Peptides 52 (2014) 23–28

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Ghrelin protects human umbilical vein endothelial cells against high glucose-induced apoptosis via mTOR/P70S6K signaling pathway Jianhua Zhu a,1 , Chenghong Zheng a,b,1 , Jie Chen a , Jing Luo c , Bintao Su a , Yan Huang a , Wen Su a , Zixi Li a , Tianpen Cui a,∗ a Laboratory of Clinical Immunology, Wuhan No. 1 Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), Wuhan, Hubei, PR China b Department of Endocrinology, Wuhan No. 1 Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), Wuhan, Hubei, PR China c Department of Biochemistry & Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology (HUST), Wuhan, Hubei, PR China

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Article history: Received 3 September 2013 Received in revised form 17 November 2013 Accepted 18 November 2013 Available online 25 November 2013 Keywords: Ghrelin Apoptosis mTOR/P70S6K GHS-R1a

a b s t r a c t Ghrelin exhibits its biological effect through binding to the growth hormone secretagogue 1a receptor (GHS-R1a). Recently, it has been reported that ghrelin has an anti-apoptotic effect in several cell types. However, the molecule mechanisms underlying the anti-apoptotic effect of ghrelin remain poorly understood. In this study, we investigated the intracellular mechanisms responsible for anti-apoptotic effect of ghrelin on human umbilical vein endothelial cells (HUVEC). Treatment of HUVEC with ghrelin inhibited high glucose-induced cell apoptosis. Ghrelin stimulated the rapid phosphorylation of mammalian target of rapamycin (mTOR), P70S6K and S6. The GHS-R1a-specific antagonist [D-Lys3]-GHRP-6 abolished the anti-apoptotic effect and inhibited the activation of mTOR, P70S6K, S6 induced by ghrelin. Pretreatment of cells with specific inhibitor of mTOR blocked the anti-apoptotic effect of ghrelin. In addition, ghrelin protected HUVECs against high glucose induced apoptosis by increasing Bcl-2/Bax ratio. Taken together, our results demonstrate that ghrelin produces a protective effect on HUVECs through activating GHSR1a and mTOR/P70S6K signaling pathway mediates the effect of ghrelin. These observations suggest that ghrelin may act as a survival factor in preventing HUVECs apoptosis caused by high glucose. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Ghrelin is a 28-amino acid peptide hormone mainly secreted by the stomach. Ghrelin has been identified as an endogenous ligand for the GH secretagogue receptor (GHS-R) [14]. Several study demonstrated that ghrelin stimulates GH release and induces a positive energy balance by promoting food intake while decreasing energy expenditure [14,21,23,25]. In addition to its direct effects on exocrine and endocrine pancreatic functions, ghrelin exerts a protective effect on the cardiovascular system [26,31], specifically by regulating vascular endothelial cell function including inhibiting vascular endothelial cells apoptosis [32], increasing contractility and cardio-protection [13,15,22], and promoting vasodilation

∗ Corresponding author at: Laboratory of Clinical Immunology, Wuhan No. 1 Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), 215 Zhongshan Dadao, Wuhan, Hubei 430022, PR China. Tel.: +86 27 85332628. E-mail addresses: [email protected], [email protected] (T. Cui). 1 These authors contributed equally to this work. 0196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2013.11.015

[3,27]. It has been reported that ghrelin inhibited the apoptosis of endothelial cell induced by high glucose through activating ERK pathway [32]. A study has reported that ghrelin protected cortical neuronal cells against oxygen–glucose deprivation-induced apoptosis in primary rat cortical neuronal cells through activating PI3K/Akt/GSK-3␤ and ERK1/2 pathways [10]. The mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine-threonine kinase, it has two multi-protein complexes: mTORC1 and mTORC2. mTORC1 phosphorylates and modulates the activity of the serine/threonine ribosomal protein S6 kinase 1 (S6K1). In turn S6K1 phosphorylates and activates S6 [18,28,29]. Activation of S6 selectively increases the translation of mRNA transcripts which encode ribosomal proteins and other translational regulators [18]. mTOR serves as a central regulator which involved in a wide array of cellular processes, including cell proliferation, metabolism, growth and survival [2,6,7,19,28]. A study reported that mTOR/P70S6K signaling pathway mediated ghrelin-induced proliferation of neural stem cells [8]. The observations suggest mTOR pathway plays an important role in ghrelin biological effects. However, in spite of this signaling pathway has been identified, it is still unclear whether the mTOR/P70S6K

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pathway or if alternative pathways might mediate the antiapoptosis effect of ghrelin. Based on these observations, we hypothesized that ghrelin may protect HUVECs from high glucose induced apoptosis via mTOR/P70S6K pathway. Therefore, this study was aimed to investigate the putative role of mTOR/P70S6K in ghrelin mediated anti-apoptosis effect under high glucose in HUVECs. 2. Materials and methods 2.1. Cell culture Human umbilical venous endothelial cells (HUVECs) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were cultured in ATCC formulated of F12K medium, supplemented with 0.05 mg/ml endothelial cell growth supplement (Sigma–Aldrich Co., St. Louis, MO, USA), 10% fetal bovine serum (Gibco, Invitrogen, NY, USA), 1% penicillinstreptomycin. HUVECs were used for the experiments between passages 2 and 6. 2.2. Cell treatment HUVECs were seeded equally into culture plates. To determine whether anti-apoptosis effects of ghrelin and phosphorylation were mediated via its receptor, GHS-R1a, cells were pretreated with [D-Lys3]-GHRP-6 (100 ␮M) (Phoenix Pharmaceutical, USA) or a vehicle (saline) for 1 h before treatment with acylated ghrelin (100 nM) (Phoenix Pharmaceutical, USA). Then cells were exposed to normal glucose (NG, 5.5 mM) or high glucose (HG, 33.3 mM) for 48 h. To investigate the effect of ghrelin on the phosphorylation of mammalian target of rapamycin (mTOR), P70S6K and S6, cells were treated with ghrelin (100 nM) for 5, 15, 30, and 60 min and assayed by Western blot analysis as described below. To further investigate the role of mTOR in ghrelin mediated effects in HUVECs. Experiments were performed by incubating the cells with the inhibitor rapamycin (100 nM) (Santa Cruz, CA) for 1 h. All experiments were performed three times in duplicate. 2.3. Apoptosis assay by flowcytometry (FCM) HUVECs were harvested and incubated with 0.2 mg/ml Annexin V-FITC (Pharmingen, San Diego, CA, USA) and 50 mg/ml propidium iodide (PI) (Sigma, USA) for 15 min at room temperature. The cells were analyzed by flow-cytometry. The percentage of cells staining positive was recorded. 2.4. TUNEL assay Apoptotic cells were also detected in situ by the terminal deoxynucleotidyl transferase-mediated dUTP-biotinnickendlabeling (TUNEL) assay using an in situ cell death detection kit (Roche Applied Science). DAPI (Sigma, USA) staining was used to identify the cells in the field. The cells for the TUNEL assay were grown on glass coverslips. Cells were fixed for 30 min in 4% paraformaldehyde and the TUNEL reaction was performed according to the manufacturer’s instructions. The percentage of apoptotic cells was determined by counting the number of apoptotic TUNEL-positive cells and dividing by the total number of cells. 2.5. Western blot analysis Cells were lysed in a buffer containing 20 mM Tris–HCl (pH 7.4), 1 mM EDTA, 140 mM NaCl, 1% (w/v) Nonidet P-40, 1 mM Na3VO4,

Fig. 1. Effect of ghrelin on high glucose-induced apoptosis in HUVECs. (A) GHS-R1a protein expression assessed by Western blot analysis. Total protein extracted from human T cells was included as a positive control. Total protein from HepG2 cells was used as a negative control. HUVECs were pre-incubated with vehicle or [DLys3]-GHRP-6 (100 ␮M) for 1 h and then treated with vehicle or ghrelin (100 nM) for 24 h. Then cells were exposed to normal glucose (NG, 5.5 mM) or high glucose (HG, 33.3 mM) for 48 h. (B) Flow cytometric analysis of apoptotic cells stained with Annexin-V and propidium iodide. (C) Apoptosis was determined using the TUNEL method, the results are expressed as apoptotic cell number (%). The data are expressed as the mean ± SEM of three different experiments (each experiment was repeated twice). *P < 0.05 vs the vehicle-treated control and + P < 0.05 vs ghrelintreated cells.

1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, and 10 mg/ml aprotinin. Cell lysates were separated by 8–12% SDS–PAGE and electrotransferred onto polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked in 5% nonfat dry milk for 1 h and incubated overnight at 4 ◦ C with the primary antibodies

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Fig. 2. Ghrelin activates mTOR/P70S6K pathway in HUVECs. Cells were treated with 100 nM ghrelin for 5, 15, 30, and 60 min (A, C, and E). Cells were pre-incubated with a vehicle or the GHS-R1a antagonist [D-Lys3]-GHRP-6 (100 ␮M) for 1 h followed by treatment with the vehicle or ghrelin (100 nM) for 30 min (B, D, and F). Protein lysates were prepared and assayed by Western blot analysis using specific anti-p-mTOR (Ser2448) and anti-mTOR antibodies (A and B), and anti-p-P70S6K (Thr389) and anti-P70S6K antibodies (C and D), and anti-p-S6K (Ser235/236) and anti-S6K antibodies (E and F). The band intensities of phosphor-forms were normalized to the band intensities of total-forms respectively, and they are expressed as relative band intensities. The data are expressed as the mean ± SEM of three different experiments (each experiment was repeated twice). *P < 0.05 vs the vehicle-treated control and + P < 0.05 vs ghrelin-treated cells.

against mTOR, p-mTOR on Ser2448, P70S6K, p-P70S6K on Thr389, S6, p-S6 on Ser235/236, Bcl-2, Bax (Cell Signaling, Danvers, MA, USA; 1:1000), GHS-R1a and ␤-actin (Santa Cruz Biotechnology; 1:1000). Blots were developed using a peroxidase-conjugated anti-rabbit IgG and a chemiluminescent detection system (Santa Cruz Biotechnology). The bands were visualized using a ChemicDoc XRS system (Bio-Rad) and quantified using Quantity One imaging software (Bio-Rad). 2.6. Statistical analyses Quantitative data were expressed as means ± SEM for at least three independent experiments. Statistical analyses were performed with SPSS version 16.0 (SSPS Inc, Chicago, IL, USA) using the unpaired t-test. A value of P < 0.05 was considered to be statistically significant.

3. Results 3.1. Ghrelin protects HUVECs against high glucose-induced apoptosis through binding to GHS-R1a Ghrelin exerts biological function through binding to its receptor GHS-R1a, we examined the protein expression of GHS-R1a in cultured HUVECs. The Western blot analysis revealed that GHS-R1a expressed in HUVECs (Fig. 1A). Total protein extracted from the human T cells was used as a positive control and HepG2 cells as a negative control. We then investigated the effect of ghrelin on high glucose-induced apoptosis in HUVECs. The flowcytometry analysis indicated that the percentage of apoptosis was significantly increased after high glucose treatment (Fig. 1B). Pretreatment of cells with ghrelin inhibited the apoptosis induced by high glucose (Fig. 1B). Similarly, high glucose increased

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TUNEL-positive cells, which were inhibited by pretreatment with ghrelin (Fig. 1C). To determine whether the anti-apoptotic effect of ghrelin is mediated by its receptor GHS-R1a, HUVECs were pretreated with the ghrelin receptor-specific antagonist and then treated by ghrelin. The exposure of cells to [D-Lys3]-GHRP-6 nearly completely blocked the protective effect of ghrelin (Fig. 1B and C). 3.2. Ghrelin activates mTOR/P70S6K signaling pathway Ghrelin activates multiple signal transduction pathways, including ERK1/2, PI3K/Akt, and Jak2/STAT3 pathways, mTOR and P70S6K are downstream effector of PI3K/Akt, which is an important signaling pathway for the proliferation and survival of some types of cell. Therefore, we investigated whether mTOR/P70S6K signaling pathway was activated by ghrelin in HUVECs. The phosphorylation of mTOR, P70S6K and S6 was examined by Western blot analysis after ghrelin treatment. The treatment of cells with ghrelin activated mTOR, P70S6K and S6 in a time-dependent manner (Fig. 2A, C and E). Ghrelin-induced activation of mTOR, P70S6K and S6 peaked between 15 and 30 min and lasted for 60 min. In order to determine whether ghrelin-induced phosphorylation of mTOR, P70S6K and S6 is mediated by its receptor GHS-R1a, HUVECs were treated with the ghrelin receptor-specific antagonist. The treatment of cells with [D-Lys3]-GHRP-6 significantly inhibited the stimulatory effects of ghrelin on the phosphorylation of mTOR, P70S6K and S6 (Fig. 2B, D, and F). 3.3. mTOR inhibitor blocks the anti-apoptotic effect of ghrelin in HUVECs To further determine whether the activation of mTOR/P70S6K signaling pathways mediates the effect of ghrelin on the survival of HUVECs, the cells were pretreated with mTOR inhibitor rapamycin before ghrelin treatment. Flowcytometry analysis and TUNEL assay showed that pretreatment of rapamycin increased the apoptosis of HUVECs, which were attenuated by ghrelin (Fig. 3A and B). These results suggested that mTOR inhibitor block the ghrelin’s anti-apoptotic effect in HUVECs. 3.4. Ghrelin increases Bcl-2/Bax ratio We then further investigated the effect of ghrelin on levels of Bcl-2 and Bax. The Western blot analysis showed that high glucose increased the level of Bax and decreased the level of Bcl-2. Ghrelin treatment inhibited the high glucose-induced increase in Bax and increased the level of Bcl-2, thereby significantly increasing the Bcl2/Bax ratio, and the ghrelin receptor-specific antagonist blocked the effects of ghrelin on the Bcl-2 and Bax levels (Fig. 4). 4. Discussion Ghrelin has been reported as an important multi-functions peptide, which promotes GH release, food intake, regulates body weight and glucose homeostasis. Numerous studies reported that ghrelin has protective effects against several types of extracellular stimulation, including oxygen deprivation, serum starvation [17], and ischemia/reperfusion [12]. In the present study, we examined the potential effects of ghrelin on high glucose-induced apoptosis in vitro and investigated its underlying molecular mechanism in HUVECs. Our results showed that ghrelin had an inhibitory effect on high glucose-induced apoptosis. The protective effect of ghrelin was accompanied by phosphorylation of mTOR, P70S6K and S6. In addition, this protective effect was blocked by antagonist of ghrelin specific receptor GHS-R1a and mTOR inhibitor rapamycin, we also

Fig. 3. Effect of inhibiting mTOR/P70S6K signaling pathway on anti-apoptosis effect of ghrelin. Cells were pre-incubated with 100 nM rapamycin for 1 h, and then treated with a vehicle or ghrelin (100 nM) for 24 h. Then cells were exposed to normal glucose (NG, 5.5 mM) or high glucose (HG, 33.3 mM) for 48 h. (A) Flow cytometric analysis of apoptotic cells stained with Annexin-V and propidium iodide. (B) Apoptosis was determined using the TUNEL method, the results are expressed as apoptotic cell number (%). The data are expressed as the mean ± SEM of three different experiments (each experiment was repeated twice). *P < 0.05 vs the vehicle-treated control and + P < 0.05 vs ghrelin-treated cells.

demonstrated that ghrelin exert its protective effect through activating GHS-R1a and mTOR/P70S6K signaling pathway. It is well-known that many biological actions of ghrelin are initiated by binding of ghrelin to its cognate cell surface receptor GHSR1a [5,14,21,25,30], which is a G-protein-coupled 7 transmembrane receptor. GHS-R1a was first cloned from the pituitary gland and hypothalamus [9]. It is highly expressed in some types of neuronal cells and endothelial cells. In our study, we verified the GHS-R1a expressed in HUVECs by Western blot. [D-Lys3]GHRP-6 is widely applied as selective GHS-R1a antagonist in different cell types and organs [20,25,34]. Wang L showed that

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Fig. 4. Effect of ghrelin on Bax and Bcl-2 protein levels. HUVECs were pre-incubated with vehicle or [D-Lys3]-GHRP-6 (100 ␮M) for 1 h and then treated with vehicle or ghrelin (100 nM) for 24 h. Then cells were exposed to normal glucose (NG, 5.5 mM) or high glucose (HG, 33.3 mM) for 48 h. Bax and Bcl-2 protein levels in HUVECs assessed by Western blot. Bax and Bcl-2 band intensities were normalized to ␤-actin band intensity. The data are expressed as the Mean ± SEM of three different experiments (each experiment was repeated twice). *P < 0.05 vs the vehicle-treated control and + P < 0.05 vs ghrelin-treated cells.

[D-Lys3]-GHRP-6 abolished the ghrelin-induced phosphorylation of ERK [30]. Kim et al. found that application of [D-Lys3]-GHRP-6, a selective antagonist for GHS-R1a, almost blocked the ghrelininduced depolarization [16]. In addition, Hyunju showed that the GHS-R1a-specific antagonist [D-Lys3]-GHRP-6 abolished the proliferative effect of ghrelin [8]. In order to determine whether the protective effect of ghrelin against high glucose insult was mediated by GHS-R1a, we treated the GHSR1a antagonist [D-Lys3]GHRP-6 along with ghrelin in HUVECs. In our study, the GHS-R1a antagonist efficiently blocked ghrelin-induced phosphorylation of mTOR, P70S6K, S6 and anti-apoptosis effect, furthermore, [DLys3]-GHRP-6 alone showed no effect on HUVECs, suggesting that the protective effect of ghrelin is initiated by binding of ghrelin to its receptor GHS-R1a. The GHS-R1a antagonist experiments also supported the expression of ghrelin receptor in HUVECs. However, it should be noted that ghrelin exhibits an anti-apoptotic activity in cardiomyocytes through binding to an unidentified receptor that is distinct from GHS-R1a [1]. Several studies reported that ghrelin exerted its functions in a GHS-R1a independent way [4,11]. Although ghrelin may bind to other receptors, its main target is GHS-R1a [20], especially in cells which strongly express GHS-R1a. The PI3K/Akt and ERK1/2 signaling pathways play a critical role in cell survival. Akt can phosphorylate its downstream effector proteins, such as mTOR and P70S6K, which are involved in cell proliferation, differentiation, and survival. Activation of P70S6K initializes through phosphorylation of one essential site Thr389, a target of the TORC1. Activated P70S6K regulates protein synthesis by phosphorylating the ribosomal protein S6 kinase 1 and eIF-4E binding protein 1 which regulate protein translation [24]. In the present study, we found that ghrelin caused a rapid phosphorylation of the mTOR/P70S6K signaling pathway in HUVECs. We then examined the functional involvement of mTOR/P70S6K signal pathways in ghrelin induced anti-apoptosis. Chemical inhibition of

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mTOR/P70S6K by rapamycin nearly completely blocked the antiapoptotic effect of ghrelin in HUVECs. These results suggested that activation of mTOR, P70S6K and S6 mediates the protective effect of ghrelin. It has been reported that the Bcl-2 family members are crucial regulators in intracellular apoptotic signal transduction by regulating the mitochondrial membrane permeability. Bcl-2 inhibits apoptosis by preventing mitochondrial membrane depolarization [33], whereas Bax promotes apoptosis by inducing mitochondrial membrane depolarization. Bcl-2/Bax ratio is a determining regulator of cell death. Our data showed that the high glucose decreases the Bcl-2/Bax ratio, whereas ghrelin treatment up-regulates Bcl2 levels and down-regulates Bax levels, resulting in significant increase of the Bcl-2/Bax ratio, which indicated that the mitochondrial apoptotic pathway was involved in the protective effect of ghrelin in HUVECs. In summary, we have demonstrated that acylated ghrelin protected against apoptosis in HUVECs induced by high glucose through activating its receptor GHS-R1a. We also have shown that ghrelin strongly activated mTOR, P70S6K, S6 and the protective effect of ghrelin was mediated by the mTOR/P70S6K pathway in HUVECs. Ghrelin up-regulated the Bcl-2/Bax ratio, which favored cell survival and inhibited the apoptotic cascade. Therefore, our findings provide supporting evidence for the potential therapeutic application of ghrelin in the prevention and treatment of endothelial injury caused by high glucose in diabetic patients. Acknowledgments This work was supported by the National Nature Science Foundation of China (81170035) and Wuhan Municipal Human Resources and Social Security Bureau (no. 2009-97). References [1] Baldanzi G, Filigheddu N, Cutrupi S, Catapano F, Bonissoni S, Fubini A, et al. 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. [2] Chiang GG, Abraham RT. Targeting the mTOR signaling network in Cancer. Trends Mol Med 2007;13:433–42. [3] Dinca M, Dumitriu IL, Gurzu MB, Slatineanu SM, Foia L, Vata L. Ghrelin and Ang1-7 have cumulative vasodilatory effects on pulmonary vessels. Rev Med Chir Soc Med Nat Iasi 2010;114:803–7. [4] Delhanty PJ, vander Eerden BC, vander Velde M, Gauna C, Pols HA, Jahr H, et al. Ghrelin and unacylated ghrelin stimulate human osteoblast growth via mitogen-activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K) pathways in the absence of GHS-R1a. J Endocrinol 2006;188:37–47. [5] Feng DD, Yang SK, Loudes C, Simon A, Al-Sarraf T, Culler M, et al. Ghrelin and obestatin modulate growth hormone-releasing hormone release and synaptic inputs onto growth hormone-releasing hormone neurons. Eur J Neurosci 2011;34:732–44. [6] Faivre S, Kroemer G, Raymond E. Current development of mTOR inhibitors as anticancer agents. Nat Rev Drug Discov 2006;5:671–88. [7] Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell 2007;12:9–22. [8] Chung H, Li E, Kim Y, Kim S, Park S. Multiple signaling pathways mediate ghrelin-induced proliferation of hippocampal neural stem cells. J Endocrinol 2013;218:49–59. [9] Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, et al. A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 1996;273:974–7. [10] Chung H, Seo S, Moon M, Park S. Phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-3b and ERK1/2 pathways mediate protective effects of acylated and unacylated ghrelin against oxygen–glucose deprivation-induced apoptosis in primary rat cortical neuronal cells. J Endocrinol 2008;198:511–21. [11] Johansson I, Destefanis S, Aberg ND, Aberg MA, Blomgren K, Zhu C, et al. Proliferative and protective effects of growth hormone secretagogues on adult rat hippocampal progenitor cells. Endocrinology 2008;149:2191–9. [12] Konturek PC, Brzozowski T, Walter B, Burnat G, Hess T, Hahn EG. Ghrelininduced gastroprotection against ischemia–reperfusion injury involves an activation of sensory afferent nerves and hyperemia mediated by nitric oxide. Eur J Pharmacol 2006;536:171–81. [13] King MK, Gay DM, Pan LC, McElmurray 3rd JH, Hendrick JW, Pirie C, et al. Treatment with a growth hormone secretagogue in a model of developing heart failure: effects on ventricular and myocyte function. Circulation 2003;103:308–13.

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P70S6K signaling pathway.

Ghrelin exhibits its biological effect through binding to the growth hormone secretagogue 1a receptor (GHS-R1a). Recently, it has been reported that g...
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