Figure 1.
Effects of exenatide and piragliatin on beta cell death in INS-1.
(A) INS-1 cells, serum-starved overnight, were pre- and post-treated with each drug with or without 2 mM STZ for 1 h. (B) INS-1 cells serum-starved overnight were treated with or without 0.8 mM palmitate for 24 h. (C) INS-1 cells serum-starved overnight were treated in the absence or presence of each drug, with or without 0.3 µM Tg for 6 h. At the end of each experiment, cell viability was assessed by cytosolic ATP levels and data from three individual experiments were presented as mean ± S.E. *, P<0.05 vs. control in the absence of each death inducer; #, both P<0.05 vs. control in the presence of palmitate by Bonferroni's post hoc analysis.
Figure 2.
Contribution of increased glucose utilization by piragliatinin the STZ-induced beta cell death.
(A–C) Intracellular NAD(P)H levels were assessed by MTT assay for 2 h starting from 0.5 h after recovery to STZ- and serum-free and 5.6 mM glucose-containing media. (D) Cellular ATP levels were measured 2.5 h after recovery at 5.6 mM glucose. (E) Eleven hours after recovery at 5.6 mM or 16.7 mM glucose in the absence (white column) or presence (black column) of 2 mM STZ, cellular ATP levels were assessed. Data from 5 to 10 determinants are presented as mean ± S.E. ** and ***, P<0.01 and P<0.001 vs. each control, respectively by Bonferroni's post hoc analysis.
Figure 3.
Beta cell protection by exenatide and piragliatin in the presence of various kinase inhibitors.
(A) In Tg-induced cell death, INS-1 cells were pretreated with 10 µM of various kinase inhibitors for 1 h. The cells were then treated with 0.3 µM Tg and 10 µM kinase inhitor in the presence or absence of either 10 nM exenatide or 10 µM piragliatin for 6 h. (B) In STZ-induced cell death, INS-1 cells were pretreated with 10 µM of various kinase inhibitors in the presence or absence of either 10 nM exenatide or 10 µM piragliatin, and then were transiently treated with 2 mM STZ in glucose- and serum-free media for 1 h. Thereafter, cells were recovered in serum-free and 5.6 mM glucose-containing media in the presence of kinase inhibitor and each drug for 17 h. At the end of each experiment, cell viability was assessed by cytosolic ATP levels. Data from 3 to 5 individual determinants were denoted as mean ± S.E. †, P<0.05 vs. each control in a same treatment set by Bonferroni's post hoc analysis; *, P<0.05 between two treatments by Student's t-test.
Figure 4.
Differential effects of exenatide and piragliatin on the activation of Akt and JNK.
(A, C, D) In Tg-induced cell death, INS-1 cells were harvested 1 h (for pAkt and Akt) and 3 h (for pJNK and JNK) after treating with 0.3 µM Tg in the presence or absence of either 10 nM exenatide or 10 µM piragliatin. (B, E, F) In STZ-induced cell death, INS-1 cells were pretreated with either 10 nM exenatide or 10 µM piragliatin. Following 2 mM STZ treatment for 1 h in the absence of glucose and serum, cells were harvested 0.5 h after recovery to STZ-free media with 5.6 mM glucose and each drug. In each experiment, the cell lysates were separated on 4–12% precast Bis-Tris gels, transferred onto PVDF membranes, and immunostained with phosphoAkt, Akt, phosphoJNK, and JNK antibodies. Beta actin was a representative image from JNK treatment set. (C–F) Quantified results of A and B (N = 2).
Figure 5.
Identification of protein spots appearing in exenatide and piragliatin treated INS-1 cells by differential 2D analysis.
Six hours after treatment with 0.3 µM Tg in the presence or absence of either 10 nM exenatide or 10 µM piragliatin, INS-1 cells were harvested and lysed for 2D analysis. After electrofocusing on strip gels (18 cm, pH 4–7), samples were separated on 10% acrylamide gels, silver stained, and scanned for image analysis (A). Representative images of six protein spots are shown in (B). (C) Each protein spot was cut out, destained and in-gel digested by trypsin. Digested samples were extracted, separated, and identified using nanoUPLC-ESI-q-TOF tandem MS.
Figure 6.
Identification of post-translationally produced potein 14-3-3 isoforms.
(A) Appearances of three spots of 14-3-3 isoforms accorded with beta cell death regardless of the type of death stimulus. (B) After pretreatment of 10 µg/ml cycloheximide in INS-1 cells for 0.5 h, cells were treated with 0.3 µM Tg in the presence or absence of cylcloheximide and either 10 nM exenatide or 10 µM piragliatin. (C) In a separate experiment, cycloheximide-induced cytotoxicity was assessed by cellular ATP levels 6.5 h after cycloheximide treatment. (D) A representative image of related MS spectrum and identified modifications of 14-3-3β, ε, and θ. (E) After 20 µM JNK inhibitor was pretreated for 1 h and treated with 0.3 µM Tg, three spots of 14-3-3 proteins were determined on 2D gels.
Figure 7.
Levels of protein 14-3-3θ negatively correlate with the viability of INS-1 cells.
(A–C) Levels of protein 14-3-3 isoforms were assessed by immunoblotting at the end of treatment. (A) A representative image, (B) quantification data for Tg treatment and (C) for STZ treatment denoting mean ± S.E. from three different samples. Control, Tg or STZ alone, Tg or STZ plus 10 nM exenatide, and Tg or STZ plus 10 µM piragliatin; *, P<0.05 vs. each control by Bonferroni's post hoc analysis. (D–E) INS-1 cells were transfected with specific siRNA for 14-3-3θ, control scrambed siRNA, or without siRNA for 48 h. (D) Knockdown of 14-3-3θ was confirmed by immunoblotting anti-14-3-3θ antibody. *, P<0.05 by Student's t-test. (E) At the end of treatment in a same experiment, cellular viability was assessed by adding CCK-8 reagent (Dojindo Laboratories, Kumamoto, Japan) in supernatant and incubating cells for 1 h at 37°C followed by measuring optical density at 450 nm according to the manufacturer's instruction. Different alphabet characters indicate different statistical significance (P<0.05 by Bonferroni's post hoc analysis).
Figure 8.
A proposed scheme of protection effects of exenatide and piragliatin in beta cell death.