in IRE1α overexpressing INS‐1 cells treated with (a) doxycycline hyclate (Dox; 1 μg/mL) with or without nicotine (1 mmol/L) or (b) PNU (10 nmol/L) for 72 h (n = 4). (c) Caspase‐3/7 fluorescence (n = 4) or (d) percentage of annexin V‐positive cells (n = 6) in INS‐1 cells treated with tunicamycin (Tm; 100 ng/mL) with or without nicotine (1 mmol/L) for 48 h. All data are expressed as the mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001 versus control.