Proteomic Analysis of INS-1 Rat Insulinoma Cells: ER Stress Effects and the Protective Role of Exenatide, a GLP-1 Receptor Agonist

<div><p>Beta cell death caused by endoplasmic reticulum (ER) stress is a key factor aggravating type 2 diabetes. Exenatide, a glucagon-like peptide (GLP)-1 receptor agonist, prevents beta cell death induced by thapsigargin, a selective inhibitor of ER calcium storage. Here, we report on our proteomic studies designed to elucidate the underlying mechanisms. We conducted comparative proteomic analyses of cellular protein profiles during thapsigargin-induced cell death in the absence and presence of exenatide in INS-1 rat insulinoma cells. Thapsigargin altered cellular proteins involved in metabolic processes and protein folding, whose alterations were variably modified by exenatide treatment. We categorized the proteins with thapsigargin initiated alterations into three groups: those whose alterations were 1) reversed by exenatide, 2) exaggerated by exenatide, and 3) unchanged by exenatide. The most significant effect of thapsigargin on INS-1 cells relevant to their apoptosis was the appearance of newly modified spots of heat shock proteins, thimet oligopeptidase and 14-3-3β, ε, and θ, and the prevention of their appearance by exenatide, suggesting that these proteins play major roles. We also found that various modifications in 14-3-3 isoforms, which precede their appearance and promote INS-1 cell death. This study provides insights into the mechanisms in ER stress-caused INS-1 cell death and its prevention by exenatide.</p></div

Classification of proteins altered by thapsigargin treatment based on their biological process or molecular functions using PANTHER classification system (PANTHER; www.pantherdb.org).

<p>Classification of proteins altered by thapsigargin treatment based on their biological process or molecular functions using PANTHER classification system (PANTHER; <a href="http://www.pantherdb.org" target="_blank">www.pantherdb.org</a>).</p

Differential protein expression during INS-1 cell death induced by ER stress and its prevention by exenatide on 2D-PAGE separation.

<p>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.</p

MS/MS Spectrum of 14–3–3θ peptide phosphorylated at Ser⁹² (⁹²SICTTVLELLDK¹⁰³ of spot No. 13).

<p>MS/MS Spectrum of 14–3–3θ peptide phosphorylated at Ser⁹² (⁹²SICTTVLELLDK¹⁰³ of spot No. 13).</p

Effects of exenatide on glucose-stimulated insulin secretion and INS-1 cell death caused by thapsigargin-induced ER-stress.

<p>(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. ***<i>p</i>< 0.001 vs. untreated control by Bonferroni’s t-test. Each experiment was run in triplicate.</p

Protein networks; (A) proteins altered by thapsigargin-induced INS-1 cell death, (B) predicted protein interaction by String analysis (www.string-db.org).

<p>Proteins altered by thapsigargin in our experiment were denoted as bold and italic.</p

Changes in post-translational modifications of 14–3–3 proteins during thapsigargin and thapsigargin plus exenatide treatments.

<p>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. ***<i>p</i>< 0.001 vs. control by Bonferroni’s t-test.</p

Changes in post-translational modifications of thimet oligopeptidase in response to thapsigargin and thapsigargin plus exenatide.

<p>Protein spots at a different modification status were quantified and data were presented as mean ± SE from three individual determinants. *<i>p</i>< 0.05 vs. control; #<i>p</i>< 0.05 vs. thapsigargin alone by Bonferroni’s t-test.</p

Effects of exenatide on proteins which were up- or down-regulated during thapsigargin-induced INS-1 cell death.

<p>(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. *<i>p</i>< 0.05 vs. control; #<i>p</i>< 0.05 vs. thapsigargin alone by Bonferroni’s t-test.</p

Identification of 14–3–3 family proteins by 2-D PAGE.

<p>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.</p