Exendin-4, a glucagon-like peptide-1 receptor agonist, inhibits cell apoptosis induced by lipotoxicity in pancreatic β-cell line

Peptides 37 (2012) 18–24

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Exendin-4, a glucagon-like peptide-1 receptor agonist, inhibits cell apoptosis induced by lipotoxicity in pancreatic ␤-cell line Qian Wei a , Yu Qiang Sun b , Jin Zhang a,∗ a b

Department of Endocrinology, The First Affiliated Hospital of China Medical University, No. 155, Nanjing North Street, Heping District, Shenyang, Liaoning 110001, China Department of Emergency Medicine, The First Affiliated Hospital of China Medical University, No. 155, Nanjing North Street, Heping District, Shenyang, Liaoning 110001, China

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Article history: Received 14 May 2012 Received in revised form 28 June 2012 Accepted 28 June 2012 Available online 7 July 2012 Keywords: Glucagon-like peptide-1 Exendin-4 Lipotoxicity Pancreatic ␤-cells Diabetes

a b s t r a c t Lipotoxicity plays an important role in the underlying mechanism of type 2 diabetes mellitus. Prolonged exposure of pancreatic ␤-cells to elevated concentrations of fatty acid is associated with ␤-cell apoptosis. Recently, glucagon-like peptide-1 (GLP-1) receptor agonists have been reported to have direct beneficial effects on ␤-cells, such as anti-apoptotic effects, increased ␤-cell mass, and improvement of ␤-cell function. The mechanism of GLP-1 receptor agonists’ protection of pancreatic ␤-cells against lipotoxicity is not completely understood. We investigated whether the GLP-1 receptor agonist exendin-4 promoted cell survival and attenuated palmitate-induced apoptosis in murine pancreatic ␤-cells (MIN6). Exposure of MIN6 cells to palmitate (0.4 mM) for 24 h caused a significant increase in cell apoptosis, which was inhibited by exendin-4. Exposure of MIN6 cells to exendin-4 caused rapid activation of protein kinase B (PKB) under lipotoxic conditions. Furthermore, LY294002, a PI3K inhibitor, abolished the anti-lipotoxic effect of exendin-4 on MIN6 cells. Exendin-4 also inhibited the mitochondrial pathway of apoptosis and down-regulated Bax in MIN6 cells. Exendin-4 enhanced glucose-stimulated insulin secretion in the presence of palmitate. Our findings suggest that exendin-4 may prevent lipotoxicity-induced apoptosis in MIN6 cells through activation of PKB and inhibition of the mitochondrial pathway. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Lipotoxicity plays an important role in the underlying mechanism of type 2 diabetes mellitus [24,25]. Under physiological conditions, free fatty acids (FFAs) sustain basal insulin secretion in the fasting state and potentiate hormone release acutely in the presence of glucose [22]. However, prolonged exposure of pancreatic ␤-cells to elevated concentrations of FFAs may contribute to the impairment of insulin gene expression [9], inhibition of insulin synthesis and secretion [32], and induction of ␤-cells apoptosis [14,19]. The serine/threonine kinase Akt, also known as protein kinase B (PKB), plays a vital role in regulating mass and function of pancreatic ␤-cells [8]. A growing body of evidence suggests that activation of PKB phosphorylation is able to protect pancreatic ␤-cells against lipotoxicity [2,15,29]. To pancreatic ␤-cells, PKB is not only a unique central node in cell signaling downstream of growth factors and cytokines, but also a key target of the insulin signal system [8,20]. Glucagon-like peptide-1 (GLP-1), an incretin hormone secreted by intestinal L cells, is a promising therapeutic agent in the treatment of diabetes. A major target for GLP-1 actions is the pancreatic

∗ Corresponding author. Tel.: +86 24 8328 2203; fax: +86 24 8325 0145. E-mail addresses: [email protected] (Q. Wei), [email protected] (J. Zhang). 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.06.018

␤-cell. One of the main physiological roles of this endocrine hormone is to enhance insulin secretion in a glucose-dependent manner [5,6,12]. In addition, it was recently reported that GLP-1 blood levels far in excess of physiologic levels induced by a GLP-1 receptor agonist exhibited many beneficial effects, including antiapoptotic and proliferative effects on ␤-cells [1,27]. However, it is not known whether a GLP-1 receptor agonist protects ␤-cells against lipotoxicity-induced apoptosis in type 2 diabetes. Therefore, the aim of the present study was to investigate whether exendin-4, a GLP-1 receptor agonist, protects pancreatic ␤-cells from lipotoxicity, and to explore the precise signal transduction pathway of PI3K/PKB that mediates the protective action.

2. Materials and methods 2.1. Materials Exendin-4 was purchased from Prospec (Rehovot, Israel); fatty acid-free bovine serum albumin (BSA, fraction V), palmitate, Hoechst33258, CCK-8 (cell counting kit-8), and LY294002 were purchased from Sigma–Aldrich (St. Louis, MO, USA). The antibodies anti-phospho-ser473-PKB and anti-total PKB were obtained from Cell Signaling Technology (Danvers, MA, USA). The TUNEL (in situ cell death detection kit, POD) kit was from Roche (Basel,

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Switzerland). The Caspase-3 activity assay kit was from R&D Systems (Minneapolis, MN, USA). The FITC Annexin V apoptosis detection kit was from BD (Franklin Lakes, NJ, USA). The SYBR PrimeScript RT-PCR kit was from TaKaRa (Otsu, Japan). The Rat Insulin Radioimmunoassay (RIA) kit was from Linco Research (St. Charles, MO, USA). 2.2. Cell culture Mouse pancreatic ␤-cells (MIN6) were cultured in Dulbecco modified Eagle medium (DMEM) containing 25 mM glucose, supplemented with 15% fetal bovine serum (FBS), 100 U/mL penicillin, 100 mg/mL streptomycin, 100 mg/mL l-glutamine, and 5 ␮L/L ␤mercaptoethanol in humidified 5% CO2 , and 95% air at 37 ◦ C. During FFA stimulation, the above medium was used but in the absence of FBS. All experiments were performed between passages 10 and 30. 2.3. Fatty acid, exendin-4 and inhibitor administration of MIN6 cells For experiments, MIN6 cells were cultured in high glucose (25 mM) serum-free DMEM with 0.5% BSA alone or 0.4 mM palmitate complexed to 0.5% BSA, with or without inhibitor, in the presence or absence of exendin-4. The 0.4 mM palmitate fatty acid solution was prepared as previously described [31]. Briefly, a 20 mM solution of palmitate in 0.01 mol/L NaOH was incubated at 70 ◦ C for 30 min. Then 330 ␮L of 30% BSA and 400 ␮L of the palmitate/NaOH were mixed together and filter-sterilized with 20 mL of the DMEM in total. Concentration of BSA is 0.5% in all medium. We used a 25 mM glucose concentration for all experiments, because an increased glucose concentration was essential for MIN6 cell growth, and compared with 5 mM glucose, 25 mM glucose protected MIN6 cells from apoptosis [21]. Graded doses of exendin-4 (1, 10, 100 and 500 nM) were prepared daily before experiments. The PI3K inhibitor LY294002 (50 ␮M), was used in the study. 2.4. Cell viability assay Cell viability was assessed by CCK-8. Briefly, cells were seeded on 96-well plates at a density of 5000 cells per well and treated with a broad range of each reagent for 24 h. Subsequently, 10 ␮L of the CCK-8 solution was added to each well of the culture plate, which contained 100 ␮L medium. After a 1-h incubation, absorbance was measured at a wavelength of 450 nm using a 96-well plate reader. 2.5. Apoptosis assay Apoptosis was detected by three different methods: Hoechst33258 staining, flow cytometric detection of FITC Annexin V and PI co-staining, and TdT-mediated dUTP nick-end labeling (TUNEL). Apoptosis indexes were assessed by counting Hoechstpositive cells (chromatin condensation or fragmented nuclear membrane), FITC Annexin V-positive cells (FITC Annexin Vpositive and PI-negative) and TUNEL-positive cells (apoptotic nucleus was brown-stained). Hoechst staining was performed by exposing the cell slides to 10 ␮g/mL Hoechst33258 for 10 min at room temperature. The cells were counted by fluorescence microscopy following staining. FITC Annexin V and PI co-staining was performed according to the FITC Annexin V apoptosis detection kit instructions. Cells were centrifuged and resuspended with cold binding buffer at concentration 1.0 × 106 mL−1 . A 100 ␮L aliquot of the cell suspension mixed with 5 ␮L Annexin V-FITC and 5 ␮L propidium iodide (PI) was added to the flow pipe. After incubation in the dark for 15 min at room temperature, flow cytometry was used to detect apoptotic cells. TUNEL staining was performed according to the manufacturer’s

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instructions with few modifications. Cells on slides were fixed in 4% paraformaldehyde for 1 h at room temperature and stained with TUNEL reaction mixture and 50 ␮L converter-horse-radish peroxidase (POD), followed by adding DAB substrate and hematoxylin. 2.6. Caspase-3 activity assay Caspase-3 activity was measured in triplicate using the Caspase-3 Colorimetric Assay kit according to the manufacturer’s instructions. Briefly, harvested cells were centrifuged at 15,000 rpm for 10 min, followed by the addition of 1 ␮L DTT and 100 ␮L lysis buffer. Cell lysates in a 96-well microplate were incubated at 37 ◦ C with 10 ␮L of Caspase-3 Colorimetric substrate (DEVD-pNA) for 2 h. Absorbances were read by a microplate reader at a wavelength of 405 nm. 2.7. Western blot Western blotting was performed as described previously [21]. In brief, protein was extracted with a cell lysis buffer. Protein samples (100 ␮g for p-PKB (ser473), 50 ␮g for total PKB) were separated by SDS-electrophoresis on 10% gradient polyacrylamide gels and transferred onto nitrocellulose membranes, followed by electroblotting using all primary antibodies according to the manufacturer’s instructions. Immuno-detection was developed with ECL advance, and the resulting images were analyzed by Scion Image software version 4.0.3.2 (Scion Corporation, Frederick, MD). 2.8. Isolation of RNA and quantitative real-time polymerase chain reaction To evaluate the effect of the aforementioned treatments on mRNA expression, levels of Bax and Bcl-2 in MIN6 cells, and total mRNAs were isolated using 1 mL Trizol reagent according to the instructions provided by the manufacturer. mRNA (20 ␮L) was reverse transcripted into cDNA. A reaction mixture with a total volume of 25 ␮L was used for real-time PCR. ␤-Actin was used as control. The primers sequences for real-time RT-PCR were: mouse ␤-actin, forward 5 -CAT CCG TAA AGA CCT CTA TGC CAA C-3 and reverse 5 -ATG GAG CCA CCG ATC CAC A-3 ; mouse Bax, forward 5 CAG GAT GCG TCC ACC AAG AA-3 and reverse 5 -GTT GAA GTT GCC ATC AGC AAA CA-3 ; mouse Bcl-2, forward 5 -TGA AGC GGT CCG GTG GAT A-3 and reverse 5 -TTC AGT CCA GCA TTT GCA GAA GTC3 . The reaction was carried out in triplicate under the following conditions: 60 min at 37 ◦ C; 5 min at 95 ◦ C for enzyme inactivation; 30 s at 95 ◦ C, 40 cycles of 5 s at 95 ◦ C and 30 s at 60 ◦ C, 15 s at 95 ◦ C, 30 s at 60 ◦ C and 15 s at 95 ◦ C. The Ct value of each sample was defined as the cycle number when the fluorescence intensity reached the threshold. The relative RNA level was normalized to ␤-actin and quantified using Ct− . 2.9. Insulin secretion MIN6 cells were plated onto 24-well culture plates and incubated in culture medium for 48 h. Then the culture medium was removed, and cells were incubated in high glucose (25 mM) serum-free DMEM with 0.5% BSA alone or 0.4 mM palmitate complexed to 0.5% BSA, in the presence of exendin-4 at different doses (0–500 nM) for 24 h. Before the experiments, MIN6 cells were preincubated for 1 h in Krebs-Ringer bicarbonate HEPES buffer (KRBH; 135 mM NaCl, 3.6 mM KCl, 2 mM NaHCO3 , 0.5 mM NaH2 PO4 , 0.5 mM MgCl2 , 1.5 mM CaCl2 , and 10 mM HEPES) and then incubated in KRBH containing 3.3 or 16.7 mM glucose for 1 h. The culture medium was extracted and insulin was determined in the supernatant with a RIA kit.

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Fig. 1. Effects of exendin-4 on cell viability and apoptosis in MIN6 cells under lipotoxicity. MIN6 cells were incubated with either 0.5% BSA (BSA) or 0.4 mM palmitate complexed to 0.5% BSA (PA), in the presence or absence of exendin-4 (1–500 nM) (Ex), in serum-free medium for 24 h. (A) CCK-8 assay for cell viability. Data, expressed as percentage of control (cells with BSA alone), are the mean ± SE. §§ p < 0.01 vs. BSA; *p < 0.05, **p < 0.01, vs. PA. Apoptosis evaluated by Hoechst33258 staining (B), FITC Annexin V and PI co-staining (C), and TUNEL technique (D). Ex at 100 nM was used in these experiments. Values are expressed as percentage of apoptotic cells and are the mean ± SE from three independent experiments. *p < 0.05, **p < 0.01.

2.10. Statistical analysis All data are expressed as mean ± standard error (SE). Statistical analyses were performed with SPSS software version 13.0 (SPSS Inc., Chicago, IL). Comparisons among multiple groups were made by one-way analysis of variance (ANOVA) followed by post hoc testing and correction by least significant difference/Dunnett’s t-test. If the F ratio was statistically significant, a Tukey’s post hoc test was considered. A p-value of less than 0.05 was considered statistically significant. 3. Results 3.1. Exendin-4 protects MIN6 cells from lipotoxicity To evaluate the potential effects of exendin-4 on ␤-cell survival, MIN6 cells were incubated with either 0.5% BSA (BSA) or 0.4 mM palmitate complexed to 0.5% BSA (PA), in the presence or absence of increasing concentrations of exendin-4 (1–500 nM) (Ex) for 24 h and then cell viabilities were determined by the CCK-8 assay. As shown in Fig. 1A, cell viability was decreased by 21% (p < 0.01 vs. BSA) after palmitate treatment. Treatment with exendin-4 prevented palmitate-induced toxicity in a dose-dependent manner, and the greatest effect

was a reduction of 29% at 100 nM (p < 0.01 vs. PA), with 10 nM being the lowest concentration with significant activity. Exendin-4 alone promoted cell growth in a dose-dependent manner. Treatment at 100 nM produced the highest potentiation by 106%, while no significant difference was observed as compared with BSA alone. Therefore, we chose the highest preventive concentration of exendin-4 (100 nM) for all subsequent cell culture experiments. To evaluate the preventive effect of exendin-4 on palmitateinduced ␤-cell apoptosis, MIN6 cells were incubated with or without palmitate (0.4 mM), in the presence or absence of exendin4 (100 nM). Twenty-four hours after incubation, the apoptosis was determined by Hoechst33258 staining, FITC Annexin V and PI costaining, and TUNEL. The percentage of apoptosis reached with these methods was 38.7%, 40.0% and 40.2%, respectively, after palmitate treatment as shown in Fig. 1B–D. All were significantly increased when compared with BSA alone, (p < 0.05 vs. BSA). With Hoechst33258 staining, FITC Annexin V and PI co-staining, and TUNEL, treatment with exendin-4 at 100 nM reversed the rate of apoptosis by 13.9%, 12.1% and 14.3%, respectively, after palmitate treatment (p < 0.01, PA + Ex vs. PA) and 2.3%, 2.0% and 3.3%, respectively, after treatment with BSA alone (p 0.068, 0.341, 0.072, BSA + Ex vs. BSA). There were no statistically significant differences in the results obtained with the three different methods.

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Fig. 2. Exendin-4 prevents MIN6 cells from apoptosis induced by palmitate via PI3K/PKB pathway. (A) Total PKB and p-PKB (ser473) evaluated by western blot analysis. MIN6 cells were incubated with either 0.5% BSA (BSA) or 0.4 mM palmitate complexed to 0.5% BSA (PA), in serum-free medium for 24 h, then 100 nM exendin-4 (Ex) was added to the medium with PA for 0–60 min. Values are expressed as relative intensity ratio and are the mean ± SE of three independent experiments. *p < 0.05, **p < 0.01, vs. PA + Ex at 0 min. (B) Total PKB and p-PKB (ser473) expression after 30 min of stimulation with graded concentrations of Ex (1–500 nM). Data are expressed as relative intensity ratio and are the mean ± SE of three independent experiments. *p < 0.05, **p < 0.01, vs. PA without Ex. (C) Total PKB and p-PKB (ser473) expression after 30 min of stimulation with 100 nM Ex, with or without inhibitor. Before Ex stimulation, cells with PA were pretreated for 30 min with PI3K inhibitor, 50 ␮M LY294002 (LY). Data are mean ± SE from three independent experiments. *p < 0.05, **p < 0.01. Apoptosis evaluated by Hoechst33258 staining (D) and TUNEL technique (E). MIN6 cells were incubated with BSA or PA, with or without LY, in the presence or absence of 100 nM Ex, in serum-free medium for 24 h. Values are expressed as percentage of apoptotic cells and are the mean ± SE of 10 random fields of vision from three independent experiments. *p < 0.05, **p < 0.01.

3.2. Exendin-4 protects MIN6 cells from apoptosis induced by palmitate via PI3K/PKB pathway To study the possible mechanism for the effect of exendin4 on preservation of ␤-cells against lipotoxicity, we investigated the PKB signal pathway and its phosphorylation on ser473 under lipotoxic conditions in MIN6 cells. As shown in Fig. 2A, exposure of the cells to 0.4 mM palmitate for 24 h resulted in significant inhibition of PKB phosphorylation (p < 0.01, PA + Ex at 0 min vs. BSA), while treatment with 100 nM exendin-4 induced rapid activation of PKB which was greatest at 30 min (p < 0.05, vs. PA + Ex at 0 min). As exendin-4 concentration increased, an apparent doserelated increase in PKB phosphorylation activation was observed, as shown in Fig. 2B. This was most evident with 100 nM exendin4 (p < 0.01, PA + Ex at 100 nM vs. PA). To confirm whether the PI3K/PKB pathway was involved in exendin-4’s antiapoptotic action on MIN6 cells exposed to palmitate, we used LY294002, a PI3K-specific inhibitor. The data in Fig. 2C indicate that in the presence of 50 ␮M LY294002, exendin-4-induced activation of PKB was

blocked markedly (p < 0.05, Ex + LY + PA vs. Ex + PA). Furthermore, LY294002 abolished exendin-4’s cytoprotective activity against palmitate-induced apoptosis; no effect was observed when using inhibitors alone (Fig. 2D and E). 3.3. Exendin-4 antiapoptotic effect on ˇ-cells in mitochondrial pathways To assess the contribution of mitochondria in the apoptotic process, the antiapoptotic protein Bcl-2 and proapoptotic protein Bax were measured. Palmitate significantly down-regulated Bcl-2 expression (Fig. 3A, p < 0.01 vs. BSA), decreased the ratio of Bcl2 to Bax (Fig. 3C, p < 0.01 vs. BSA), up-regulated Bax expression (Fig. 3B, p < 0.01 vs. BSA), and activated caspase-3 in ␤-cells (Fig. 3D, p < 0.01 vs. BSA). However, exendin-4 at 100 nM down-regulated Bax expression (Fig. 3B, p < 0.05 vs. PA), in keeping with decreasing the caspase-3 activity (Fig. 3D, p < 0.01 vs. PA), and up-regulated Bcl-2 expression (Fig. 3A, p < 0.05 vs. BSA), increasing the ratio of Bcl-2 to Bax (Fig. 3C, p < 0.05 vs. PA).

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Fig. 3. Exendin-4 anti-apoptotic effect on ␤-cells in mitochondrial pathways. MIN6 cells were incubated with either 0.5% BSA (BSA) or 0.4 mM palmitate complexed to 0.5% BSA (PA), with or without 100 nM exendin-4 (Ex), in serum-free medium for 24 h. Bcl-2 mRNA (A) and Bax mRNA (B) assessed by RT-PCR in MIN6 cells, normalized to ␤-actin mRNA level. Data are expressed as relative mRNA to control (cells with BSA alone), and are the mean ± SE of three independent experiments. (C) Ratio of Bcl-2 to Bax. Data are expressed as relative mRNA to control (cells with BSA alone), and are the mean ± SE of three independent experiments. (D) Colorimetric Assay for caspase-3 activity. Data are expressed as percentage of control (cells with BSA alone) and are the mean ± SE of three independent experiments. *p < 0.05, **p < 0.01.

3.4. Exendin-4 stimulates insulin secretion of ˇ-cells in the presence of palmitate We measured the amount of insulin released in response to glucose under lipotoxic conditions. The insulin secretion induced by 3.3 or 16.7 mM glucose was determined after a 24-h incubation in high glucose (25 mM) serum-free DMEM with 0.5% BSA alone or 0.4 mM palmitate complexed to 0.5% BSA, in the presence of exendin-4 at different doses (0–500 nM). As shown in Fig. 4, 0.4 mM palmitate inhibited insulin secretion from MIN6 cells under the stimulation with either 3.3 or 16.7 mM glucose (p < 0.01, vs. BSA). Treatment with exendin-4 enhanced insulin secretion from MIN6 cells in the present of palmitate under stimulation with 16.7 mM glucose in a dose-dependent manner, 10 nM being the lowest significantly effective concentration (p < 0.01, vs. PA). At concentrations of 10 nM and higher, exendin-4 also increased insulin secretion in response to stimulation with 3.3 mM glucose in the present of palmitate, while there was no significant increase compared with PA alone. 4. Discussion In the present study, we investigated the effects of exendin-4 on ␤-cell function and survival in a lipotoxic environment, and the mechanism by which these effects were produced. Our results suggest that exendin-4 promoted ␤-cell survival in a dose-dependent

Fig. 4. Effects of exendin-4 on glucose-stimulated insulin secretion from MIN6 cells in the presence of palmitate. MIN6 cells were incubated with either 0.5% BSA (BSA) or 0.4 mM palmitate complexed to 0.5% BSA (PA), in the presence or absence of exendin-4 (1–500 nM) (Ex), in serum-free medium for 24 h. Before the experiments, MIN6 cells were preincubated for 1 h in KRBH and then incubated in KRBH containing 3.3 () or 16.7 mM () glucose for 1 h. All values are expressed as the means ± SE of three independent experiments. *p < 0.05, vs. stimulation with 3.3 mM glucose in the presence of PA. § p < 0.05, §§ p < 0.01 vs. stimulation with 16.7 mM glucose in the presence of PA.

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manner, attenuated ␤-cell apoptosis, and enhanced glucosestimulated insulin secretion in a lipotoxic environment. Moreover, the data indicate that exendin-4 exerted its anti-apoptotic effect partly via the PI3K/PKB pathway and the mitochondrial pathway. Free fatty acids (FFAs) act as potent signaling molecules in several cellular processes, and experimental evidence indicates that prolonged exposure to high concentrations of FFAs has detrimental effects on ␤-cells, including reduced glucose-stimulated insulin release [22], suppressed proinsulin biosynthesis, as well as ␤-cell loss by apoptosis [9,14,19]. Thus, lipotoxicity-mediated ␤-cell dysfunction and apoptosis may be relevant in the development of type 2 diabetes [3]. For these reasons, therapies that increase ␤-cell survival in the presence of FFAs could have considerable clinical impact, as they might prevent type 2 diabetes or attenuate the progression of the disease [4,13]. More recently, a growing body of evidence indicates that GLP-1 mimetics and enhancers improve ␤-cell function and mass through several signaling pathways. These agents constitute a novel class of anti-diabetes medications that will have a major impact in the treatment of type 2 diabetes [6,11,28]. Treatment with GLP1 agonists promoted islet neogenesis in association with ␤-cell proliferation and protection from cellular apoptosis in mice [18]. However, until now the protective effects of the GLP-1 receptor agonist exendin-4 on pancreatic ␤-cells in a lipotoxic environment in vitro have remained unclear. In this study, we demonstrated that exendin-4 significantly promoted pancreatic ␤-cell survival by inhibiting apoptosis (Fig. 1), and eliminated caspase-3 activity under palmitate incubation (Fig. 3). In addition, activation of the GLP-1 receptor agonist exendin-4 induced PI3K/PKB signaling. The protein kinase PKB is a multifunctional regulator of cell survival, cell cycle, priming of apoptosis and other effects, and is a downstream effector of PI3K. It has been shown previously that ␤-cell apoptosis induced by FFAs was associated with inhibition of PKB phosphorylation [7,10,27,31]. The anti-apoptotic effects of GLP-1 in pancreatic ␤-cells [29,30] were indeed mediated through PI3K/PKB signaling. Furthermore, evidence indicates that the protective effects of exendin-4 against cytokine-induced ␤-cell death require activation of PKB [17]. We therefore investigated whether exendin-4 repelled lipotoxicity in MIN6 cells via this pathway. Our results demonstrated that exposure of MIN6 cells to palmitateinduced lipotoxicity clearly inhibited PKB phosphorylation, while exendin-4 reversed it and caused rapid activation of PKB under lipotoxicity. By using the PI3K inhibitor LY294002 we observed a decrease in the protective effect of exendin-4 in MIN6 cells (Fig. 2), which indicates that exendin-4 protects pancreatic ␤-cells against lipotoxicity at least in part via the PI3K/PKB pathway. BCL-2 is a large family of apoptosis-regulator gene products members of which may either facilitate cell survival (Bcl-2, BclXL, Bcl-w, and others) or promote cell death (Bax, Bak, Bad, and others), and the relative amount of these proteins is a regulator that functions by selective protein-protein interactions [19,23]. In rodent islets, FFA-induced apoptosis may be attributed to the dysfunction of mitochondria, which is characterized by a decrease of Bcl-2 mRNA and an increase of Bax mRNA expression [19]. Similar to rodent results, our data clearly showed that apoptosis of pancreatic ␤-cells exposed to FFAs was accompanied by a marked increase of Bax mRNA, and decrease of Bcl-2 mRNA, in keeping with increased activity of caspase-3. After treatment with exendin-4 in a lipotoxic environment, we observed a decrease of caspase-3 activity, significant down-regulation of Bax mRNA, and up-regulation of Bcl-2 mRNA. Our experiments also showed the increased ratio of Bcl-2 over Bax. These results imply that the protective effect of exendin-4 is associated with the mitochondrial pathway accompanied by inhibition of Bax mRNA expression and promotion of Bcl-2 mRNA expression. However, the detailed mechanism by which the other BCL-2 family members regulate caspase activity and the effect

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of exendin-4 on mitochondria under lipotoxic remain to be determined. Finally, we studied the effect of exendin-4 on insulin secretion from MIN6 cells. It is well known that ␤-cell dysfunction is involved in the development of type 2 diabetes mellitus. The longterm exposure to a high lipid concentrations plays a vital role in the impairment of ␤-cell function [24,25]. Palmitate, as a commonly used saturated fatty acid, could blunt glucose-stimulated insulin secretion due to its lipotoxicity [22,32]. Previous studies have shown that GLP-1 and exendin-4 can enhance insulin secretion when stimulated with high glucose in mouse islet [16] and MIN6 cells [16,26]. Our experiment further confirmed that exendin4 improves ␤-cell function by significant enhancement of high glucose-stimulated insulin secretion in a lipotoxic environment, but only at concentrations of at least 10 nM. These results suggest that exendin-4 may have a promising therapeutic potential for treating type 2 diabetes. However, it is still not known whether exendin-4’s effect on glucose-stimulated insulin secretion from MIN6 cells under lipotoxic conditions is linked to its antiapoptotic action. The detailed mechanism remains to be clarified in the future. 5. Conclusions Our data suggest that exendin-4 exerts a protective effect against lipotoxicity in pancreatic ␤-cell by suppressing apoptosis and improves ␤-cell function by enhancement of glucosestimulated insulin secretion in a lipotoxic environment. In addition, the protective effect of exendin-4 on ␤-cells requires activation of PKB, which also is involved in mitochondrial pathways. Because lipotoxicity is thought to be responsible for ␤-cell decompensation in the development of obesity-associated type 2 diabetes, an antilipotoxic effect of exendin-4 may increase interest with respect to its utilization as a therapeutic agent in the treatment of type 2 diabetes. Acknowledgment We thank the Biochemical Department of China Medical University for valuable suggestions and help in the experiment. References [1] Bulotta A, Farilla L, Hui H, Perfetti R. The role of GLP-1 in the regulation of islet cell mass. Cell Biochem Biophys 2004;40:65–78. [2] Buteau J, El-Assaad W, Rhodes CJ, Rosenberg L, Joly E, Prentki M. Glucagon-like peptide-1 prevents beta cell lipotoxicity. Diabetologia 2004;47:806–15. [3] Cnop M. Fatty acids and glucolipotoxicity in the pathogenesis of type 2 diabetes. Biochem Soc Trans 2008;36:348–52. [4] Cnop M, Vidal J, Hull RL, Utzschneider KM, Carr DB, Schraw T, et al. Progressive loss of ␤-cell function leads to worsening glucose tolerance in first-degree relatives of subjects with type 2 diabetes. Diabetes Care 2007;30:677–82. [5] Doyle ME, Egan JM. Mechanisms of action of glucagon-like peptide 1 in the pancreas. Pharmacol Ther 2007;113:546–93. [6] Drucker DJ, Nauck MA. The incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet 2006;368:1696–705. [7] Eitel K, Staiger H, Rieger J, Mischak H, Brandhorst H, Brendel MD, et al. Protein kinase C ␦ activation and translocation to the nucleus are required for fatty acid-induced apoptosis of insulin-secreting cells. Diabetes 2003;52:991–7. [8] Elghazi L, Balcazar N, Bernal-Mizrachi E. Emerging role of protein kinase B/Akt signaling in pancreatic ␤-cell mass and function. Int J Biochem Cell Biol 2006;38:157–63. [9] Elks ML. Chronic perfusion of rat islets with palmitate suppresses glucose stimulated insulin release. Endocrinology 1993;133:208–14. [10] Granata R, Settanni F, Biancone L, Trovato L, Nano R, Bertuzzi F, et al. Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic beta-cells and human islets: involvement of 30,50-cyclic adenosine monophosphate/protein kinase A, extracellular signal-regulated kinase 1/2, and phosphatidyl inositol 3-Kinase/Akt signaling. Endocrinology 2007;148:512–29. [11] Holst JJ, Orskov C. The incretin approach for diabetes treatment: modulation of islet hormone release by GLP-1 agonism. Diabetes 2004;53(Suppl. 3):S197–204.

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[12] Holst JJ. The physiology of glucagon-like peptide 1. Physiol Rev 2007;87:1409–39. [13] Kahn SE. The relative contributions of insulin resistance and beta-cell dysfunction to the pathophysiology of Type 2 diabetes. Diabetologia 2003;46:3–19. [14] Kharroubi I, Ladriere L, Cardozo AK, Dogusan Z, Cnop M, Eizirik DL. Free fatty acids and cytokines induce pancreatic ␤-cell apoptosis by different mechanisms: role of nuclear factor-кB and endoplasmic reticulum stress. Endocrinology 2004;145:5087–96. [15] Kim SJ, Winter K, Nian C, Tsuneoka M, Koda Y, McIntosh CH. Glucose-dependent insulinotropic polypeptide (GIP) stimulation of pancreatic beta-cell survival is dependent upon phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signaling, inactivation of the forkhead transcription factor Foxo1, and downregulation of bax expression. J Biol Chem 2005;280:22297–307. [16] Kitani K, Oguma S, Nishiki T, Ohmori I, Galons H, Matsui H, et al. A Cdk5 inhibitor enhances the induction of insulin secretion by exendin-4 both in vitro and in vivo. J Physiol Sci 2007;57:235–9. [17] Li L, El-Kholy W, Rhodes CJ, Brubaker PL. Glucagon-like peptide-1 protects beta cells from cytokine-induced apoptosis and necrosis: role of protein kinase B. Diabetologia 2005;48:1339–49. [18] Li Y, Hansotia T, Yusta B, Ris F, Halban PA, Drucker DJ. Glucagon-like peptide-1 receptor signaling modulates beta cell apoptosis. J Biol Chem 2003;278:471–8. [19] Lupi R, Dotta F, Marselli L, Guerra SD, Masini M, Santangelo C, et al. Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that beta-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated. Diabetes 2002;51:1437–42. [20] Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell 2007;129:1261–74. [21] Martinez SC, Tanabe K, Cras-Méneur C, Abumrad NA, Bernal-Mizrachi E, Permutt MA. Inhibition of Foxo1 protects pancreatic islet beta-cells against fatty acid and endoplasmic reticulum stress-induced apoptosis. Diabetes 2008;57:846–59.

[22] McGarry JD, Dobbins RL. Fatty acid, lipotoxicity and insulin secretion. Diabetologia 1999;42:128–38. [23] Mizuno N, Yoshitomi H, Ishida H, Kuromi H, Kawaka J, Seino Y, et al. Altered bcl2 and bax expression and intracellular Ca2+ signaling in apoptosis of pancreatic cells and the impairment of glucose-induced insulin secretion. Endocrinology 1998;139:1429–39. [24] Poitout V, Robertson RP. Glucolipotoxicity: fuel excess and beta-cell dysfunction. Endocr Rev 2008;29:351–66. [25] Prentki M, Nolan CJ. Islet beta cell failure in type 2 diabetes. J Clin Invest 2006;116:1802–12. [26] Shigeto M, Katsura M, Matsuda M, Ohkuma S, Kaku K. Low, but physiological, concentration of GLP-1 stimulates insulin secretion independent of the cAMP-dependent protein kinase pathway. J Pharmacol Sci 2008;108: 274–9. [27] Tews D, Lehr S, Hartwig S, Osmers A, Paslack W, Ecke J. Anti-apoptotic action of exendin-4 in INS-1 beta cells: comparative protein pattern analysis of isolated mitochondria. Horm Metab Res 2009;41:294–301. [28] Wang Q, Brubaker PL. Glucagon-like peptide-1 treatment delays the onset of diabetes in 8 week-old db/db mice. Diabetologia 2002;45:1263–73. [29] Wang Q, Li L, Xu E, Wong V, Rhodes C, Brubaker PL. Glucagon-like peptide1 regulates proliferation and apoptosis via activation of protein kinase B in pancreatic INS-1 beta cells. Diabetologia 2004;47:478–87. [30] Widenmaier SB, Sampaio AV, Michael T, Mclntosh C. Noncanonical activation of Akt/protein kinase B in ␤-cells by the incretin hormone glucose-dependent insulinotropic polypeptide. J Biol Chem 2009;284:10764–73. [31] Wrede CE, Dickson LM, Lingohr MK, Briaud I, Rhodes CJ. Protein kinase B/Akt prevents fatty acid-induced apoptosis in pancreatic beta-cells (INS-1). J Biol Chem 2002;277:49676–84. [32] Zhou YP, Grill VE. Long-term exposure of rat pancreatic islets to fatty acid inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty acid cycle. J Clin Invest 1994;93:870–6.