In vitro beta cell protection from STZ by PPAG.

<p>INS-1E cells were pre-treated with 30 μM PPAG for 16 hours, exposed to 1 mM streptozotocin for 1 hour and then cultured for 23 hours in medium containing 11 or 33 mM glucose with or without PPAG (n = 3). The percentage of apoptotic (A) and necrotic cells (B) was determined following staining with the nuclear dyes propidium iodide and Hoechst 33342. A minimum of 500 cells was counted for each condition. Percentage of apoptotic (A) and necrotic cells (B). *p<0.05 against control (CTRL). #p<0.05 against STZ-treated cells in 11 mM glucose. §p<0.05 as indicated.</p

Effect of STZ and PPAG on beta cell apoptosis/proliferation and the expression of BCL2 and phospho H2AX.

<p>Shaded bars: untreated controls, CTRL (n = 5); filled bars: STZ-treated animals, STZ (n = 4); open bars: STZ-treated animals that were treated with PPAG, STZ+PPAG (n = 4). (A) Percentage of TUNEL stained cells in the insulin-positive cell population in pancreatic tissue sections. (B) Percentage of Ki67 stained cells in the insulin-positive cell population in pancreatic tissue sections. Values are mean ± SEM. Significance compared to the control was determined by one-way ANOVA with Dunnett post-test. **P<0.01; NS (not significant). For the effect of PPAG on STZ induced apoptosis a one-sample t-test was used. #P<0.05. (C) Densitometric quantification of BCL2 expression in the islets. Values are mean ± SEM. Significance compared with the control was determined using an one-way ANOVA with a Dunnett post-test. **P<0.01; NS (not significant). (D) Representative images of the BCL2 staining in pancreatic islets, 30 hours post-STZ. (E) Beta cell DNA damage/repair. Shaded bars: untreated controls, CTRL (n = 5); filled bars: STZ-treated animals, STZ (n = 4 for 30 hours and n = 3 for 11 days); open bars: STZ-treated animals that were treated with PPAG, STZ+PPAG (n = 4 for 30 hours and n = 4 for 11 days). No positive cells were detected in control animals. Results from STZ and STZ+PPAG were not statistically different (p≥0.05). **p<0.01 between control group and STZ.</p

In vitro beta cell protection from hydrogen peroxide by PPAG and the effect of PPAG on oxidative stress.

<p>Cell death in INS-1E cells pre-treated with 30 μM PPAG for 16 hours, exposed to 30 μM hydrogen peroxide (H₂O₂) for 30 min, and then cultured for 24 hours in control medium with or without PPAG (n = 4). The percentage of apoptotic (A) and necrotic cells (B) was determined following staining with the nuclear dyes propidium iodide and Hoechst 33342. A minimum of 500 cells was counted for each condition. Percentage of apoptotic (A) and necrotic cells (B).*p<0.05 against untreated cells. #p<0.05 against H₂O₂-treated cells. (C) INS-1E cells were pre-treated for 16 hours with 30 μM PPAG, exposed to 1 mM STZ for 1 hour, and further cultured for 2 hours in the presence of absence of PPAG (n = 3). Oxidative stress was measured by dichlorofluorescein (DCF) oxidation. *p<0.05 against untreated cells.</p

Effect of PPAG on lipotoxic cell death in human islet cells.

<p>Human islets were treated with 0.5 mM palmitate and 30 μM PPAG or vehicle for 3 days (n = 4). The percentage of apoptotic (A) and necrotic cells (B) was determined following staining with the nuclear dyes propidium iodide and Hoechst 33342. A minimum of 500 cells was counted for each condition. *p<0.05 against untreated cells, #p< 0.05 as indicated.</p

Effect of STZ and PPAG on blood glucose levels and beta cell mass in vivo.

<p>Shaded bars: untreated controls, CTRL (n = 10); filled bars: STZ-treated animals, STZ (n = 7); open bars: STZ-treated animals that were treated with PPAG, STZ+PPAG (n = 8). (A) Non-fasting blood glucose levels immediately before and 30 hours after a single injection of STZ. (B) Daily follow up of non-fasting blood glucose from immediately before a single injection of STZ until 11 days. Values are mean ± SEM. ANOVA with Bonferroni post test was used to determine the significance between groups on each day. **p<0.01; ***p<0.001 between control and STZ group. #p<0.05; ## p<0.01; ###p< 0.001 between control and STZ + PPAG group. § p<0.05; §§ p<0.01; §§§ p< 0.001 between STZ and STZ + PPAG group. (C) Percentage of beta cell mass normalized to the control 30 hours (C) or 11 days (D) after STZ injection. Data shown are means ± SEM. **p<0.01; #p<0.05; NS: not significant.</p

Expression of caspases and BCL2.

<p>Western blot and densitometry for BCL2 and cleaved caspase 9 and 3 in INS-1E cells pre-treated with 30 μM PPAG for 16 hours, exposed to 1 mM STZ for 1 hour and then cultured for 7 hours in medium containing 11 or 33 mM glucose with or without PPAG (n = 3).*p<0.05 against control (CTRL). §p<0.05 as indicated.</p

C/EBPδ silencing increases cytokine-induced chemokine production by enhancing STAT1 activation.

<p>INS-1E cells were transfected with either siCtrl (white dots/bars), siC/EBPδ #1 (black triangles/bars) or siC/EBPδ #2 (grey squares/bars) and subsequently left untreated, or treated with IL-1β+IFN-γ for the indicated time points; (A–E) C/EBPδ, CXCL1, 9, 10 & CCL20 mRNA expressions were assayed by RT-PCR and normalized for the housekeeping gene GAPDH; (F–G) CXCL1 and CXCL9 protein secretions were evaluated by ELISA. (H) C/EBPδ, phospho-STAT1, total STAT1, IRF-1 and α-tubulin expressions were evaluated by Western blot. (I) 24 h after siRNA transfection, cells were transfected with a STAT1 luciferase reporter+pRL-CMV and subsequently left untreated or exposed to cytokines for 16 h as indicated. Results are mean Relative Luciferase Unit (R.L.U.) ± SEM. (J–L) Mean optical density measurements of IRF1, phospho-STAT1 and total STAT1 Western blots corrected for α-tubulin (representative figure in H). (G) SOCS-1 mRNA expression was assayed by RT-PCR and normalized for the housekeeping gene GAPDH. Results are mean ± SEM of 4–6 experiments; *: <i>p</i><0.05, **: <i>p</i><0.01 and ***: <i>p</i><0.001 vs untreated transfected with the same siRNA; §: <i>p</i><0.05, §§: <i>p</i><0.01 and §§§: <i>p</i><0.001 vs siCtrl treated with cytokines at the same time point; ANOVA followed by Student's <i>t</i> test with Bonferroni correction.</p

Amplified CHOP expression contributes to the exacerbation of apoptosis in cytokine-treated C/EBPδ-deficient INS-1E cells.

<p>(A–E) INS-1E cells were transfected with siCtrl (white dots), siC/EBPδ #1 (black triangles) or siC/EBPδ #2 (grey squares) and subsequently left untreated, or treated with IL-1β+IFN-γ for the indicated time points. (A–C) C/EBPδ, CHOP and GADD34 mRNA expressions were assayed by RT-PCR and normalized for the housekeeping gene GAPDH; (D) C/EBPδ, CHOP and α-tubulin expressions were evaluated by Western blot. (E) Mean optical density measurements of CHOP Western blots corrected for α-tubulin (representative figure in D). (F–G) INS-1E cells were transfected with siCtrl (white bars), siC/EBPδ #1 (black bars), siCHOP (grey bars) or siC/EBPδ #1+siCHOP (hatched grey bars) and subsequently left untreated, or treated with IL-1β+IFN-γ for 24 h as indicated. (F) Apoptosis was assessed by HO/PI staining. (G) Cleaved caspase 3, C/EBPδ, CHOP and α-tubulin expressions were evaluated by Western blot. Results are mean ± SEM of 4–5 experiments; *: <i>p</i><0.05, **: <i>p</i><0.01 and ***: <i>p</i><0.001 vs untreated transfected with the same siRNA; §: <i>p</i><0.05, §§: <i>p</i><0.01 and §§§: <i>p</i><0.001 vs siCtrl treated with cytokines at the same time point; ##: <i>p</i><0.01 vs siC/EBPδ #1 treated with cytokines at the same time point; ANOVA followed by Student's <i>t</i> test with Bonferroni correction.</p

C/EBPδ-silencing increases the expression of the pro-apoptotic Bcl-2 family member BIM.

<p>(A–C) INS-1E cells were transfected with siCtrl (white dots), siC/EBPδ #1 (black triangles) or siC/EBPδ #2 (grey squares) and subsequently left untreated, or treated with IL-1β+IFN-γ for the indicated time points. (A) BIM mRNA expression was assayed by RT-PCR and normalized for the housekeeping gene GAPDH; (B) BIM, C/EBPδ and α-tubulin expressions were evaluated by Western blot. (C) Mean optical density measurements of BIM Western blots corrected for α-tubulin (representative figure in B). (D) INS-1E cells were transfected with siCtrl (white bars), or with either siC/EBPδ #1 (black bars), siBIM (grey bars) or siC/EBPδ #1+siBIM (hatched grey bars) and subsequently left untreated, or treated with IL-1β+IFN-γ for 24 h as indicated. Apoptosis was then assessed by HO/PI staining. Results are mean ± SEM of 4–6 experiments; *: <i>p</i><0.05, **: <i>p</i><0.01 and ***: <i>p</i><0.001 vs untreated transfected with the same siRNA; §: <i>p</i><0.05, §§: <i>p</i><0.01 and §§§: <i>p</i><0.001 vs siCtrl treated with cytokines at the same time point; ###: <i>p</i><0.001 vs siC/EBPδ #1 treated with cytokines at the same time point; ANOVA followed by Student's <i>t</i> test with Bonferroni correction.</p

Suggested model for the role of C/EBPδ signalling in cytokine-treated β-cells.

<p>Transcription factors are highlighted in circle, thin arrows represents positive (inducing) effects, while bold T-shaped lines represent inhibitory effects. Upon IL-1β+IFN-γ exposure, C/EBPδ is up-regulated downstream of the transcription factors NF-κB and STAT1. Once synthesized, it inhibits CHOP and BIM expression, hampering cytokine-induced caspase 9 and 3 cleavage and apoptosis. C/EBPδ also exerts a positive role on cytokine-induced IRF-1 expression, which, via the induction of the regulator SOCS-1, down-regulates STAT1 activation and STAT1-driven chemokine production.</p