Attenuation of Olanzapine-Induced Endoplasmic Reticulum Stress Improves Insulin Secretion in Pancreatic Beta Cells

Abstract

Second-generation antipsychotics (SGAs), in particular, olanzapine and clozapine, have been associated with the development of type 2 diabetes mellitus (T2D) and metabolic syndrome in individuals with schizophrenia. In this context, beta cell dysfunction is a plausible mechanism by which SGAs cause T2D. Herein, we analyzed the direct effects of olanzapine, a commonly prescribed SGA with diabetogenic properties, on the INS-1 (821/13) beta cell line and isolated pancreatic islets. Treatment of INS-1 beta cells with non-toxic concentrations of olanzapine (3-6 μM) during 4 h activated endoplasmic reticulum (ER) stress-mediated signaling by increasing PERK/eIF2α phosphorylation, IRE-1 phosphorylation and XBP-1 splicing. Moreover, glucose-stimulated insulin secretion (GSIS) was inhibited when olanzapine was present for 16 h. The insulin secretory function of INS-1 cells was restored by inhibiting olanzapine-induced ER stress with tauroursodeoxycholic acid (TUDCA). Similar effects of olanzapine with or without TUDCA on ER-stress-mediated signaling and GSIS were found in pancreatic islets from female mice. Our results indicate that early activation of ER stress in pancreatic beta cells is a potential mechanism behind the alterations in glucose homeostasis induced by olanzapine.

Keywords: ER stress; beta cell; olanzapine; schizophrenia; second-generation antipsychotics; type 2 diabetes.

Figure 1

Effect of olanzapine on the cellular viability of INS-1 cells after 24 h of treatment. (a) Representative crystal violet staining images captured with a light microscope at 10× magnification. (b) Cell viability monitored by crystal violet assay, represented as the percentages of viability compared to control cells. (c) Cell viability measured by the MTT assay relative to control cells. Data are shown as percentages of viability compared to control cells. Control cells were treated with DMSO (0.01%). (d) Representative images of confocal immunofluorescence of islets upon treatment with 6 μM olanzapine for 24 h. Insulin (in red) and glucagon (in green). 40× magnification. Scale bar = 50 μm. Experiments were performed in 20 islets per condition. Data are presented as means ± SEM, n = 3 independent experiments (3 replicates/condition), passage 25–31. p-values were determined by two-way ANOVA and Bonferroni post-hoc test. ** p < 0.01, *** p < 0.001, comparison between treatments.

Figure 2

Effects of olanzapine treatment on ER-stress-mediated signaling in INS-1 beta cells and pancreatic islets. (a) Western blot analysis of the effect of olanzapine in ER stress markers in INS-1 beta cells after 4 h of treatment showing phosphorylation levels of PERK, eIF2α and IRE1α. Control: 0.01% DMSO was used as a vehicle. Representative Western blot images are shown. P-PERK, p-IRE1α and tubulin correspond to the same gel, and p-eiF2α and eiF2α correspond to the same gel. The graphs show the fold increases versus control cells. Tubulin and total eIF2α were used as loading controls. The experiments were repeated six times, independently, with INS-1 cells in different passages, with triplicate conditions in each one. Data are presented as mean ± SEM of n = 6 independent experiments (3 replicates/condition), passage 25–35. p-values were determined by one-way ANOVA and Bonferroni Post-hoc test. * p < 0.05, *** p < 0.001 versus control (non-treated) cells. (b) XBP1 splicing analyzed in INS-1 beta cells treated with olanzapine under similar experimental conditions. Data are presented as mean ± SEM of n = 2 independent experiments (3 replicates/condition). Differences between 2 groups were compared using the Mann–Whitney U test ** p < 0.01 versus control (non-treated) cells. (c) Pancreatic islets from female mice were incubated with 6 μM olanzapine for 4 h, and phosphorylation levels of PERK, IRE1α and eIF2α were analyzed by Western blot. Representative Western blot images from 2 independent experiments are shown. P-PERK and GAPDH correspond to the same gel, p-IRE1α and Vinculin correspond to the same gel and p-eiF2α and eiF2α correspond to the same gel. Each lane corresponds to a cell lysate from a pool of 80–100 islets isolated from one mouse. Similar results were obtained in pools of islets from 2 independent mice.

Figure 3

Effects of 4 or 24 h treatment with olanzapine on GSIS and insulin content in INS-1 beta cells. (a) GSIS in INS-1 beta cells upon olanzapine treatment for 4 h. (b) Insulin content at 4 h of olanzapine treatment. (c) GSIS in INS-1 beta cells upon olanzapine treatment for 24 h. (d) Insulin content at 24 h. Control: 0.01% DMSO was used as vehicle. Data are presented as means ± SEM of n = 3 independent experiments (3 replicates/condition), passage 30–36. In (a,c), p-values were analyzed by two-way ANOVA and Bonferroni post-hoc test. ns (non-significant), ** p < 0.01 and *** p < 0.001 versus control cells with 16.7 mM glucose. In (d), p-values were determined by one-way ANOVA and Bonferroni Post-hoc test. * p < 0.05, versus control (non-treated) cells.

Figure 4

Alleviation of olanzapine-induced ER-stress-mediated signaling by TUDCA in INS-1 cells and pancreatic islets. (a) PERK, IRE1α and eIF2α phosphorylation in INS-1 beta cells upon co-treatment with olanzapine and 250 μM TUDCA. Control: 0.01% DMSO was added as vehicle. Quantification and statistical analysis of p-PERK, p-eIF2α and p-IRE1α levels. Representative Western blot images are shown. P-PERK, p-IRE1α and tubulin correspond to the same gel, and p-eiF2α and eiF2α correspond to the same gel. The graphs show the fold increase versus control cells. Tubulin and total eIF2α were used as loading controls. The experiments were repeated six times, independently, with INS-1 cells in different passages, with triplicate conditions in each one. Data are presented as mean ± SEM of n = 3 independent experiments (3 replicates/condition), passage 25–35. p-values were determined by one-way ANOVA and Bonferroni post-hoc test * p < 0.05, *** p < 0.001 compared to INS-1 treated with DMSO; ^††† p < 0.001 compared to co-treatment with the same dose of olanzapine and TUDCA. (b) XBP1 splicing in INS-1 beta cells upon co-treatment with 6 μM olanzapine and 250 μM TUDCA for 3 h. Data are presented as mean ± SEM of n = 2 independent experiments (3 replicates/condition). Differences between 2 groups were compared using Mann–Whitney U test: ^†† p < 0.01 compared with olanzapine plus TUDCA. (c) Pancreatic islets from female mice were incubated with 6 μM olanzapine and 250 μM TUDCA for 4 h, and phosphorylation levels of PERK and IRE1α were analyzed by Western blot. Representative Western blot images from 2 independent experiments are shown. P-PERK, PERK and vinculin correspond to the same gel; p-IRE1α and IRE1 and GAPDH correspond to the same gel; and p-eiF2α and eiF2α correspond to the same gel. Each lane corresponds to a cell lysate from a pool of 80–100 islets isolated from one mouse. Similar results were obtained in pools of islets from 2 independent experiments.

Figure 5

Restoration of insulin secretion in INS-1 cells and pancreatic islets by co-treatment with olanzapine and TUDCA. (a) GSIS in INS-1 beta cells co-treated with olanzapine (6 μM) in the absence or presence of TUDCA (250 μM) for 24 h. Data are presented as mean ± SEM of n = 3 independent experiments (3 replicates/condition), passage 25–35. (b) GSIS in pancreatic islets under the same experimental conditions described in (a). Experiments were performed in 3–4 replicates per condition in islets from n = 6 mice. p-values were determined by two-way ANOVA and Bonferroni post-hoc test. * p < 0.05, compared to INS-1 or islets treated with DMSO and stimulated with 16.7 mM glucose. ^†† p < 0.01, compared to INS-1 cells or islets treated with olanzapine plus TUDCA and stimulated with 16.7 mM glucose.