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Ghrelin inhibits cell apoptosis induced by lipotoxicity in pancreatic β-cell line
Regulatory Peptides 161 (2010) 43–50
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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / r e g p e p
Ghrelin inhibits cell apoptosis induced by lipotoxicity in pancreatic β-cell line Wei Wang a,b, Dan Zhang a, Hong Zhao c, Ying Chen a, Ying Liu d, Cuiping Cao a, Lingling Han a, Guoliang Liu a,⁎ a
Department of Endocrinology, First Hospital of China Medical University, Shenyang 110001, China Department of Endocrinology, Second Hospital of Haerbin Medical University, Haerbin 150086, China c Department of Endocrinology, General Hospital of Daqing Oil-Field, Daqing 163001, China d Department of Endocrinology, Siping Central Hospital, Changchun 136000, China b
a r t i c l e
i n f o
Article history: Received 16 July 2009 Received in revised form 20 December 2009 Accepted 30 December 2009 Available online 14 January 2010 Keywords: Ghrelin Lipotoxicity MIN6 cells Protein kinase B (PKB) C-Jun N-terminal kinase (JNK) Diabetes
a b s t r a c t Lipotoxicity plays an important role in underlying mechanism of type 2 diabetes. Prolonged exposure of pancreatic βcells to elevated levels of fatty acid is associated with β-cell apoptosis. Ghrelin is a 28-amino acid peptide, mainly secreted from X/A like cells of gastric fungus. The effects of ghrelin are considered to be broadly including cell protection. However, the mechanism of ghrelin protecting pancreatic β-cells against lipotoxicity is unknown. Our study showed that ghrelin promoted cell survival and attenuated palmitate-induced apoptosis in pancreatic β-cells (MIN6). Exposure of MIN6 cells to palmitate (0.4 mM) for 24 h caused a significant increase in cell apoptosis, which could be protected by ghrelin. Exposure of MIN6 cells to ghrelin caused a rapid activation of protein kinase B (PKB) and inhibition of c-Jun N-terminal kinase (JNK) under lipotoxic state. Furthermore, LY294002, a PI3K inhibitor, abolished the anti-lipotoxic effect of ghrelin, as well as ghrelin-induced inhibition of JNK, while JNK inhibitor, SP600125 enhanced protective effect of ghrelin on MIN6 cells. Ghrelin also inhibited the mitochondrial pathway of apoptosis and it down-regulated Bax in MIN6 cells. For secretion experiment, ghrelin suppressed insulin release under palmitate-incubated state. Our findings suggest that ghrelin may prevent lipotoxicity-induced apoptosis in MIN6 cells through activation of PKB, inhibition of JNK and mitochondrial pathway. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Lipotoxicity plays an important role in underlying mechanism of type 2 diabetes [1,2]. In principle, physiologic levels of glucose and lipids are not toxic but essential to normal β-cell function. However, the prolonged exposure of pancreatic β-cells to elevated levels of glucose and/or fat signal is associated with impairment of insulin gene expression [3], inhibition of insulin synthesis and secretion [4] and induction of β-cell apoptosis [5–7]. A number of studies have shown that fatty acids can induce β-cell apoptosis in the presence of high glucose [7,8]. The serine/ threonine kinase Akt, also known as protein kinase B (PKB), plays a vital role in regulating mass and function of pancreatic β-cell [9]. There are growing evidence that activation of PKB phosphorylation is able to prevent pancreatic β-cell from lipotoxicity [6,10,11]. To pancreatic β-cells, PKB is not only a unique central node in cell signaling downstream of growth factors and cytokines, but also a key target of insulin signal system as well [9,12]. Ghrelin is a 28-amino acid peptide acylated at the serine 3 position with an octanoyl group, and is mainly secreted from X/A like cells of gastric fungus, as a natural endogenous ligand of the orphan growth hormone secretagogue receptor type 1a (GHS-R1a), through which it acts as a growth hormone releasing peptide and food intake modulator [13,14]. The effects of ghrelin are considered to be broadly including ⁎ Corresponding author. Tel.: +86 15204515822; fax: +86 24 23242525. E-mail address: [email protected] (G. Liu). 0167-0115/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2009.12.017
energy and glucose homeostasis, regulation of proliferation, apoptosis and differentiation of various normal and neoplastic cell lines, etc. [14]. Recently, the relationship between ghrelin and pancreatic β-cells has attracted much attention. Some studies have showed ghrelin-secreting cells named ε cells could be detected in the verges of pancreatic islets, suggesting that ghrelin affects pancreatic β-cells via both endocrine and paracrine pathways [15,16]. It becomes increasingly clear that the effects of ghrelin on the regulation of pancreatic β-cell proliferation, apoptosis and function, are mediated by mechanisms dependent and independent of GHS-R1a [17,18]. Ghrelin could promote cell proliferation and inhibit pancreatic β-cell apoptosis induced by interferon-r/tumor necrosis factora (IFN-r/TNF-a) synergism, as well as doxorubicin-induced β-cells apoptosis [17,18]. PI3K/PKB is supposed as the main signal pathway that mediated protective effect of ghrelin in type 1 diabetes [14,17,18]. However, it remains to be elucidated whether ghrelin protects β-cells against lipotoxicity-induced apoptosis in type 2 diabetes. Therefore, the aim of the present study is to investigate whether ghrelin prevents β-cells from lipotoxicity and explore the mechanism behind. 2. Materials and methods 2.1. Materials Ghrelin was purchased from Phoenix Pharmaceuticals, Inc. (Burlingame, CA, USA); fatty acid-free bovine serum albumin (BSA,
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fraction V), palmitate, hoechst33258, 3-(4, 5-dimethylthiazol-2-yl)2, 5-diphenyltetrazolium bromides (MTT), LY294002 and SP600125 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies used were anti-phospho-ser473-PKB, anti-total PKB, anti-phosphoJNK1/2, anti-total JNK, were from cell signaling technology (Danvers, MA, USA). TUNEL (in situ cell death detection kit, POD) kit was from Roche (Basel, Switzerland). Caspase-3 activity assay kit was purchased from R&D Systems (Minneapolis, MN, USA). Insulin detection ELISA kit was from Linco Research (St. Charles, MO, USA). 2.2. Cell culture of mouse insulinoma cells MIN6 cells, a widely used pancreatic β-cell line(passages 11–30), were cultured in Dulbecco's modified eagle's medium (DMEM) containing 25 mM glucose, with 15% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μg/mL L-glutamine, 5 μL/L β-mercaptoethanol in humidified 5% CO2, and 95% air at 37 °C.
protocol with few modifications, cell slides were fixed in 4% paraformaldehyde and stained with TUNEL reaction mixture and 50 μL converter-horse-radish peroxidase (POD), followed by adding DAB substrate and hematoxylin. 2.7. Western blot Western blotting was carried out as described previously [21]. Briefly, protein was extracted with a cell lysis buffer. Protein samples (100 µg for p-PKB (ser473) and p-JNK1/2 or 50 µg for total PKB, JNK) were separated by SDS-electrophoresis through either 10% gradient polyacrylamide gels and transferred to nitrocellulose membranes, followed by immunoblotting 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. 2.8. RNA isolation and reverse-transcription polymerase chain reaction
2.3. Fatty acid, ghrelin 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 ghrelin. Preparation of the 0.4 mM palmitate fatty acid media was carried out as previously described [19]. Briefly, a 20 mM solution of palmitate in 0.01 mol/L NaOH was incubated at 70 °C for 30 min. Then 330 μL of 30% BSA and 400 μL of the palmitate/NaOH were mixed together and filter-sterilized with 20 mL of the DMED in total. Concentration of BSA is 0.5% in all medium. We selected sole glucose (25 mM) for all the experiments, because the sole glucose was essential for MIN6 cell growth, and compared with 5 mM glucose, 25 mM glucose protected MIN6 cells from apoptosis [19]. Graded doses of ghrelin (1, 10, 100 and 500 nM) were prepared daily before experiments. 50 μM LY294002, an inhibitor of PI3K, and 300 nM SP600125, an inhibitor of JNK were used in the study. 2.4. Cell viability assay Cell viability was assessed by MTT as described previously [20]. Cells were seeded on 96-well plates at a density of 5000 cells per well. For detection, cells were incubated with 1 mg/mL MTT for approximately 1 h. The medium was removed and the formazan product was solubilized with 150 μL dimethylsulfoxide. Viability was assessed by spectrophotometry at 570 nm absorbance using a 96-well plate reader. 2.5. Caspase-3 activity assay The caspase-3 amount was measured in triplicate by Caspase-3 Colorimetric Assay kit according to the manufacturer's protocol. Briefly, harvested cells were centrifuged at 10,000 rpm for 1 min, followed by the addition of 1 μL DTT and 100 μL lysis buffer. Cell lysates in a 96 well microplate were incubated at 37 °C with 5 μL of Caspase-3 Colorimetric substrate (DEVD-pNA) for 2 h. Absorbances were read by a microplate reader at 405 nm wavelength.
Reverse-transcription polymerase chain reaction was performed to semi-quantify mRNA expression of BAX, BCL-2 gene. Total RNA was isolated and first strand cDNA was synthesized using MMLV reverse transcriptase and hexamers as described previously [22] from MIN6 cells. The primer sequences were devised by primer5 software and were the following: mouse β-actin, forward 5′-CTGTCCCTGTATGCCTCTG-3′ and reverse5′-TGTCACGCACGATTTCC-3; mouse BAX, forward 5′-GCAGCGGCAGTGATGGAC-3′ and reverse 5′-GCAAAGTAGAAGAGGGCAACC-3′; mouse BCL-2, forward 5′-GCTACCGTCGTGACTTCGC-3′ and reverse 5′-CTACCCAGCCTCCGTTATCC-3′.cDNA (9 μL) was amplified by PCR in a 50 μL volume with amplitaq gold polymerase following conditions: 35 cycles in all, one cycle of 94 °C for 30 s, annealing for 30 s,72 °C for 30 s, and 72 °C for 7 min for elongation. The annealing temperature was adjusted for each target: 60 °C for BAX, 60 °C for BCL-2, and 57 °C for β-actin. The PCR products (362 bp for BAX, 270 bp for BCL-2, 217 bp for β-actin) were separated by 1.5% agarose gel electrophoresis and visualized by ethidiumbromide staining. The resulting images were analyzed by Scion Image software. 2.9. ELISA for insulin For secretion experiments, MIN6 cells were plated onto 24-well culture plates and incubated at 37 °C in 5% CO2, in culture medium for 48 h. Then the media were removed, and cells were incubated in 1 mL of 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 ghrelin at different doses (0–500 nM). After 6 h or 24 h of incubation, the media were extracted and insulin concentrations were determined by an ELISA kit. Insulin release was corrected for the amount of living cells (2 × 105 cells) through flow cytometer. 2.10. Statistical analysis Data are presented as means ± SE. Statistical analyses were performed with SPSS using ANOVA. Differences between groups were assessed for significance by Dunnett's test. A p value of less than 0.05 was considered significant.
2.6. Apoptosis assay 3. Results Apoptosis was evaluated by two different methods, hoechst33258 staining and TdT-mediated dUTP nick-end labeling (TUNEL) technique. Apoptosis indexes were assessed respectively by counting Hoechst positive cells (chromatin condensation or fragmented nuclear membrane) and TUNEL positive cells (apoptotic nucleus was brown-stained). Hoechst staining was performed by exposing the cell slides to 10 µg/mL hoechst33258 for 10 min at room temperature. TUNEL staining was performed according to the manufacturer's
3.1. Ghrelin inhibits lipotoxicity in MIN6 cells To test the potential effects of ghrelin on β-cell, MIN6 cells were incubated with either 0.5% BSA or 0.4 mM palmitate complexed to 0.5% BSA (PA), in the presence or absence of ghrelin (AG, added to the medium every 8 h) for 24 h. As shown in Fig. 1A, cell viability was decreased by 21% (p < 0.05, vs. BSA) after PA treatment. Treatment
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with AG dose-dependently prevented PA-induced toxicity, most effectively by 34% at 100 nM (p<0.01, vs. PA), 10 nM being the lowest significantly active concentration, while AG alone dose-dependently promoted cell growth, treatment at 100 nM significantly produced best potentiation by 128% (p<0.05, vs. BSA), as compared with BSA alone. Cell apoptosis was significantly increased in PA group by 61% when compared to BSA (Fig. 1B), while treatment with AG at 100 nM alone decreased by 15% (p<0.05, vs. BSA). PA/AG (at 100 nM) synergism decreased caspase 3 activity by 27% when compared to PA alone (p<0.05, vs. PA). Fig. 1C showed the apoptosis evaluated by hoechst33258 staining. The percentage of apoptosis reached to 47% after PA treatment for 24 h, which was significantly increased when compared with BSA alone. Treatment with
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AG at 100 nM reversed apoptosis rate by 16% (p<0.01, PA+AG vs. PA) or 1.7% (p<0.05, BSA+AG vs. BSA), respectively. 3.2. Ghrelin prevents MIN6 cells from apoptosis induced by lipotoxicity via PI3K/PKB pathway To explore the mechanism behind ghrelin-induced anti-lipotoxicity effect, we utilized MIN6 cells and investigated PKB signal pathway and its phosphorylation on ser473 under lipotoxic condition. As shown in Fig. 2A, exposure of the cells to 0.4 mM palmitate for 24 h caused significant inhibition of PKB phosphorylation (p < 0.01, PA+ AG at 0 min vs. BSA), while ghrelin at 100 nM induced rapid activation of PKB and reached the maximal effect at 30 min (p < 0.01, vs. PA+ AG at 0 min). Fig. 2B showed that ghrelin dose-dependently stimulated activation of PKB phosphorylation and treatment at 100 nM produced most effective potentiation (p < 0.01, PA+ AG at 100 nM vs. PA). To confirm relationship between PI3K/PKB pathway and apoptosis, we investigated whether AG prevented palmitate-induced apoptosis in the presence of specific inhibitor of PI3K (LY294002). As indicated in Fig. 2C, ghrelin-induced activation of PKB was markedly blocked by PI3K inhibitor, LY294002 (p < 0.05, AG + LY + PA vs. AG + PA). Fig. 2D showed that PA group had much more brown-stained apoptotic cells than other groups. Moreover, LY294002 abolished ghrelin cytoprotective activity against palmitate-induced apoptosis, no effect was observed when using inhibitors alone (Fig. 2E and F). 3.3. Ghrelin attenuates activation of palmitate-induced JNK phosphorylation and JNK inhibitor, SP600125, enhances anti-apoptotic effects of ghrelin in MIN6 cells The c-Jun N-terminal kinase, JNK plays important roles in pancreatic β-cell proliferation and apoptosis. As shown in Fig 3A, MIN6 cells were incubated with PA for 24 h and produced activation of JNK phosphorylation (p < 0.01, PA + AG at 0 min vs. BSA), while ghrelin at 100 nM resulted in a transient decrease at 30 min (p < 0.05, vs. PA + AG at 0 min). Then we examined the relationship between JNK with PKB pathway. We found that ghrelin-induced inhibition of JNK phosphorylation was blocked by PI3K inhibitor, LY294002 (p < 0.05, AG + LY + PA vs. AG + PA) (Fig. 3B). To further determine whether JNK pathway was pivotal to ghrelin anti-apoptotic action, we examined the effect of SP600125, a specific inhibitor of JNK pathway. Our results showed that SP600125 alone can significantly prevent palmitate-induced apoptosis. Combination of SP600125 with AG at 100 nM could enhance ghrelin's anti-apoptotic effect in MIN6 cells (Fig. 3C and D). 3.4. Ghrelin antiapoptosis effect involve mitochondrial pathways
Fig. 1. Effects of ghrelin on cell viability, caspase-3 activity and apoptosis in MIN6 cells under lipotoxicity state. 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 ghrelin (AG, added to the medium every 8 h) at different doses (1–500 nM), in serum-free medium for 24 h. A, An MTT assay for cell viability. Data, expressed as percentage of control (cells with BSA alone), are the means ± SE of 13 replicates. , p < 0.05, vs. BSA; §, p < 0.05, §§, p < 0.01, vs. PA. B, Caspase-3 activity assay by ELISA. AG at 100 nM was used in this experiment. Results are expressed as percentage of control (cells with BSA alone) and are the means± SE of three independent experiments. C, Apoptosis evaluated by counting Hoechst positive cells. AG at 100 nM was used in this experiment. Values are expressed as percentage of Hoechst positive cells and are the means ± SE of 10 random fields of vision from three independent experiments. , p < 0.05; , p < 0.01.
We detected important members of BCL-2 protein family, BAX and BCL-2 in mitochondria pathways. Consistent with caspase 3 activity assay (Fig. 4D), palmitate significantly up-regulated BAX (Fig. 4A, p < 0.01, vs. BSA), increased the ratio of BAX over BCL-2 (Fig. 4C, p < 0.01, vs. BSA) and down-regulated BCL-2 (Fig. 4B, p < 0.05, vs. BSA). However, ghrelin at 100 nM down-regulated BAX mRNA expression (Fig. 4A, p < 0.01, vs. PA), in line with decreasing the ratio of BAX over BCL-2 (Fig. 4C, p < 0.05, vs. PA), but did not affect BCL-2 mRNA expression (Fig. 4B). 3.5. Ghrelin inhibits insulin secretion from MIN6 cells under palmitateincubated state Conflicting results have been reported concerning the effect of ghrelin on insulin secretion from pancreatic β-cells [23]. We therefore studied the influence of ghrelin on insulin secretion from MIN6 cells under lipotoxic condition. As shown in Fig. 5A, after 6 h of incubation, ghrelin dose-dependently inhibited insulin secretion from MIN6 cells
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in the absence of palmitate (ghrelin 0 nM, 1437 ± 298 pM; 1 nM, 1344 ± 285 pM; 10 nM, 1121 ± 367 pM; 100 nM, 823 ± 327 pM; 500 nM, 836 ± 303 pM), as well as in the presence of palmitate (ghrelin 0 nM, 594 ± 188 pM; 1 nM, 514 ± 232 pM; 10 nM, 466 ±
129 pM; 100 nM, 413 ± 153 pM; 500 nM, 426 ± 130 pM). Ghrelin at no less than 10 nM, but not at 1 nM, significantly inhibited insulin secretion from MIN6 cells under either BSA- or palmitate-incubated state. However, as data seen in Fig. 5B, there were no significant
Fig. 2. Ghrelin prevent MIN6 cells from apoptosis induced by lipotoxicity 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 ghrelin (AG) was added to the medium with PA for 0–60 min. Values are expressed as means ± SE of three independent experiments. , p < 0.05, , p < 0.01, vs. PA + AG at 0 min. B, Total PKB and p-PKB (ser473) expression after 30 min of stimulation with graded concentrations of AG (1–500 nM). Data are expressed as relative intensity ratio and are the means ± SE of three independent experiments. , p < 0.05, , p < 0.01, vs. PA without AG. C, Total PKB and p-PKB (ser473) expression after 30 min of stimulation with 100 nM AG, with or without inhibitor. Before AG stimulation, cells with PA were pretreated for 30 min with PI3K inhibitor, 50 μM LY294002 (LY). Results are means ± SE from three independent experiments. , p < 0.05. D, magnification, × 400 (by TUNEL); E and F, Apoptosis evaluated respectively by TUNEL technique and Hoechst-staining method. MIN6 cells were incubated with BSA or PA, with or without LY, in the presence or absence of 100 nM AG (added to the medium every 8 h), in serum-free medium for 24 h. Values are expressed as percentage of apoptotic cells and are the means ± SE of 10 random fields of vision from three independent experiments. , p < 0.05; , p < 0.01.
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Fig. 3. Ghrelin antiapoptosis effects involve JNK pathways. A, Total JNK and p-JNK1/2 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 ghrelin (AG) was added to the medium with PA for 0–60 min. Values are expressed as means ± SE of three independent experiments. , p < 0.05, , p < 0.01,vs. PA + AG at 0 min; ＃, p < 0.05, vs. PA + AG at 15 min. B. Total JNK and p-JNK1/2 expression after 30 min of stimulation with 100 nM AG, with or without inhibitor. Before AG stimulation, cells with PA were pretreated for 30 min with PI3K inhibitor, 50 μM LY294002 (LY). Results are means ± SE from three experiments. , p < 0.05; C and D, Apoptosis evaluated respectively by TUNEL technique and Hoechst-staining method. MIN6 cells were incubated with BSA or PA, with or without 300 nM SP600125 (SP), in the presence or absence of 100 nM AG (added to the medium every 8 h), in serum-free medium for 24 h. Values are expressed as percentage of apoptotic cells and are the means ± SE of 10 random fields of vision from three independent experiments. , p < 0.05; , p < 0.01.
effects of ghrelin on insulin release under lipotoxic pressure after 24 h of incubation (ghrelin 0 nM, 430 ± 126 pM; 1 nM, 403 ± 91 pM; 10 nM, 393 ± 122 pM; 100 nM, 385 ± 149 pM; 500 nM, 306± 87 pM), although ghrelin at no less than 500 nM markedly inhibited insulin release from MIN6 cells in the absence of palmitate (ghrelin 0 nM, 1211 ± 321 pM; 1 nM, 1153 ± 383 pM; 10 nM, 1135 ± 279 pM; 100 nM, 1092 ± 387 pM; 500 nM, 1014 ± 287 pM). 4. Discussion Our present study demonstrated that ghrelin promoted β-cell survival and partially inhibited apoptosis induced by lipotoxicity in MIN6 cells. Granata R et al. [17] had confirmed that ghrelin prevented pancreatic β-cell line (HIT-T15) from IFN-r/TNF-a induced apoptosis. Zhang Y et al. [18] also attained similar results under doxorubicininduced condition. Our results certified the protective action of ghrelin on pancreatic β-cells (MIN6) in lipotoxic environment. Lipotoxicity and glucolipotoxicity play important roles in patients with type 2 diabetes. Apart from the deterioration role of lipotoxicity and glucolipotoxicity in β-cell function, elevation of plasma fatty acid level also plays a pathogenic role in the early stages of the disease. Survival of pancreatic cells is obviously of importance for maintaining normal glucose metabolism and islet function. Growing evidence have showed that prolonged exposure of isolated islets or insulin-secreting
cells to elevated levels of fatty acids is associated with β-cell apoptosis [6,10,11,24]. Thus, blockade of apoptosis through inhibition of lipotoxicity may be a novel approach for protecting pancreatic islets. Interestingly, ghrelin was recently shown to protect pancreatic β-cells from apoptosis induced by IFN-r/TNF-a synergism or doxorubicin [17,18]. However, there were no reports of protective effects of ghrelin on pancreatic β-cells in lipotoxic environment. Our study had proved that ghrelin significantly promoted pancreatic β-cell survival by inhibiting apoptosis and eliminating caspase-3 activity under palmitate-incubated condition (Fig. 1). Moreover, we determined that PI3K/PKB signaling pathway was involved in ghrelin's anti-apoptotic effect. It was becoming increasingly evident that β-cell apoptosis induced by palmitate was associated with inhibition of PKB phosphorylation [6,10,11,25]. Ghrelin's anti-apoptotic effects in many cell lines [21,26–28], including pancreatic β-cells [17,18], were indeed mediated through PI3K/PKB signaling. We considered whether ghrelin counteract lipotoxicity in MIN6 cells through this pathway. Our findings showed that exposure of MIN6 cells to palmitate-induced lipotoxicity markedly inhibited PKB phosphorylation, while ghrelin caused rapid activation of PKB under lipotoxicity condition. PI3K inhibitor (LY294002) blocked protective effect of ghrelin in MIN6 cells (Fig. 2). These data indicated that ghrelin exerted an anti-apoptotic effect on pancreatic β-cells under lipotoxic pressure, at least in part via PI3K/PKB pathway.
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Fig. 4. Ghrelin antiapoptosis effects involve 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 ghrelin (AG), in serum-free medium for 6 h. A and B, BAX mRNA and BCL-2 mRNA respectively assessed by RT-PCR in MIN6 cells, normalized to β-actin mRNA level. C, The ratio of BAX over BCL-2. Result expressed as relative intensity ratio, and are the means ± SE of three independent experiments; D, Caspase-3 activity assay by ELISA. Results are expressed as percentage of control (cells with PA) and are the means ± SE of three independent experiments. , p < 0.05; , p < 0.01.
Fig. 5. Effect of ghrelin on insulin secretion from MIN6 cells under palmitate-incubated state. Medium insulin concentration was measured by ELISA after 6 h or 24 h incubation of MIN6 cells in the presence (■) or absence (□) of 0.4 mM palmitate (PA)with 1–500 nM ghrelin (AG) or without ghrelin. A, Medium insulin after 6 h of incubation. B, Medium insulin after 24 h of incubation. All values are expressed as the means ± SE of four independent experiments. , p < 0.05, , p < 0.01, vs. nonstimulation with AG in the absence of PA; §, p < 0.05, vs. nonstimulation with AG in the presence of PA.
The c-Jun NH2-terminal kinase, JNK is classic stress-activated protein kinase and many cellular stresses have been shown to stimulate JNK activation. Recent studies demonstrated that treatment with the saturated fatty acid palmitic acid resulted in sustained JNK activation in pancreatic β-cells [19,29]. Early induction of JNK plays an important role in fatty acid-induced beta-cell apoptosis [19], and there existed a cross-talking between JNK and PKB pathways [19,30]. The role of JNK pathway in palmitate-induced pancreatic β-cell apoptosis and association with ghrelin's anti-apoptotic action need to be determined. Thus, we detected the change of JNK proteins expression before and after treatment with ghrelin under lipotoxicity state. In line with previous studies [19], we demonstrated that palmitate produced an activation of JNK1/2 phosphorylation in MIN6 cells, and treatment with SP600125 alone significantly blocked palmitate-induced apoptosis (Fig. 3). We also found that ghrelin could inhibit activation of JNK1/2 phosphorylation induced by palmitate. These results suggested that ghrelin antiapoptosis effects involved JNK pathways. In previous studies, PKB can directly phosphorylate the amino acid residue S83 on apoptosis signal-regulated kinase1 (ASK1, also known as MAPKKK5), which is an upstream activating kinase within the JNK and p38 pathways [31]. Therefore, a balance between PI3K/PKB survival signaling and JNK apoptotic signaling can be established through such crosstalk [12]. Our data showed that, under lipotoxic state, ghrelin-induced inhibition of JNK pathway can be partly blocked by PKB inhibitor, LY294002 (Fig. 3B), whereas JNK inhibitor, SP600125 had no effect on ghrelin-stimulated activation of PKB (data not shown). These data indicated that JNK protein may be one downstream target of PI3K/PKB pathway in palmitate-incubated MIN6 cells. Ghrelin-induced inhibition of JNK pathway may be partly dependent on the activation of PI3K/PKB pathway. Then we incubated MIN6 cells with SP600125/ghrelin synergism, which resulted in enhancing ghrelin protective effects. These results showed that ghrelin's anti-lipotoxicity effect was associated with JNK pathway, and change of JNK may be partly ascribed
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to ghrelin-induced activation of PKB pathway. Moreover, ghrelin could not fully suppress palmitate-induced activation of JNK phosphorylation. In mammalian cells, apoptosis may involve disruption of mitochondrial function, which is regulated by members of the BCL-2 protein family. Palmitate-induced apoptosis typically involves the mitochondrial pathway [5,7]. For these reasons, we determined whether induction of apoptosis in MIN6 cells by palmitate and effect of ghrelin were associated with BCL-2 protein family, pro-apoptotic gene BAX or anti-apoptotic gene BCL-2. We found an increase in Bax, and a decrease in Bcl-2, consistent with increased activity of caspase-3 after MIN6 cells were incubated with palmitate for 6 h. After treatment with AG in environment of lipotoxicity, we detected a decrease of caspase-3 activity and significant downregulation of BAX. Although ghrelin may not significantly influence mRNA expression of BCL-2, the result also demonstrated the slight increase of BCL-2 mRNA. These results indicated that protective effect of ghrelin was associated with mitochondrial pathway by inhibiting BAX mRNA expression. Finally, we detected the effect of ghrelin on insulin secretion from MIN6 cells. Previous studies had certified that ghrelin inhibited glucose-stimulated insulin secretion in mouse islet [32] and MIN6 cells [33]. Our experiment further confirmed that ghrelin prevents MIN6 cells from secreting insulin in lipotoxic environment, although our results showed that inhibitory effect of ghrelin on insulin secretion required higher concentrations at no less than 10 nM, and could not last 24 h in lipotoxic environment. It is still debated whether ghrelin stimulates or suppresses insulin secretion in pancreatic β-cells [34,35]. Some report had identified that ghrelin can promote insulin secretion in other pancreatic β-cell line, such as HIT-T15 [17]. These different conclusions were possibly due to different experimental objects or design. It is still unknown that whether ghrelin effect on insulin release from MIN6 cells participate in its anti-apoptotic action, and detailed mechanism remains to be clarified in the future. Pancreatic β-cell apoptosis induced by lipotoxicity was thought of as a main mechanism in type 2 diabetes with obesity. Reducing impairment of lipotoxicity on pancreatic β-cells may be one key point of delaying progress and deterioration of diabetes. But clinical medicines used as counteracting lipotoxicity were so limited. The finding that ghrelin protecting β-cells against lipotoxicity may lead to novel therapeutic approaches aimed at enhancing the survival of βcells, thereby reducing or delaying lipotoxicity-mediated β-cell destruction in the development of type 2 diabetes. In summary, the results of the present study demonstrated that the protective effects of ghrelin against palmitate-induced apoptosis required activation of PKB, in the meantime, also involved JNK and mitochondria pathways. Acknowledgment We thank the Biochemical Department of China Medical University for valuable suggestions and help. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.regpep.2009.12.017. References [1] Poitout V, Robertson RP. Lipotoxicity: fuel excess and beta-cell dysfunction. Endocr Rev 2008;29:351–66. [2] Prentki M, Nolan CJ. Islet beta cell failure in type 2 diabetes. J Clin Invest 2006;116(7): 1802–12. [3] Elks ML. Chronic perfusion of rat islets with palmitate suppresses glucose stimulated insulin release. Endocrinology 1993;133:208–14. [4] Kelpe CL, Moore PC, Parazzoli SD, Wicksteed B, Rhodes CJ, Poitout V. Palmitate inhibition of insulin gene expression is mediated at the transcriptional level via ceramide synthesis. J Biol Chem 2003;278:30015–21.
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[5] Lupi R, Dotta F, Marselli L, Del Guerra S, Masini M, Santangelo C, Patane G, Boggi U, Piro S, Anello M, Bergamini E, Mosca F, Di Mario U, Del Prato S, Marchetti P. 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. [6] 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. [7] Maestre I, Jordan J, Calvo S, Reig JA, Cena V, Soria B, Prentki M, Roche E. Mitochondrial dysfunction is involved in apoptosis induced by serum withdrawal and fatty acids in the beta-cell line INS-1. Endocrinology 2003;144:335–45. [8] Maedler K, Oberholzer J, Bucher P, Spinas GA, Donath MY. Monounsaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human pancreatic beta cell turnover and function. Diabetes 2003;52:726–33. [9] Elghazi L, Balcazar N, Bernal-Mizrachi E. Emerging role of protein kinase B/Akt signaling in pancreatic β-cell mass and function. Int J Biochem Cell Biol 2006;38: 157–63. [10] 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. [11] 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. [12] Manning Brendan D, Cantley Lewis C. AKT/PKB signaling: navigating downstream. Cell 2007;129:1261–74. [13] Soares JB, Leite-Moreira AF. Ghrelin, des-acyl ghrelin and obestatin: three pieces of the same puzzle. Peptides 2008;29:1255–70. [14] Camiña JP. Cell biology of the ghrelin receptor. J Neuroendocrinol 2006;18:65–76. [15] Prado CL, Pugh-Bernard AE, Elghazi L, Sosa-Pineda B, Sussel L. Ghrelin cells replace insulin producing β cells in two mouse models of pancreas development. Proc Natl Acad Sci 2004;101:2924–9. [16] Wierup N, Svensson H, Mulder H, Sundler F. The ghrelin cell: a novel developmentally regulated islet cell in the human pancreas. Regul Pept 2002;107:63–9. [17] Granata R, Settanni F, Biancone L, Trovato L, Nano R, Bertuzzi F, Destefanis S, Annunziata M, Martinetti M, Catapano F, Ghè C, Isgaard J, Papotti M, Ghigo E, Muccioli G. Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic beta-cells and human islets: involvement of 3′, 5′-cyclic adenosine monophosphate/protein kinase A, extracellular signal-regulated kinase 1/2, and phosphatidyl inositol 3-Kinase/Akt signaling. Endocrinology 2007;148: 512–29. [18] Zhang Y, Ying B, Shi L, Fan H, Yang D, Xu D, Wei Y, Hu X, Zhang Y, Zhang X, Wang T, Liu D, Dou L, Chen G, Jiang F, Wen F. Ghrelin inhibit cell apoptosis in pancreatic beta cell line HIT-T15 via mitogen-activated protein kinase/phosphoinositide 3kinase pathways. Toxicology 2007;237:194–202. [19] 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. [20] Baldanzi Gianluca, Filigheddu Nicoletta, Cutrupi Santina, Catapano Filomena, Bonissoni Sara, Fubini Alberto, Malan Daniela, Baj Germano, Granata Riccarda, Broglio Fabio, Papotti Mauro, Surico Nicola, Bussolino Federico, Isgaard Jorgen, Deghenghi Romano, Sinigaglia Fabiola, Prat Maria, Muccioli Giampiero, Ghigo Ezio, Graziani. Andrea. Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3-kinase/AKT. J Cell Biol 2002;159:1029–37. [21] Nakae J, Biggs III WH, Kitamura T, Cavenee WK, Wright CV, Arden KC, Accili D. Regulation of insulin action and pancreatic beta-cell function by mutated alleles of the gene encoding forkhead transcription factor Foxo1. Nat Genet 2002;32:245–53. [22] Roduit R, Morin J, Massé F, Segall L, Roche E, Newgard CB, Assimacopoulos-Jeannet F, Prentki M. Glucose downregulates the expression of the peroxisome proliferator-activated receptor-alpha gene in the pancreatic beta-cell. J Biol Chem 2000;275:35799–806. [23] Yada T, Dezaki K, Sone H, Koizumi M, Damdindorj B, Nakata M, Kakei M. Ghrelin regulates insulin release and glycemia: physiological role and therapeutic potential. Curr Diabetes Rev 2008;4:18–23. [24] Karaskov Elizabeth, Scott Cameron, Zhang Liling, Teodoro Tracy, Ravazzola Mariella, Volchuk Allen. Chronic palmitate but not oleate exposure induces endoplasmic reticulum stress, which may contribute to INS-1 pancreatic β-cell apoptosis. Endocrinology 2006;147:3398–407. [25] Eitel K, Staiger H, Rieger J, Mischak H, Brandhorst H, Brendel MD, Bretzel RG, Häring HU, Kellerer M. Protein kinaseCδ activation and translocation to the nucleus are required for fatty acid-induced apoptosis of insulin-secreting cells. Diabetes 2003;52:991–7. [26] Hong Zhao, GuoLiang Liu, QiuYue Wang, LiYing Ding, Hui Cai, HaiHong Jiang, ZhongQiu Xin. Effect of ghrelin on human endothelial cells apoptosis induced by high glucose. Biochem Biophys Res Commun 2007;362:677–81. 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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / r e g p e p
Ghrelin inhibits cell apoptosis induced by lipotoxicity in pancreatic β-cell line Wei Wang a,b, Dan Zhang a, Hong Zhao c, Ying Chen a, Ying Liu d, Cuiping Cao a, Lingling Han a, Guoliang Liu a,⁎ a
Department of Endocrinology, First Hospital of China Medical University, Shenyang 110001, China Department of Endocrinology, Second Hospital of Haerbin Medical University, Haerbin 150086, China c Department of Endocrinology, General Hospital of Daqing Oil-Field, Daqing 163001, China d Department of Endocrinology, Siping Central Hospital, Changchun 136000, China b
a r t i c l e
i n f o
Article history: Received 16 July 2009 Received in revised form 20 December 2009 Accepted 30 December 2009 Available online 14 January 2010 Keywords: Ghrelin Lipotoxicity MIN6 cells Protein kinase B (PKB) C-Jun N-terminal kinase (JNK) Diabetes
a b s t r a c t Lipotoxicity plays an important role in underlying mechanism of type 2 diabetes. Prolonged exposure of pancreatic βcells to elevated levels of fatty acid is associated with β-cell apoptosis. Ghrelin is a 28-amino acid peptide, mainly secreted from X/A like cells of gastric fungus. The effects of ghrelin are considered to be broadly including cell protection. However, the mechanism of ghrelin protecting pancreatic β-cells against lipotoxicity is unknown. Our study showed that ghrelin promoted cell survival and attenuated palmitate-induced apoptosis in pancreatic β-cells (MIN6). Exposure of MIN6 cells to palmitate (0.4 mM) for 24 h caused a significant increase in cell apoptosis, which could be protected by ghrelin. Exposure of MIN6 cells to ghrelin caused a rapid activation of protein kinase B (PKB) and inhibition of c-Jun N-terminal kinase (JNK) under lipotoxic state. Furthermore, LY294002, a PI3K inhibitor, abolished the anti-lipotoxic effect of ghrelin, as well as ghrelin-induced inhibition of JNK, while JNK inhibitor, SP600125 enhanced protective effect of ghrelin on MIN6 cells. Ghrelin also inhibited the mitochondrial pathway of apoptosis and it down-regulated Bax in MIN6 cells. For secretion experiment, ghrelin suppressed insulin release under palmitate-incubated state. Our findings suggest that ghrelin may prevent lipotoxicity-induced apoptosis in MIN6 cells through activation of PKB, inhibition of JNK and mitochondrial pathway. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Lipotoxicity plays an important role in underlying mechanism of type 2 diabetes [1,2]. In principle, physiologic levels of glucose and lipids are not toxic but essential to normal β-cell function. However, the prolonged exposure of pancreatic β-cells to elevated levels of glucose and/or fat signal is associated with impairment of insulin gene expression [3], inhibition of insulin synthesis and secretion [4] and induction of β-cell apoptosis [5–7]. A number of studies have shown that fatty acids can induce β-cell apoptosis in the presence of high glucose [7,8]. The serine/ threonine kinase Akt, also known as protein kinase B (PKB), plays a vital role in regulating mass and function of pancreatic β-cell [9]. There are growing evidence that activation of PKB phosphorylation is able to prevent pancreatic β-cell from lipotoxicity [6,10,11]. To pancreatic β-cells, PKB is not only a unique central node in cell signaling downstream of growth factors and cytokines, but also a key target of insulin signal system as well [9,12]. Ghrelin is a 28-amino acid peptide acylated at the serine 3 position with an octanoyl group, and is mainly secreted from X/A like cells of gastric fungus, as a natural endogenous ligand of the orphan growth hormone secretagogue receptor type 1a (GHS-R1a), through which it acts as a growth hormone releasing peptide and food intake modulator [13,14]. The effects of ghrelin are considered to be broadly including ⁎ Corresponding author. Tel.: +86 15204515822; fax: +86 24 23242525. E-mail address: [email protected] (G. Liu). 0167-0115/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2009.12.017
energy and glucose homeostasis, regulation of proliferation, apoptosis and differentiation of various normal and neoplastic cell lines, etc. [14]. Recently, the relationship between ghrelin and pancreatic β-cells has attracted much attention. Some studies have showed ghrelin-secreting cells named ε cells could be detected in the verges of pancreatic islets, suggesting that ghrelin affects pancreatic β-cells via both endocrine and paracrine pathways [15,16]. It becomes increasingly clear that the effects of ghrelin on the regulation of pancreatic β-cell proliferation, apoptosis and function, are mediated by mechanisms dependent and independent of GHS-R1a [17,18]. Ghrelin could promote cell proliferation and inhibit pancreatic β-cell apoptosis induced by interferon-r/tumor necrosis factora (IFN-r/TNF-a) synergism, as well as doxorubicin-induced β-cells apoptosis [17,18]. PI3K/PKB is supposed as the main signal pathway that mediated protective effect of ghrelin in type 1 diabetes [14,17,18]. However, it remains to be elucidated whether ghrelin protects β-cells against lipotoxicity-induced apoptosis in type 2 diabetes. Therefore, the aim of the present study is to investigate whether ghrelin prevents β-cells from lipotoxicity and explore the mechanism behind. 2. Materials and methods 2.1. Materials Ghrelin was purchased from Phoenix Pharmaceuticals, Inc. (Burlingame, CA, USA); fatty acid-free bovine serum albumin (BSA,
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fraction V), palmitate, hoechst33258, 3-(4, 5-dimethylthiazol-2-yl)2, 5-diphenyltetrazolium bromides (MTT), LY294002 and SP600125 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies used were anti-phospho-ser473-PKB, anti-total PKB, anti-phosphoJNK1/2, anti-total JNK, were from cell signaling technology (Danvers, MA, USA). TUNEL (in situ cell death detection kit, POD) kit was from Roche (Basel, Switzerland). Caspase-3 activity assay kit was purchased from R&D Systems (Minneapolis, MN, USA). Insulin detection ELISA kit was from Linco Research (St. Charles, MO, USA). 2.2. Cell culture of mouse insulinoma cells MIN6 cells, a widely used pancreatic β-cell line(passages 11–30), were cultured in Dulbecco's modified eagle's medium (DMEM) containing 25 mM glucose, with 15% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μg/mL L-glutamine, 5 μL/L β-mercaptoethanol in humidified 5% CO2, and 95% air at 37 °C.
protocol with few modifications, cell slides were fixed in 4% paraformaldehyde and stained with TUNEL reaction mixture and 50 μL converter-horse-radish peroxidase (POD), followed by adding DAB substrate and hematoxylin. 2.7. Western blot Western blotting was carried out as described previously [21]. Briefly, protein was extracted with a cell lysis buffer. Protein samples (100 µg for p-PKB (ser473) and p-JNK1/2 or 50 µg for total PKB, JNK) were separated by SDS-electrophoresis through either 10% gradient polyacrylamide gels and transferred to nitrocellulose membranes, followed by immunoblotting 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. 2.8. RNA isolation and reverse-transcription polymerase chain reaction
2.3. Fatty acid, ghrelin 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 ghrelin. Preparation of the 0.4 mM palmitate fatty acid media was carried out as previously described [19]. Briefly, a 20 mM solution of palmitate in 0.01 mol/L NaOH was incubated at 70 °C for 30 min. Then 330 μL of 30% BSA and 400 μL of the palmitate/NaOH were mixed together and filter-sterilized with 20 mL of the DMED in total. Concentration of BSA is 0.5% in all medium. We selected sole glucose (25 mM) for all the experiments, because the sole glucose was essential for MIN6 cell growth, and compared with 5 mM glucose, 25 mM glucose protected MIN6 cells from apoptosis [19]. Graded doses of ghrelin (1, 10, 100 and 500 nM) were prepared daily before experiments. 50 μM LY294002, an inhibitor of PI3K, and 300 nM SP600125, an inhibitor of JNK were used in the study. 2.4. Cell viability assay Cell viability was assessed by MTT as described previously [20]. Cells were seeded on 96-well plates at a density of 5000 cells per well. For detection, cells were incubated with 1 mg/mL MTT for approximately 1 h. The medium was removed and the formazan product was solubilized with 150 μL dimethylsulfoxide. Viability was assessed by spectrophotometry at 570 nm absorbance using a 96-well plate reader. 2.5. Caspase-3 activity assay The caspase-3 amount was measured in triplicate by Caspase-3 Colorimetric Assay kit according to the manufacturer's protocol. Briefly, harvested cells were centrifuged at 10,000 rpm for 1 min, followed by the addition of 1 μL DTT and 100 μL lysis buffer. Cell lysates in a 96 well microplate were incubated at 37 °C with 5 μL of Caspase-3 Colorimetric substrate (DEVD-pNA) for 2 h. Absorbances were read by a microplate reader at 405 nm wavelength.
Reverse-transcription polymerase chain reaction was performed to semi-quantify mRNA expression of BAX, BCL-2 gene. Total RNA was isolated and first strand cDNA was synthesized using MMLV reverse transcriptase and hexamers as described previously [22] from MIN6 cells. The primer sequences were devised by primer5 software and were the following: mouse β-actin, forward 5′-CTGTCCCTGTATGCCTCTG-3′ and reverse5′-TGTCACGCACGATTTCC-3; mouse BAX, forward 5′-GCAGCGGCAGTGATGGAC-3′ and reverse 5′-GCAAAGTAGAAGAGGGCAACC-3′; mouse BCL-2, forward 5′-GCTACCGTCGTGACTTCGC-3′ and reverse 5′-CTACCCAGCCTCCGTTATCC-3′.cDNA (9 μL) was amplified by PCR in a 50 μL volume with amplitaq gold polymerase following conditions: 35 cycles in all, one cycle of 94 °C for 30 s, annealing for 30 s,72 °C for 30 s, and 72 °C for 7 min for elongation. The annealing temperature was adjusted for each target: 60 °C for BAX, 60 °C for BCL-2, and 57 °C for β-actin. The PCR products (362 bp for BAX, 270 bp for BCL-2, 217 bp for β-actin) were separated by 1.5% agarose gel electrophoresis and visualized by ethidiumbromide staining. The resulting images were analyzed by Scion Image software. 2.9. ELISA for insulin For secretion experiments, MIN6 cells were plated onto 24-well culture plates and incubated at 37 °C in 5% CO2, in culture medium for 48 h. Then the media were removed, and cells were incubated in 1 mL of 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 ghrelin at different doses (0–500 nM). After 6 h or 24 h of incubation, the media were extracted and insulin concentrations were determined by an ELISA kit. Insulin release was corrected for the amount of living cells (2 × 105 cells) through flow cytometer. 2.10. Statistical analysis Data are presented as means ± SE. Statistical analyses were performed with SPSS using ANOVA. Differences between groups were assessed for significance by Dunnett's test. A p value of less than 0.05 was considered significant.
2.6. Apoptosis assay 3. Results Apoptosis was evaluated by two different methods, hoechst33258 staining and TdT-mediated dUTP nick-end labeling (TUNEL) technique. Apoptosis indexes were assessed respectively by counting Hoechst positive cells (chromatin condensation or fragmented nuclear membrane) and TUNEL positive cells (apoptotic nucleus was brown-stained). Hoechst staining was performed by exposing the cell slides to 10 µg/mL hoechst33258 for 10 min at room temperature. TUNEL staining was performed according to the manufacturer's
3.1. Ghrelin inhibits lipotoxicity in MIN6 cells To test the potential effects of ghrelin on β-cell, MIN6 cells were incubated with either 0.5% BSA or 0.4 mM palmitate complexed to 0.5% BSA (PA), in the presence or absence of ghrelin (AG, added to the medium every 8 h) for 24 h. As shown in Fig. 1A, cell viability was decreased by 21% (p < 0.05, vs. BSA) after PA treatment. Treatment
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with AG dose-dependently prevented PA-induced toxicity, most effectively by 34% at 100 nM (p<0.01, vs. PA), 10 nM being the lowest significantly active concentration, while AG alone dose-dependently promoted cell growth, treatment at 100 nM significantly produced best potentiation by 128% (p<0.05, vs. BSA), as compared with BSA alone. Cell apoptosis was significantly increased in PA group by 61% when compared to BSA (Fig. 1B), while treatment with AG at 100 nM alone decreased by 15% (p<0.05, vs. BSA). PA/AG (at 100 nM) synergism decreased caspase 3 activity by 27% when compared to PA alone (p<0.05, vs. PA). Fig. 1C showed the apoptosis evaluated by hoechst33258 staining. The percentage of apoptosis reached to 47% after PA treatment for 24 h, which was significantly increased when compared with BSA alone. Treatment with
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AG at 100 nM reversed apoptosis rate by 16% (p<0.01, PA+AG vs. PA) or 1.7% (p<0.05, BSA+AG vs. BSA), respectively. 3.2. Ghrelin prevents MIN6 cells from apoptosis induced by lipotoxicity via PI3K/PKB pathway To explore the mechanism behind ghrelin-induced anti-lipotoxicity effect, we utilized MIN6 cells and investigated PKB signal pathway and its phosphorylation on ser473 under lipotoxic condition. As shown in Fig. 2A, exposure of the cells to 0.4 mM palmitate for 24 h caused significant inhibition of PKB phosphorylation (p < 0.01, PA+ AG at 0 min vs. BSA), while ghrelin at 100 nM induced rapid activation of PKB and reached the maximal effect at 30 min (p < 0.01, vs. PA+ AG at 0 min). Fig. 2B showed that ghrelin dose-dependently stimulated activation of PKB phosphorylation and treatment at 100 nM produced most effective potentiation (p < 0.01, PA+ AG at 100 nM vs. PA). To confirm relationship between PI3K/PKB pathway and apoptosis, we investigated whether AG prevented palmitate-induced apoptosis in the presence of specific inhibitor of PI3K (LY294002). As indicated in Fig. 2C, ghrelin-induced activation of PKB was markedly blocked by PI3K inhibitor, LY294002 (p < 0.05, AG + LY + PA vs. AG + PA). Fig. 2D showed that PA group had much more brown-stained apoptotic cells than other groups. Moreover, LY294002 abolished ghrelin cytoprotective activity against palmitate-induced apoptosis, no effect was observed when using inhibitors alone (Fig. 2E and F). 3.3. Ghrelin attenuates activation of palmitate-induced JNK phosphorylation and JNK inhibitor, SP600125, enhances anti-apoptotic effects of ghrelin in MIN6 cells The c-Jun N-terminal kinase, JNK plays important roles in pancreatic β-cell proliferation and apoptosis. As shown in Fig 3A, MIN6 cells were incubated with PA for 24 h and produced activation of JNK phosphorylation (p < 0.01, PA + AG at 0 min vs. BSA), while ghrelin at 100 nM resulted in a transient decrease at 30 min (p < 0.05, vs. PA + AG at 0 min). Then we examined the relationship between JNK with PKB pathway. We found that ghrelin-induced inhibition of JNK phosphorylation was blocked by PI3K inhibitor, LY294002 (p < 0.05, AG + LY + PA vs. AG + PA) (Fig. 3B). To further determine whether JNK pathway was pivotal to ghrelin anti-apoptotic action, we examined the effect of SP600125, a specific inhibitor of JNK pathway. Our results showed that SP600125 alone can significantly prevent palmitate-induced apoptosis. Combination of SP600125 with AG at 100 nM could enhance ghrelin's anti-apoptotic effect in MIN6 cells (Fig. 3C and D). 3.4. Ghrelin antiapoptosis effect involve mitochondrial pathways
Fig. 1. Effects of ghrelin on cell viability, caspase-3 activity and apoptosis in MIN6 cells under lipotoxicity state. 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 ghrelin (AG, added to the medium every 8 h) at different doses (1–500 nM), in serum-free medium for 24 h. A, An MTT assay for cell viability. Data, expressed as percentage of control (cells with BSA alone), are the means ± SE of 13 replicates. , p < 0.05, vs. BSA; §, p < 0.05, §§, p < 0.01, vs. PA. B, Caspase-3 activity assay by ELISA. AG at 100 nM was used in this experiment. Results are expressed as percentage of control (cells with BSA alone) and are the means± SE of three independent experiments. C, Apoptosis evaluated by counting Hoechst positive cells. AG at 100 nM was used in this experiment. Values are expressed as percentage of Hoechst positive cells and are the means ± SE of 10 random fields of vision from three independent experiments. , p < 0.05; , p < 0.01.
We detected important members of BCL-2 protein family, BAX and BCL-2 in mitochondria pathways. Consistent with caspase 3 activity assay (Fig. 4D), palmitate significantly up-regulated BAX (Fig. 4A, p < 0.01, vs. BSA), increased the ratio of BAX over BCL-2 (Fig. 4C, p < 0.01, vs. BSA) and down-regulated BCL-2 (Fig. 4B, p < 0.05, vs. BSA). However, ghrelin at 100 nM down-regulated BAX mRNA expression (Fig. 4A, p < 0.01, vs. PA), in line with decreasing the ratio of BAX over BCL-2 (Fig. 4C, p < 0.05, vs. PA), but did not affect BCL-2 mRNA expression (Fig. 4B). 3.5. Ghrelin inhibits insulin secretion from MIN6 cells under palmitateincubated state Conflicting results have been reported concerning the effect of ghrelin on insulin secretion from pancreatic β-cells [23]. We therefore studied the influence of ghrelin on insulin secretion from MIN6 cells under lipotoxic condition. As shown in Fig. 5A, after 6 h of incubation, ghrelin dose-dependently inhibited insulin secretion from MIN6 cells
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in the absence of palmitate (ghrelin 0 nM, 1437 ± 298 pM; 1 nM, 1344 ± 285 pM; 10 nM, 1121 ± 367 pM; 100 nM, 823 ± 327 pM; 500 nM, 836 ± 303 pM), as well as in the presence of palmitate (ghrelin 0 nM, 594 ± 188 pM; 1 nM, 514 ± 232 pM; 10 nM, 466 ±
129 pM; 100 nM, 413 ± 153 pM; 500 nM, 426 ± 130 pM). Ghrelin at no less than 10 nM, but not at 1 nM, significantly inhibited insulin secretion from MIN6 cells under either BSA- or palmitate-incubated state. However, as data seen in Fig. 5B, there were no significant
Fig. 2. Ghrelin prevent MIN6 cells from apoptosis induced by lipotoxicity 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 ghrelin (AG) was added to the medium with PA for 0–60 min. Values are expressed as means ± SE of three independent experiments. , p < 0.05, , p < 0.01, vs. PA + AG at 0 min. B, Total PKB and p-PKB (ser473) expression after 30 min of stimulation with graded concentrations of AG (1–500 nM). Data are expressed as relative intensity ratio and are the means ± SE of three independent experiments. , p < 0.05, , p < 0.01, vs. PA without AG. C, Total PKB and p-PKB (ser473) expression after 30 min of stimulation with 100 nM AG, with or without inhibitor. Before AG stimulation, cells with PA were pretreated for 30 min with PI3K inhibitor, 50 μM LY294002 (LY). Results are means ± SE from three independent experiments. , p < 0.05. D, magnification, × 400 (by TUNEL); E and F, Apoptosis evaluated respectively by TUNEL technique and Hoechst-staining method. MIN6 cells were incubated with BSA or PA, with or without LY, in the presence or absence of 100 nM AG (added to the medium every 8 h), in serum-free medium for 24 h. Values are expressed as percentage of apoptotic cells and are the means ± SE of 10 random fields of vision from three independent experiments. , p < 0.05; , p < 0.01.
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Fig. 3. Ghrelin antiapoptosis effects involve JNK pathways. A, Total JNK and p-JNK1/2 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 ghrelin (AG) was added to the medium with PA for 0–60 min. Values are expressed as means ± SE of three independent experiments. , p < 0.05, , p < 0.01,vs. PA + AG at 0 min; ＃, p < 0.05, vs. PA + AG at 15 min. B. Total JNK and p-JNK1/2 expression after 30 min of stimulation with 100 nM AG, with or without inhibitor. Before AG stimulation, cells with PA were pretreated for 30 min with PI3K inhibitor, 50 μM LY294002 (LY). Results are means ± SE from three experiments. , p < 0.05; C and D, Apoptosis evaluated respectively by TUNEL technique and Hoechst-staining method. MIN6 cells were incubated with BSA or PA, with or without 300 nM SP600125 (SP), in the presence or absence of 100 nM AG (added to the medium every 8 h), in serum-free medium for 24 h. Values are expressed as percentage of apoptotic cells and are the means ± SE of 10 random fields of vision from three independent experiments. , p < 0.05; , p < 0.01.
effects of ghrelin on insulin release under lipotoxic pressure after 24 h of incubation (ghrelin 0 nM, 430 ± 126 pM; 1 nM, 403 ± 91 pM; 10 nM, 393 ± 122 pM; 100 nM, 385 ± 149 pM; 500 nM, 306± 87 pM), although ghrelin at no less than 500 nM markedly inhibited insulin release from MIN6 cells in the absence of palmitate (ghrelin 0 nM, 1211 ± 321 pM; 1 nM, 1153 ± 383 pM; 10 nM, 1135 ± 279 pM; 100 nM, 1092 ± 387 pM; 500 nM, 1014 ± 287 pM). 4. Discussion Our present study demonstrated that ghrelin promoted β-cell survival and partially inhibited apoptosis induced by lipotoxicity in MIN6 cells. Granata R et al. [17] had confirmed that ghrelin prevented pancreatic β-cell line (HIT-T15) from IFN-r/TNF-a induced apoptosis. Zhang Y et al. [18] also attained similar results under doxorubicininduced condition. Our results certified the protective action of ghrelin on pancreatic β-cells (MIN6) in lipotoxic environment. Lipotoxicity and glucolipotoxicity play important roles in patients with type 2 diabetes. Apart from the deterioration role of lipotoxicity and glucolipotoxicity in β-cell function, elevation of plasma fatty acid level also plays a pathogenic role in the early stages of the disease. Survival of pancreatic cells is obviously of importance for maintaining normal glucose metabolism and islet function. Growing evidence have showed that prolonged exposure of isolated islets or insulin-secreting
cells to elevated levels of fatty acids is associated with β-cell apoptosis [6,10,11,24]. Thus, blockade of apoptosis through inhibition of lipotoxicity may be a novel approach for protecting pancreatic islets. Interestingly, ghrelin was recently shown to protect pancreatic β-cells from apoptosis induced by IFN-r/TNF-a synergism or doxorubicin [17,18]. However, there were no reports of protective effects of ghrelin on pancreatic β-cells in lipotoxic environment. Our study had proved that ghrelin significantly promoted pancreatic β-cell survival by inhibiting apoptosis and eliminating caspase-3 activity under palmitate-incubated condition (Fig. 1). Moreover, we determined that PI3K/PKB signaling pathway was involved in ghrelin's anti-apoptotic effect. It was becoming increasingly evident that β-cell apoptosis induced by palmitate was associated with inhibition of PKB phosphorylation [6,10,11,25]. Ghrelin's anti-apoptotic effects in many cell lines [21,26–28], including pancreatic β-cells [17,18], were indeed mediated through PI3K/PKB signaling. We considered whether ghrelin counteract lipotoxicity in MIN6 cells through this pathway. Our findings showed that exposure of MIN6 cells to palmitate-induced lipotoxicity markedly inhibited PKB phosphorylation, while ghrelin caused rapid activation of PKB under lipotoxicity condition. PI3K inhibitor (LY294002) blocked protective effect of ghrelin in MIN6 cells (Fig. 2). These data indicated that ghrelin exerted an anti-apoptotic effect on pancreatic β-cells under lipotoxic pressure, at least in part via PI3K/PKB pathway.
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Fig. 4. Ghrelin antiapoptosis effects involve 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 ghrelin (AG), in serum-free medium for 6 h. A and B, BAX mRNA and BCL-2 mRNA respectively assessed by RT-PCR in MIN6 cells, normalized to β-actin mRNA level. C, The ratio of BAX over BCL-2. Result expressed as relative intensity ratio, and are the means ± SE of three independent experiments; D, Caspase-3 activity assay by ELISA. Results are expressed as percentage of control (cells with PA) and are the means ± SE of three independent experiments. , p < 0.05; , p < 0.01.
Fig. 5. Effect of ghrelin on insulin secretion from MIN6 cells under palmitate-incubated state. Medium insulin concentration was measured by ELISA after 6 h or 24 h incubation of MIN6 cells in the presence (■) or absence (□) of 0.4 mM palmitate (PA)with 1–500 nM ghrelin (AG) or without ghrelin. A, Medium insulin after 6 h of incubation. B, Medium insulin after 24 h of incubation. All values are expressed as the means ± SE of four independent experiments. , p < 0.05, , p < 0.01, vs. nonstimulation with AG in the absence of PA; §, p < 0.05, vs. nonstimulation with AG in the presence of PA.
The c-Jun NH2-terminal kinase, JNK is classic stress-activated protein kinase and many cellular stresses have been shown to stimulate JNK activation. Recent studies demonstrated that treatment with the saturated fatty acid palmitic acid resulted in sustained JNK activation in pancreatic β-cells [19,29]. Early induction of JNK plays an important role in fatty acid-induced beta-cell apoptosis [19], and there existed a cross-talking between JNK and PKB pathways [19,30]. The role of JNK pathway in palmitate-induced pancreatic β-cell apoptosis and association with ghrelin's anti-apoptotic action need to be determined. Thus, we detected the change of JNK proteins expression before and after treatment with ghrelin under lipotoxicity state. In line with previous studies [19], we demonstrated that palmitate produced an activation of JNK1/2 phosphorylation in MIN6 cells, and treatment with SP600125 alone significantly blocked palmitate-induced apoptosis (Fig. 3). We also found that ghrelin could inhibit activation of JNK1/2 phosphorylation induced by palmitate. These results suggested that ghrelin antiapoptosis effects involved JNK pathways. In previous studies, PKB can directly phosphorylate the amino acid residue S83 on apoptosis signal-regulated kinase1 (ASK1, also known as MAPKKK5), which is an upstream activating kinase within the JNK and p38 pathways [31]. Therefore, a balance between PI3K/PKB survival signaling and JNK apoptotic signaling can be established through such crosstalk [12]. Our data showed that, under lipotoxic state, ghrelin-induced inhibition of JNK pathway can be partly blocked by PKB inhibitor, LY294002 (Fig. 3B), whereas JNK inhibitor, SP600125 had no effect on ghrelin-stimulated activation of PKB (data not shown). These data indicated that JNK protein may be one downstream target of PI3K/PKB pathway in palmitate-incubated MIN6 cells. Ghrelin-induced inhibition of JNK pathway may be partly dependent on the activation of PI3K/PKB pathway. Then we incubated MIN6 cells with SP600125/ghrelin synergism, which resulted in enhancing ghrelin protective effects. These results showed that ghrelin's anti-lipotoxicity effect was associated with JNK pathway, and change of JNK may be partly ascribed
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to ghrelin-induced activation of PKB pathway. Moreover, ghrelin could not fully suppress palmitate-induced activation of JNK phosphorylation. In mammalian cells, apoptosis may involve disruption of mitochondrial function, which is regulated by members of the BCL-2 protein family. Palmitate-induced apoptosis typically involves the mitochondrial pathway [5,7]. For these reasons, we determined whether induction of apoptosis in MIN6 cells by palmitate and effect of ghrelin were associated with BCL-2 protein family, pro-apoptotic gene BAX or anti-apoptotic gene BCL-2. We found an increase in Bax, and a decrease in Bcl-2, consistent with increased activity of caspase-3 after MIN6 cells were incubated with palmitate for 6 h. After treatment with AG in environment of lipotoxicity, we detected a decrease of caspase-3 activity and significant downregulation of BAX. Although ghrelin may not significantly influence mRNA expression of BCL-2, the result also demonstrated the slight increase of BCL-2 mRNA. These results indicated that protective effect of ghrelin was associated with mitochondrial pathway by inhibiting BAX mRNA expression. Finally, we detected the effect of ghrelin on insulin secretion from MIN6 cells. Previous studies had certified that ghrelin inhibited glucose-stimulated insulin secretion in mouse islet [32] and MIN6 cells [33]. Our experiment further confirmed that ghrelin prevents MIN6 cells from secreting insulin in lipotoxic environment, although our results showed that inhibitory effect of ghrelin on insulin secretion required higher concentrations at no less than 10 nM, and could not last 24 h in lipotoxic environment. It is still debated whether ghrelin stimulates or suppresses insulin secretion in pancreatic β-cells [34,35]. Some report had identified that ghrelin can promote insulin secretion in other pancreatic β-cell line, such as HIT-T15 [17]. These different conclusions were possibly due to different experimental objects or design. It is still unknown that whether ghrelin effect on insulin release from MIN6 cells participate in its anti-apoptotic action, and detailed mechanism remains to be clarified in the future. Pancreatic β-cell apoptosis induced by lipotoxicity was thought of as a main mechanism in type 2 diabetes with obesity. Reducing impairment of lipotoxicity on pancreatic β-cells may be one key point of delaying progress and deterioration of diabetes. But clinical medicines used as counteracting lipotoxicity were so limited. The finding that ghrelin protecting β-cells against lipotoxicity may lead to novel therapeutic approaches aimed at enhancing the survival of βcells, thereby reducing or delaying lipotoxicity-mediated β-cell destruction in the development of type 2 diabetes. In summary, the results of the present study demonstrated that the protective effects of ghrelin against palmitate-induced apoptosis required activation of PKB, in the meantime, also involved JNK and mitochondria pathways. Acknowledgment We thank the Biochemical Department of China Medical University for valuable suggestions and help. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.regpep.2009.12.017. References [1] Poitout V, Robertson RP. Lipotoxicity: fuel excess and beta-cell dysfunction. Endocr Rev 2008;29:351–66. [2] Prentki M, Nolan CJ. Islet beta cell failure in type 2 diabetes. J Clin Invest 2006;116(7): 1802–12. [3] Elks ML. Chronic perfusion of rat islets with palmitate suppresses glucose stimulated insulin release. Endocrinology 1993;133:208–14. [4] Kelpe CL, Moore PC, Parazzoli SD, Wicksteed B, Rhodes CJ, Poitout V. Palmitate inhibition of insulin gene expression is mediated at the transcriptional level via ceramide synthesis. J Biol Chem 2003;278:30015–21.
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