Pdcd2l Promotes Palmitate-Induced Pancreatic Beta-Cell Apoptosis as a FoxO1 Target Gene

Abstract

Transcription factor FoxO1 is a key regulator of the insulin-signaling pathway, and is reported to play important roles in pancreatic β cell differentiation, proliferation, apoptosis and stress resistance. The multifunctional nature of FoxO1 is due to its regulation of various downstream targets. Previous studies in our lab identified potential FoxO1 target genes using the ChIP-DSL technique and one of those genes, Pdcd2l, was selected for further study. We found that the expression of Pdcd2l was increased with palmitate treatment; the luciferase assay result revealed that enhanced Pdcd2l promoter activity was responsible for the elevation of Pdcd2l expression. ChIP-PCR was performed to confirm the combination of FoxO1 to Pdcd2l promoter, result showing that FoxO1 could bind to Pdcd2l promoter and this binding was further enhanced after palmitate treatment. Overexpression of FoxO1 significantly induced Pdcd2l promoter activity, leading to increased mRNA level; consistently, interference of FoxO1 abolished the increment of Pdcd2l gene expression triggered by palmitate treatment. In addition, overexpression of Pdcd2l could further increase the percentage of apoptotic cells induced by palmitate incubation, whilst interference of Pdcd2l partially reversed the palmitate-induced apoptosis together with activated Caspase-3, indicating that the latter may play a part in this process. Therefore, in this study, we confirmed the binding of FoxO1 to the Pdcd2l gene promoter and studied the role of Pdcd2l in β cells for the first time. Our results suggested that FoxO1 may exert its activity partially through the regulation of Pdcd2l in palmitate-induced β cell apoptosis and could help to clarify the molecular mechanisms of β cell failure in type 2 diabetes.

Fig 1. Treatment of Palmitate dose-dependently increased the mRNA level and protein level of Pdcd2l in β cells.

INS-1 cells were treated with indicated dose of palmitate for 24 h, Pdcd2l mRNA level was detected by Q-PCR(A) and protein level was analyzed by western blot (B). MIN6 cells (C) and primary rat islets (D) were incubated with indicated concentrations of palmitate for 48 h, and the mRNA levels were detected by Q-PCR. Data shown are means±SEM and representative of three separate experiments. *, p<0.05 compared with control; **, p<0.01 compared with control.

Fig 2. Treatment of Palmitate induced cell apoptosis dose-dependently.

A: INS-1 cells were incubated with indicated concentration of palmitate for 24 h, cell apoptosis was detected by PI staining. PI-positive cells (red) indicate apoptotic cells and Hoechst-positive cells (blue) indicate live cells. Images shown here are representatives of three independent experiments. Scale bar, 20 μm. B: Quantification of the percentage of PI-positive INS-1 cells treated with palmitate. C: INS-1 cells were incubated with palmitate for 24 h, and expression of cleaved Caspase-3 was detected by western blot. Data shown are means±SEM and representative of three separate experiments. *, p<0.05 compared with control; **, p<0.01 compared with control.

Fig 3. Treatment of Palmitate enhanced Pdcd2l promoter activity and gene expression time-dependently.

A: INS-1 cells were transfected with pGL3-Pdcd2l luciferase reporter plasmid for 18 h and then exposure to 0.2 mol/L palmitate, cells were collected at indicated time points for luciferase reporter assays. B: INS-1 cells were incubated with 0.2 mol/L palmitae for indicated time points and gene expression was detected by Q-PCR. Data shown are means±SEM and representative of three separate experiments. *, p<0.05 compared with control; **, p<0.01 compared with control.

Fig 4. Treatment of Palmitate further increased the binding of FoxO1 to Pdcd2l promoter.

INS-1 cells (A) and MIN6 cells (B) were treated with 0.4 mol/L palmitate for 12 h, and then cells were collected for ChIP-PCR. Input chromatin from control and immunoprecipitated DNA was PCR-amplified using primers specific to suspected FOXO1 target regions. Sequence enrichment in immunoprecipitated DNA from antibody (Ab, anti-FOXO1 serum) vs IgG chromatin indicated FOXO1 binding within the genomic region. Results shown here are representatives for three independent experiments.

Fig 5. Regulation of FoxO1 on Pdcd2l promoter activity and gene expression.

A: INS-1 cells were co-transfected with pGL3-Pdcd2l luciferase reporter plasmid and pCMV5 or pCMV5-FoxO1 plasmid for 24 h, and then cells were harvested for luciferase reporter assays. B: INS-1 cells were transfected with pCMV5 or pCMV5-FoxO1 for 48 h and then Pdcd2l gene expression was detected by Q-PCR. C and D: INS-1 cells were infected with Ad-siGFP or Ad-siFoxO1 for 18 h and then incubated with or without 0.2 mol/L palmitate for another 24 h, after which gene expression of FoxO1 (C) and Pdcd2l (D) were measured by Q-PCR. Data shown are means±SEM and representative of three separate experiments. *, p<0.05 compared with control; **, p<0.01 compared with control.

Fig 6. Effect of Pdcd2l in palmitate induced β cell apoptosis.

A-G: Silencing of Pdcd2l partially reversed cell apoptosis caused by palmitate incubation. INS-1 cells were transiently transfected with Control-siRNA or si-Pdcd2l for 18 h, and then were incubated with or without 0.2 mol/L palmitate for another 24 h. A: Representative western blot and densitometric analysis of Pdcd2l relative to tubulin. B: Cell apoptosis was detected by PI staining. PI-positive cells (red) indicate apoptotic cells and Hoechst-positive cells (blue) indicate live cells. Scale bar, 20 μm. C: Apoptotic rate was calculated by counting PI-positive cells verse Hoechst-positive cells. G: Representative western blot and densitometric analysis of Pdcd2l and cleaved Caspase-3 relative to tubulin. D: Primary rat islets were isolated and cultured in vitro, and then transfected with Cy3-NControl (fluorescence-labeled siRNA) for 48 h to confirm the transfection efficiency. Scale bar, 100 μm. Islets were transfected with Control-siRNA or si-Pdcd2l for 24 h, and then were treated with or without 0.4 mol/L palmitate for another 48 h. E: Representative western blot and densitometric analysis of Pdcd2l relative to tubulin. F: Cell apoptosis was detected by TUNEL staining. TUNEL-positive cells (green) indicate apoptotic cells and Hoechst/Insulin double positive cells indicate live β cells. Scale bar, 20 μm. H and I: Overexpression of FoxO1 or Pdcd2l further increased cell apoptosis caused by palmitate treatment. INS-1 cells were transiently transfected with pCMV5 or pCMV5-FoxO1 or pcDNA3.0-Pdcd2l for 18 h, and then were incubated with or without 0.2 mol/L palmitate for another 24 h. H: Cell apoptosis was detected by PI staining. Scale bar, 20 μm. I: Apoptotic rate was calculated by counting PI-positive cells verse Hoechst-positive cells. Images shown here are representatives for three independent experiments. Data shown are means±SEM and representative of three separate experiments. **, p<0.01 compared with control without treatment of palmitate; ##, p<0.01 or #, p<0.05 compared with control treated with palmitate.