Fig 1.
Effects of exenatide on glucose-stimulated insulin secretion and INS-1 cell death caused by thapsigargin-induced ER-stress.
(A) INS-1 cells were stimulated by exenatide at 11.1 mM glucose for insulin secretion. (B) Serum-deprived INS-1 cells were treated 0.3 μM thapsigargin in the absence or presence of various concentrations of exenatide for 6 h. At the end of experiment, cellular viability was assessed by determining intracellular ATP levels. (C) After 6 h treatment, cellular caspase-3/7 activity was determined as a sensitive apoptotic marker. (D) Caspase-3/7 activity was determined according to the treatment time of 0.3 μM thapsigargin in the absence or presence of 10 nM exenatide. ***p< 0.001 vs. untreated control by Bonferroni’s t-test. Each experiment was run in triplicate.
Fig 2.
Regulation of gene expression by exenatide treatment under thapsigargin-induced INS-1 cell death.
Serum-deprived INS-1 cells were treated 0.3 μM thapsigargin in the absence or presence of 10 nM exenatide for 6 h. Total RNA was isolated and mRNA expression of five genes related to the INS-1 cell death/survival was determined by RT-PCR. (A) Gel images of PCR products were obtained and quantified using densitometry. Expression levels of (B) Bip and (C) CHOP as ER stress-related chaperons, (D) IRS-2 as a signal mediator of INS-1 cell survival, (E) TXNIP as a glucotoxicity mediator, and (F) GLP-1 receptor genes as a target molecule of exenatide were assessed. Data were presented as mean ± SE from three individual determinants. #p< 0.05 vs. untreated control; *p< 0.05 vs. thapsigargin alone by Bonferroni’s t-test.
Fig 3.
Differential protein expression during INS-1 cell death induced by ER stress and its prevention by exenatide on 2D-PAGE separation.
After 6 h treatment of thapsigargin alone or thapsigargin plus exenatide, lysates of INS-1 cells were separated on 2D-PAGE and visualized by silver staining. Arrow denotes the differentially expressed protein spots shown in thapsigargin alone compared to untreated control. Direction of change was denoted as alphabet “D” (down) or “U” (up) in front of each spot number.
Fig 4.
Classification of proteins altered by thapsigargin treatment based on their biological process or molecular functions using PANTHER classification system (PANTHER; www.pantherdb.org).
Table 1.
Proteins differentially altered by thapsigargin treatment (N = 3).
Fig 5.
Protein networks; (A) proteins altered by thapsigargin-induced INS-1 cell death, (B) predicted protein interaction by String analysis (www.string-db.org).
Proteins altered by thapsigargin in our experiment were denoted as bold and italic.
Fig 6.
Effects of exenatide on proteins which were up- or down-regulated during thapsigargin-induced INS-1 cell death.
(A) Eighteen protein spots altered by thapsigargin alone were not changed by addition of exenatide. (B) Change of eight protein spots among total 58 protein spots significantly altered by thapsigargin alone, was augmented by exenatide add-on. (C) Twenty protein spots decreased by thapsigargin-induced INS-1 cell death were significantly reversed by addition of exenatide. (D) Increase of twelve protein spots by thapsigargin alone was completely blocked by exenatide add-on. Data were presented as mean ± SE from three individual determinants. (E) Enlarged spot images of 14–3–3 isoforms. *p< 0.05 vs. control; #p< 0.05 vs. thapsigargin alone by Bonferroni’s t-test.
Fig 7.
Changes in post-translational modifications of thimet oligopeptidase in response to thapsigargin and thapsigargin plus exenatide.
Protein spots at a different modification status were quantified and data were presented as mean ± SE from three individual determinants. *p< 0.05 vs. control; #p< 0.05 vs. thapsigargin alone by Bonferroni’s t-test.
Table 2.
Modifications of thimet oligopeptidase identified on 2D-PAGE (Fig. 7) using peptide sequencing with MS/MS analysis.
Fig 8.
Identification of 14–3–3 family proteins by 2-D PAGE.
From lysates of thapsigargin-treated INS-1 cells, total 16 protein spots of 14–3–3 proteins were separated on 2D-PAGE and PTMs in each spot were determined by peptide sequencing with MS/MS analysis.
Fig 9.
Changes in post-translational modifications of 14–3–3 proteins during thapsigargin and thapsigargin plus exenatide treatments.
Protein spots of each 14–3–3 isoform at a different modification status were quantified and data were presented ad mean ± SE from three separate experiments. ***p< 0.001 vs. control by Bonferroni’s t-test.
Table 3.
Modifications of 14–3–3 isoforms identified on 2D-PAGE (Fig. 8) using peptide sequencing with MS/MS analysis.
Fig 10.
MS/MS Spectrum of 14–3–3θ peptide phosphorylated at Ser92 (92SICTTVLELLDK103 of spot No. 13).
Table 4.
Prediction of kinases possibly promoting the phosphorylation of 14–3–3 using GPS v2.1 (http://gps.biocuckoo.org/).