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Endocrinology, doi:10.1210/en.2006-0991
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Endocrinology Vol. 148, No. 1 148-159
Copyright © 2007 by The Endocrine Society

Ghrelin Inhibits Apoptosis in Hypothalamic Neuronal Cells during Oxygen-Glucose Deprivation

Hyunju Chung, Eunhee Kim, Dae Hee Lee, Sanghee Seo, Sunghee Ju, Dahm Lee, Hocheol Kim and Seungjoon Park

Department of Pharmacology (H.C., E.K., S.S., S.J., D.L., S.P.), Kyunghee University School of Medicine and Institute for Basic Medical Sciences, and Department of Herbal Pharmacology (D.H.L., H.K.), College of Oriental Medicine, Kyunghee University, Seoul 130-701, Korea

Address all correspondence and requests for reprints to: Seungjoon Park, M.D., Ph.D., Department of Pharmacology, Kyunghee University School of Medicine, 1 Hoiki-dong, Dongdaemun-ku, Seoul 130-701, Korea. E-mail: sjpark{at}khu.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ghrelin is an endogenous ligand for the GH secretagogue receptor, produced and secreted mainly from the stomach. Ghrelin stimulates GH release and induces positive energy balances. Previous studies have reported that ghrelin inhibits apoptosis in several cell types, but its antiapoptotic effect in neuronal cells is unknown. Therefore, we investigated the role of ghrelin in ischemic neuronal injury using primary hypothalamic neurons exposed to oxygen-glucose deprivation (OGD). Here we report that treatment of hypothalamic neurons with ghrelin inhibited OGD-induced cell death and apoptosis. Exposure of neurons to ghrelin caused rapid activation of ERK1/2. Ghrelin-induced activation of ERK1/2 and the antiapoptotic effect of ghrelin were blocked by chemical inhibition of MAPK, phosphatidylinositol 3 kinase, protein kinase C, and protein kinase A. Ghrelin attenuated OGD-induced activation of c-Jun NH2-terminal kinase and p-38 but not ERK1/2. We also investigated ghrelin regulation of apoptosis at the mitochondrial level. Ghrelin protected cells from OGD insult by inhibiting reactive oxygen species generation and stabilizing mitochondrial transmembrane potential. In addition, ghrelin-treated cells showed an increased Bcl-2/Bax ratio, prevention of cytochrome c release, and inhibition of caspase-3 activation. Finally, in vivo administration of ghrelin significantly reduced infarct volume in an animal model of ischemia. Our data indicate that ghrelin may act as a survival factor that preserves mitochondrial integrity and inhibits apoptotic pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GHRELIN IS A novel 28-amino-acid peptide esterified with octanoic acid on Ser 3 that is principally released from Gr cells in the oxyntic mucosa of the stomach (1). Ghrelin has been identified as an endogenous ligand for the GH secretagogue receptor (GHS-R) (2). Ghrelin stimulates GH release via the hypothalamus and direct pituitary pathways and induces a positive energy balance by stimulating food intake while decreasing fat use through GH-independent mechanisms (2, 3, 4, 5). Ghrelin also has numerous peripheral actions including direct effects on exocrine and endocrine pancreatic functions, carbohydrate metabolism, the cardiovascular system, gastric secretion, stomach motility, and sleep (6, 7, 8).

Stroke is the neurological condition that develops when a portion of the brain is deprived of oxygen and glucose. The damage caused to neurons during ischemia is due to a reduction in the oxygen and glucose supply, i.e. oxygen and glucose deprivation (OGD). The OGD insult, followed by reoxygenation, is thought to mimic the pathological conditions of ischemia. The precise mechanism of ischemic neuronal cell death is not clear; however, apoptosis is one of the mechanisms involved (9). Recent studies have reported that ghrelin stimulates proliferation and inhibits apoptosis in several cell types (10, 11, 12, 13). Moreover, recent reports suggest that the synthetic GHS, GH-releasing peptide (GHRP)-6, modulates IGF-I expression in the central nervous system (14) and ghrelin enhances learning and memory processes (15). Considering that the ghrelin receptor GHS-R1a expression and ghrelin binding sites are present in the hypothalamus and other brain areas (16, 17, 18), these findings suggest that ghrelin could have direct effects on brain function. Based on these observations, we hypothesized that ghrelin may protect neuronal cells from ischemic injury. Therefore, we tested this hypothesis by investigating the role of ghrelin in ischemic neuronal injury using primary cultured rat hypothalamic neuronal cells. To examine the mechanisms of ghrelin neuroprotection, selective inhibitors of MAPKs, phosphatidylinositol 3 kinase (PI3K), protein kinase C (PKC), and protein kinase A (PKA) were employed. Because mitochondria are involved in a variety of key events in apoptosis (19), we investigated the effect of ghrelin on OGD-induced reactive oxygen species (ROS) production, mitochondrial inner transmembrane potential ({Delta}{psi}M), cytochrome c release, and the Bcl-2 family of proteins. Finally, we demonstrate that in vivo administration of ghrelin decreases infarct volumes in an animal model of ischemia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Rat ghrelin was obtained from Peptides International (Louisville, KY). D-Lys-3-GHRP-6 was purchased from Bachem (Torrance, CA). Human recombinant IGF-I was obtained from Sigma Chemical Co. (St. Louis, MO). Neurobasal and RPMI 1640 media were from Life Technologies, Inc./Invitrogen (Carlsbad, CA). Primary antibodies to cytochrome c and Bcl-2 were obtained from Santa Cruz Biotechnology Inc. (Delaware, CA), and anti-BAX was from Abcam Inc. (Cambridge, UK). Anti-phospho-antibodies to ERK1/2 (Thr202/Tyr204), p-38 (Thr180/Tyr182) and stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) (Thr183/Tyr185) were purchased from Cell Signaling Technology (Danvers, MA). PD98059, wortmannin, H89, and GF109203X were from Tocris (Ellisville, MO). All tissue culture reagents were obtained from Life Technologies, Inc./Invitrogen, and all other reagents were obtained from Sigma unless otherwise indicated.

Cell cultures
Primary rat hypothalamic neuronal cultures were performed as previously described (20). Briefly, hypothalami were obtained from 1-d-old Sprague Dawley rats. The entire hypothalamus was quickly dissected and mechanically dispersed in Ca2+- and Mg2+-free buffered Hanks’ balanced salt solution. Then tissues were dissociated enzymatically (0.125% trypsin solution, 37 C for 10 min) and mechanically and filtered through a nylon mesh (pore size 40 µm). Cells were plated at a density of 2x104 cells/cm2 on 50 µg/ml poly-L-lysine-coated 100-mm culture dishes and grown in Neurobasal medium supplemented with 2% B27, 0.5 mM L-glutamine, and 2.5 ng/ml basic fibroblast growth factor (bFGF). Three days after dissociation, the medium was changed to bFGF-free neurobasal medium. Cells were used between 6 and 8 d in vitro. More than 90% of primary cultured hypothalamic cells were positive for neuronal marker NeuN antibodies, determined by immunohistochemistry and confocal microscopy (data not shown). To determine whether ghrelin protects hypothalamic cells from OGD insult, cells were pretreated with ghrelin (10–7 to 10–13 M) or vehicle (saline) for 24 h. Then cells were exposed to OGD or maintained under normoxic conditions. To examine whether the effect of ghrelin is exerted through its receptor GHS-R1a, cells were incubated with D-Lys-3-GHRP-6 (10–4 M) or vehicle (saline) for 1 h before the treatment with ghrelin. Experiments were also performed by adding the following pharmacological inhibitors to culture media, PD98059 (50 µM), wortmannin (200 nM), GF109203X (5 µM), or H89 (5 µM). Cell death and apoptosis were determined by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DNA fragmentation ELISA, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL), and flow cytometric analysis described below. To investigate the effect of ghrelin on the ERK1/2 pathway, 1) cells were treated with ghrelin (10–7 M) or vehicle for 5, 10, 30, 60, and 120 min; and 2) cells were treated with ghrelin (10–7 to 10–13 M) or vehicle for 10 min, and assayed by Western blot analysis described below.

OGD
To induce ischemia, cells were exposed to OGD as previously described, with some modifications (21). On the day of the experiment, the regular Neurobasal culture medium was replaced with OGD medium (glucose-free RPMI supplemented with 1% fetal bovine serum). Cultures were then placed in a humidified 37 C incubator within the Hypoxic Workstation (Daiki Sciences Co. Ltd. by Ruskinn Technology, Bridgend Mid Glamorgan, UK) containing a gas mixture of 0.1% O2, 5% CO2, and 94.9% N2 for 30 min to initiate the ischemic insult. OGD was terminated by replacing the OGD medium with Neurobasal medium containing 4.5 mg/ml glucose, and cultures were incubated for an additional 24 h under normoxic conditions.

Cell death and apoptosis
Cells viability was measured by the MTT assay as previously described (22). Histone-complexed DNA fragments were quantified by the Cell Death Detection ELISA (Roche, Mannheim, Germany) according to the manufacturer’s protocol. To measure the double-stranded cleavage of DNA, a TUNEL assay was performed using the APO-BrdU TUNEL Assay kit (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions. Apoptosis was also evaluated by flow cytometric analysis (FACSCalibur, Becton Dickinson, San Jose, CA) of the proportion of cells stained with Annexin-V/fluorescein isothiocyanate (FITC) (Biovision Inc., Mountain View, CA). Briefly, 5 x 105 cells were stained with 500 µl binding buffer containing Annexin-V-FITC and propidium iodide (PI) for 10–15 min at RT. Data were analyzed using flow cytometry software.

Western blot analysis
Cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 140 mM NaCl, 1% (wt/vol) Nonidet P-40, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, and 10 µg/ml aprotinin. Cell lysates were separated by 12% SDS-PAGE and electrotransferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). For the detection of Bax, Bcl-2, and cytochrome c, cells were fractionated into mitochondria and cytosol using the Mitochondria/Cytosol Fractionation Kit (BioVision, Mountain View, CA) according to the manufacturer’s instructions. The membranes were soaked in blocking buffer (1 x Tris-buffered saline, 1% BSA, 1% nonfat dry milk) for 1 h and incubated overnight at 4 C with the primary antibody. Blots were developed using a peroxidase-conjugated antirabbit IgG and a chemiluminescent detection system (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The bands were visualized using a ChemicDoc XRS system (Bio-Rad, Hercules, CA) and quantified using Quantity One imaging software (Bio-Rad). The p-ERK1/2 band intensity was normalized to ERK1/2 band intensity and the intensities of Bax, Bcl-2, and cytochrome c were adjusted by the ß-actin band intensity.

RT-PCR
Total RNA from hypothalamic neuronal cells was extracted using the QIAGEN RNeasy Mini Kit (QIAGEN Inc., Germantown, MD) according to the manufacturer’s instructions and was reverse transcribed using the Superscript II reverse transcriptase (Life Technologies, Inc., St. Louis, MO) at 42 C with random hexamer priming. A RNA control tube containing all RT reagents except reverse transcriptase was included as a negative control to monitor genomic DNA contamination. To confirm the GHS-R expression in cultured hypothalamic neuronal cells, the resultant cDNA was amplified using primers specific for GHS-R1a (sense 5'-TTC GCC ATC TGC TTC CCT CTG-3' and antisense 5'-TGT CTG CTT GTG GTT CTG GTC-3') and ß-actin (sense 5'-ATG GGT CAG AAG GAC TCC TAC G-3' and antisense 5'-AGT GGT ACG ACC AGA GGC ATA C-3') and the GeneAmp* PCR System 2700 (Applied Biosystems, Singapore).

Measurement of ROS production
Intracellular ROS generation was measured using confocal microscopy on cells stained with the ROS-sensitive fluorescent dye 2',7'-dichlorofluorescein diacetate (DCF-DA) (Sigma) as previously described (24). ROS production was assessed on four-well chamber slides. The cells were incubated with 2.5 µM DCF-DA for 30 min. Fluorescence was captured using a x40 objective lens on a Carl Zeiss LSM 510 Meta (Oberkochen, Germany) confocal microscope (485-nm excitation and 535-nm emission). DCF fluorescence was quantified from cells of interest using the measurement functions on the Carl Zeiss confocal software.

Assessment of mitochondrial transmembrane potential ({Delta}{psi}M)
The {Delta}{psi}M was monitored with the JC-1 reagent in the Mitochondrial Membrane Potential Detection kit (Stratagene, La Jolla, CA) and confocal microscopy according to the manufacturer’s instructions. Briefly, cells were incubated with 1 x JC-1 reagent solution at 37 C for 15 min. Culture slides were washed and mounted with PBS, and confocal images were acquired by the Carl Zeiss LSM 510 microscope. The ratio of red to green fluorescence was quantified from cells of interest using the measurement functions on the confocal microscopy software.

Caspase-3 activity
A caspase-3 fluorescent assay kit from Peptron (Daejon, Korea) was used for the determination of caspase-3 activity in cells according to the manufacturer’s instructions. Briefly, cell lysates were incubated with the reaction buffer containing 2.5 mM Ac-Asp-Glu-Val-Asp-AMC (Ac-DEVD-AMC) at 37 C for 120 min, and fluorescence was measured by a fluorometer (PerkinElmer, Wellesley, MA) using excitation at 360 nm and emission at 460 nm.

Transient middle cerebral artery occlusion (MCAO)
Reversible focal cerebral ischemia was generated in adult male Sprague Dawley rats weighing 250–280 g. Animals were anesthetized by inhalation of a nitrous oxide/oxygen/halothane (69%:30%:1%) mixture. After a midline incision was made, the right MCA was occluded for 2 h as previously described (25). Ghrelin was administered ip (80 or 160 µg/kg) 30 min before MCAO and at the beginning of reperfusion. The dose of ghrelin was chosen on the basis of previous reports showing that 80 µg/kg of ghrelin exerted a protective action on the stomach of rats exposed to ethanol- and stress-induced gastric damage (26, 27) In addition, we also examined whether 160 µg/kg of ghrelin showed a dose-dependent protective effect. The rats were killed by halothane overdose after 24 h of reperfusion. Their brains were removed, cut into six 2-mm coronal slices, and stained with triphenyl tetrazolium chloride (TTC) at 37 C for 30 min in the dark for the evaluation of infarct volume. TTC stains the undamaged tissue as red whereas dead cells remain white (28). Brain slices were photographed using a digital camera and quantified using an image analyzing system (Optimas 6.5; Media Cybermetics, Silver Springs, MD). The infarct area in each slice was calculated by subtracting the normal ipsilateral area from that of the contralateral hemisphere to reduce errors due to cerebral edema and was presented as the percentage of the infarct area to that of the contralateral hemisphere. All described experiments were approved by the Kyunghee University Animal Care Committee and conducted according to the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals.

Statistical analysis
Data are presented as mean ± SEM (n = 3 or 4/treatment for the in vitro experiment and n = 5–10/group for the in vivo experiment). Each experiment was repeated at least twice. Statistical analysis between groups was performed using one-way ANOVA and Holm-Sidak method for multiple comparisons using SigmaStat for Windows Version 3.10 (Systat Software, Inc., Point Richmond, CA). P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GHS-R1a expression in hypothalamic neuronal cells
We have examined the expression of GHS-R1a in hypothalamic neuronal cells compared with the pituitary. We found that GHS-R1a mRNA was present in hypothalamic cells (Fig. 1Go) as previously reported (2, 29).


Figure 1
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  		FIG. 1. Expression of GHS-R1a mRNA in hypothalamic neuronal cells. Total RNA was extracted from primary cultured hypothalamic neuronal cells and pituitary (as a positive control). The GHS-R1a expression was analyzed by RT-PCR and ß-actin was used as an internal control. RT (–) reaction containing all RT reagents except reverse transcriptase was included as a negative control to monitor genomic DNA contamination.

 
Ghrelin protect hypothalamic neuronal cells against OGD insult
To investigate whether ghrelin may act as a survival factor for neuronal cells, we assayed the effect of ghrelin on cell death induced by OGD insult, which causes apoptotic cell death of neurons in vitro (30, 31). After a 30-min exposure of cells to OGD insult followed by 24 h of reoxygenation, 50% of cells were not viable as determined by the MTT assay (Fig. 2AGo). However, OGD-induced cell death was significantly reduced in a dose-dependent manner by a 24-h pretreatment with ghrelin (10–7 to 10–13 M). The percentage of viable cells compared with normoxic controls decreased to 53.1% (OGD) and was significantly increased to 67.4 (10–13 M), 71.3 (10–11 M), 80.6 (10–9 M), and 83.7% (10–7 M) by pretreatment with ghrelin. IGF-I (10 nM) was used as a positive control and significantly inhibited OGD-induced cell death as previously described (32).


Figure 2
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  		FIG. 2. Ghrelin protects hypothalamic neuronal cells from OGD-induced apoptotic cell death. A and C, Hypothalamic cells were pretreated with vehicle or ghrelin (10–7 to 10–13 M) for 24 h. B and D, Cells were preincubated with vehicle or D-Lys-3-GHRP-6 (10–4 M) for 1 h and then treated with vehicle or ghrelin (10–7 to 10–11 M) for 24 h. E and F, Cells were pretreated with vehicle or ghrelin (10–7) for 24 h. Then cells were exposed to OGD for 30 min followed by 24 h of reoxygenation or maintained under normal conditions (normoxia). IGF-I (10–8 M) was used as a positive control. A and B, Cell viability measured by the MTT assay. C and D, DNA fragmentation, a marker of apoptosis, measured by ELISA. E, TUNEL staining of hypothalamic neuronal cells. Fragmented DNA was labeled with FITC-conjugated 2'-deoxyuridine 5'-triphosphate. FITC labels were observed using a confocal microscope. F, Flow cytometric analysis of apoptotic cells stained with Annexin-V and propidium iodide. Values are the mean ± SEM (n = 4). Each experiment was repeated twice. *, P < 0.05 vs. vehicle-treated cells exposed to OGD.

 
Neuronal cells exposed to OGD insult have been observed to undergo apoptosis (30, 31). Therefore, we investigated the effects of ghrelin on OGD-induced apoptosis in hypothalamic neuronal cells. The apoptotic DNA fraction was significantly increased from 7.5% (normoxia) to 38.0% by OGD insult (Fig. 2CGo). Pretreatment of cells with ghrelin prevented apoptosis induced by OGD insult in a concentration-dependent manner (Fig. 2CGo). The percentage of apoptotic DNA was significantly decreased to 24.9 (10–13 M), 19.7 (10–11 M), 18.6 (10–9 M), and 18.2% (10–7 M) by pretreatment with ghrelin. Similarly, OGD increased TUNEL-positive cells, which were attenuated by pretreatment with ghrelin or IGF-I (Fig. 2EGo). Flow cytometric analysis revealed that the percentage of annexin (+)/PI (–) fluorescent cells increased from 5.5% (normoxia) to 29.5% (OGD) and was significantly decreased to 6.8% by pretreatment with ghrelin (Fig. 2FGo).

To determine whether the antiapoptotic effect of ghrelin is mediated by its receptor GHS-R1a, hypothalamic neuronal cells were treated with the receptor specific antagonist D-Lys-3-GHRP-6 (33). Exposure of cells to D-Lys-3-GHRP-6 (10–6 M) abolished the protective effect of ghrelin against OGD insult (Fig. 2Go, B and D).

Ghrelin activates the ERK1/2 pathway
MAPKs include ERKs, JNK, and SAPK, also known as p-38. ERKs are known to be linked to survival/neuroprotective effects, whereas JNK and p-38 are related to neuronal apoptosis (34). It was recently reported that ghrelin can activate the ERK1/2 pathway in cardiomyocytes (10) and adipocytes (11). Therefore, we investigated the effect of ghrelin on the ERK1/2 pathway in primary cultured hypothalamic neuronal cells. Treatment of cells with ghrelin activated ERK1/2 in a time- (Fig. 3AGo) and dose-dependent (Fig. 3BGo) manner. Ghrelin-induced activation of ERK1/2 peaked between 10 and 60 min and lasted 120 min. Ghrelin concentrations ranging from 10–7 to 10–13 M resulted in rapid and strong activation of ERK1/2. We further determined which signaling pathways are involved in ghrelin-induced ERK1/2 activation. We found that pretreatment of hypothalamic cells with the MAPK inhibitor PD98059 (50 µM), the PI3K inhibitor wortmannin (200 nM), the PKC inhibitor GF109203 x (5 µM), or the PKA inhibitor H89 (5 µM) significantly attenuated ghrelin-induced phosphorylation of ERK1/2 (P < 0.05), suggesting that all of these pathways contribute to ghrelin-induced ERK1/2 activation (Fig. 3Go, C–E).


Figure 3
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  		FIG. 3. Ghrelin activates ERK1/2 in hypothalamic neuronal cells. A, Time course of ghrelin-induced phosphorylation of ERK1/2. Cells were treated with ghrelin (10–7 M) for 5, 10, 30, 60, and 120 min and assayed by Western blot using specific anti-phospho-ERK1/2 (Thr202/Tyr204) antibodies and anti-ERK1/2 antibodies. B, Dose responsiveness of ghrelin-induced phosphorylation of ERK1/2. Cells were treated with ghrelin (10–7 to 10–13 M) for 10 min and assayed by Western blot as above. C–F, MAPK, PI3K, PKC, and PKA pathways mediate ghrelin-induced phosphorylation of ERK1/2. Hypothalamic cells were preincubated with 50 µM PD98059 for 1 h, 200 nM wortmannin for 30 min, 5 µM GF109203X for 30 min, or 5 µM H89 for 30 min, then treated with ghrelin (10–7 M) for 10 min and assayed by Western blot as above. The phospho-ERK1/2 band intensity was normalized to ERK1/2 band intensity and expressed as relative band intensity. Values are the mean ± SEM (n = 4). Each experiment was repeated twice. *, P < 0.05 vs. untreated cells; {dagger}, P < 0.05 vs. ghrelin-treated cells.

 
MAPK, PI3K, PKC, and PKA pathways mediate ghrelin’s antiapoptotic effects against OGD insult
Our data suggest that ERK1/2 activation may be involved in the ghrelin-induced antiapoptotic effect in hypothalamic neuronal cells exposed to OGD insult. To further verify our hypothesis, we tested whether pretreatment of cells with PD98059 (50 µM), wortmannin (200 nM), GF109203X (5 µM), or H89 (5 µM) impaired the antiapoptotic activity of ghrelin against OGD insult. We found that all of these inhibitors significantly blocked the antiapoptotic effects of ghrelin (P < 0.05) (Fig. 4Go). These data suggest that ghrelin inhibits apoptosis induced by OGD insult via the activation of MAPK, PI3K, PKC, and PKA pathways.


Figure 4
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  		FIG. 4. MAPK, PI3K, PKC, and PKA pathways mediate antiapoptotic effect of ghrelin. Hypothalamic cells were preincubated with 50 µM PD98059 for 1 h, 200 nM wortmannin for 30 min, 5 µM GF109203X for 30 min, or 5 µM H89 for 30 min and then treated with ghrelin (10–7 M) for 24 h. Then cells were exposed to OGD for 30 min followed by 24 h of reoxygenation or maintained under normal conditions (normoxia). IGF-I (10–8 M) was used as a positive control. DNA fragmentation was measured by ELISA. Values are the mean ± SEM (n = 4). Each experiment was repeated twice. *, P < 0.05 vs. OGD-insulted, vehicle-treated cells; {dagger}, P < 0.05 vs. OGD-insulted, ghrelin-treated cells.

 
Ghrelin differentially attenuates OGD-induced activation of MAPK isoforms
Because OGD activates the MAPK isoforms (21), we investigated the effect of ghrelin on this process. Ghrelin inhibited OGD-induced activation of p-38 and JNK, whereas it had no effect on ERK1/2 (Fig. 5Go). IGF-I, used as a positive control, showed similar results. Ghrelin did not alter the activities of p-38 and JNK but significantly stimulated the ERK1/2 activity in hypothalamic cells incubated under normoxic condition. These results suggest that ghrelin differentially attenuates the activation of MAPK isoforms in hypothalamic neuronal cells exposed to OGD insult. Differential inhibition of MAPK stress kinase isoforms has also been observed in nerve growth factor (NGF)-treated PC12 cells exposed to OGD insult (35).


Figure 5
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  		FIG. 5. Ghrelin differentially attenuates OGD-induced activation of MAPK isoforms. Hypothalamic cells were pretreated with vehicle or ghrelin (10–7 M) for 24 h. Then cells were exposed to OGD for 30 min followed by 24 h of reoxygenation or maintained under normal conditions (normoxia). IGF-I (10–8 M) was used as a positive control. A, ERK1/2, JNK, and p-38 activation were assayed by Western blot using specific anti-phospho-ERK1/2 (Thr202/Tyr204), anti-phospho-SAPK/JNK (Thr183/Tyr185), and anti-phospho-p-38 (Thr180/Tyr182) antibodies, respectively. B, Phospho-ERK1/2, phospho-JNK, and phospho-p-38 band intensities were normalized to ß-actin band intensity and expressed as relative band intensity. Values are the mean ± SEM (n = 3). Each experiment was repeated twice. *, P < 0.05 vs. normoxic, vehicle-treated cells; {dagger}, P < 0.05 vs. OGD-insulted, vehicle-treated cells.

 
Ghrelin inhibits the OGD-induced increase in ROS production
ROS play important roles in neuronal apoptosis after ischemic injury (36, 37, 38). Ghrelin is known to inhibit H2O2-induced cytokine release in human endothelial cells (39) and to ameliorate oxidative gastric damage induced by alendronate (40). These results suggest that ghrelin may have antioxidant properties. To determine whether ghrelin may inhibit OGD-induced cell death by suppression of ROS generation, we examined changes in ROS levels. Compared with normoxic cells, cells treated with 30 min of OGD followed by reoxygenation showed increased ROS production. In contrast, treatment of cells with ghrelin significantly reduced the increase in DCF fluorescence induced by OGD (Fig. 6Go). Our results indicate that ghrelin may inhibit OGD-induced neuronal apoptosis through its possible antioxidant properties.


Figure 6
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  		FIG. 6. Ghrelin inhibits OGD-induced ROS generation. Hypothalamic cells were pretreated with vehicle or ghrelin (10–7 M) for 24 h. Then cells were exposed to OGD for 30 min followed by 24 h of reoxygenation or maintained under normal conditions (normoxia). A, ROS levels were determined using confocal microscopy on cells stained with the ROS-sensitive fluorescent dye 2',7'-dichlorofluorescein diacetate (DCF-DA). B, DCF fluorescence was quantified from cells of interest using the measurement functions on the Carl Zeiss LSM 510 Meta software. *, P < 0.05 vs. OGD-insulted, vehicle-treated cells.

 
Effect of ghrelin on mitochondrial transmembrane potential ({Delta}{psi}M)
In many apoptotic cells, the {Delta}{psi}M collapses due to the opening of the mitochondrial permeability transition (PT) pore (41, 42). IGF-I blocks apoptosis by preventing mitochondrial depolarization (loss of {Delta}{psi}M) (43). Therefore, we examined the effect of ghrelin on {Delta}{psi}M. JC-1 exhibits potential-dependent accumulation in the mitochondria indicated by a fluorescence emission shift from green (~525 nm) to red (~590 nm). The green fluorescence (monomeric form) represents a lower {Delta}{psi}M, whereas the red fluorescence (aggregate form) is considered to represent a high {Delta}{psi}M (44, 45). The ratio between green and red depends on {Delta}{psi}M, and mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. As shown in Fig. 7AGo, normoxic cells stained with JC-1 showed the typical distribution of red or green fluorescent signals present in the same cell. The mean value of the red/green fluorescence intensity ratio was 1.24 ± 0.04 (Fig. 7BGo). Thirty minutes of OGD followed by 8 h of reoxygenation significantly decreased the {Delta}{psi}M, as indicated by the marked increase in green fluorescence, and the normalized JC-1 fluorescence decreased to 0.35 ± 0.04. When cells were pretreated with ghrelin, OGD-induced mitochondrial depolarization was inhibited, and the mean value of the red/green fluorescence intensity ratio was significantly increased to 0.87 ± 0.07. Similar to a previous report (43), 10 nM IGF-I showed a protective effect against OGD-induced mitochondrial membrane depolarization.


Figure 7
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  		FIG. 7. Ghrelin stabilizes mitochondrial transmembrane potential ({Delta}{psi}M). Hypothalamic cells were pretreated with vehicle, ghrelin (10–7 M), or IGF-I (10–8 M) for 24 h. Then cells were exposed to OGD for 30 min followed by 8 h of reoxygenation or maintained under normal conditions (normoxia). A, The {Delta}{psi}M was determined by confocal microscopy using JC-1 dye. Mitochondria with a higher membrane potential have a red fluorescence, and those with a lower membrane potential are green. B, The ratio of red to green fluorescence was quantified in cells of interest using the measurement functions in the Carl Zeiss LSM 510 Meta software. *, P < 0.05 vs. OGD-insulted, vehicle-treated cells.

 
Ghrelin increases Bcl-2/BAX ratio
Bcl-2 inhibits apoptosis by preventing mitochondrial membrane depolarization (46), whereas Bax promotes apoptosis by inducing mitochondrial membrane depolarization and cytochrome c release (47). IGF-I is known to inhibit apoptosis by increasing Bcl-2 and the Bcl-2/BAX ratio in mesangial cells exposed to high glucose levels (48, 49). Bax protein, found predominantly in the cytosol under normoxic conditions, is translocated to the mitochondrial outer membrane by OGD insult (50). To identify changes in protein levels of Bax, a Western blot was conducted to detect Bax in cytosolic and mitochondrial fractions. Figure 8AGo shows the OGD-induced increase in Bax protein in the mitochondrial fraction of cells, suggesting a translocation from the cytosol to the mitochondria. Ghrelin treatment significantly inhibited the OGD-induced increase in Bax protein in the mitochondria. We also investigated the levels of Bcl-2 protein in the cytosol. We found that Bcl-2 protein levels were decreased by OGD insult, whereas ghrelin treatment increased Bcl-2, thereby significantly increasing the Bcl-2/Bax ratio.


Figure 8
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  		FIG. 8. Ghrelin increases Bcl-2/Bax mRNA and protein ratios, prevents cytochrome c release, and inhibits caspase-3 activation. Hypothalamic cells were pretreated with vehicle or ghrelin (10–7 M) for 24 h. Then cells were exposed to OGD for 30 min followed by 24 h of reoxygenation or maintained under normal conditions (normoxia). A, Effect of ghrelin on Bax and Bcl-2 protein levels in hypothalamic cells exposed to OGD assessed by Western blot. Bax and Bcl-2 band intensities were normalized to ß-actin band intensity. B, Effect of ghrelin on cytochrome c protein levels in the cytosolic fraction of hypothalamic cells exposed to OGD assessed by Western blot. Cytochrome c band intensity was normalized to ß-actin band intensity. C, Changes in caspase-3 activity assessed using the cell-permeable caspase-3 substrate Ac-DEVD-AMC. Values are the mean ± SEM (n = 3 or 4). Each experiment was repeated twice. *, P < 0.05 vs. normoxic cells; {dagger}, P < 0.05 vs. OGD-exposed, vehicle-treated cells.

 
Effects of ghrelin on cytochrome c release and caspase-3 activation
Cytochrome c release is regulated by the interactions of Bcl-2 family proteins (51). To determine whether ghrelin inhibits cytochrome c release, Western blots were performed on cytosolic fractions of hypothalamic cells. As shown in Fig. 8BGo, exposure of cells to OGD insult resulted in the release of cytochrome c from the mitochondria to the cytosol, which was significantly reduced by pretreatment with ghrelin. We also observed a protective effect of ghrelin at the level of caspase-3 activation and found that ghrelin and IGF-I significantly reduced the OGD-induced increase in caspase-3 activity assessed (Fig. 8CGo).

Ghrelin decreases infarct volume in MCAO rats
To determine whether administration of ghrelin may attenuate the severity of ischemia in vivo, we examined the effect of ghrelin in MCAO rats. We found a significant dose-dependent decrease in infarct volume in animals given ghrelin compared with vehicle-treated rats (Fig. 9AGo). The infarct volume of the vehicle-treated rats was 230.4 ± 25.3 mm3, whereas the volumes of the ghrelin-treated groups were 171.4 ± 31.6 mm3 (80 µg/kg, P < 0.01) and 123.8 ± 21.5 mm3 (160 µg/kg, P < 0.01) (Fig. 9BGo). It should be noted that, in the previous reports (26, 27), ip injection of 80 µg/kg of ghrelin significantly increased plasma ghrelin level.


Figure 9
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  		FIG. 9. Ghrelin decreases infarct volume in MCAO rats. Ghrelin was administered ip (80 or 160 µg/kg) 30 min before MCAO and at the beginning of reperfusion. The right MCA was occluded for 2 h. Brain was reperfused for 24 h and removed for the evaluation of infarct volume. A, Representative sections stained with triphenyl tetrazolium chloride (TTC). Undamaged tissue stains red, whereas dead cells do not pick up the dye and remain white. Vehicle-treated brain shows extensive damage. Ghrelin-treated brain shows a substantial reduction of damage when compared with the vehicle-treated brain. B, Infarct volume was calculated by measuring the infarct areas on coronal brain sections. Values are the mean ± SEM (n = 5–10/group). *, P < 0.05 vs. vehicle-treated group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrated that ghrelin treatment protects hypothalamic neuronal cells from the apoptotic stimuli of OGD insult. Neuroprotective effects of ghrelin were dependent on the activities of MAPK, PI3K, PKC, and PKA signaling pathways. We showed for the first time that ghrelin suppresses OGD-induced ROS production and stabilizes mitochondrial transmembrane potential ({Delta}{psi}M). We also demonstrated that ghrelin treatment alters the status of the Bcl-2 family of proteins, inhibiting cytochrome c release and caspase-3 activity and promoting the survival of hypothalamic neuronal cells. Finally, we presented evidence that in vivo administration of ghrelin decreased infarct volumes in an animal model of ischemia.

Ghrelin protects several cell types such as adipocytes (11), osteoblasts (13), cardiomyocytes, and endothelial cells (10) by inhibiting apoptotic stimuli. In this study, we showed for the first time that ghrelin, even at rather low doses (10–13 M), protects hypothalamic neuronal cells from OGD-induced cell death by inhibiting apoptosis. This observation mirrors a report by Kim et al. (11), in which ghrelin acted as an adipocyte mitogen at low concentrations (10–13 M). Ghrelin has been reported to have protective effects against a variety of stimuli including ischemia/reperfusion (53, 54), alendronate (40), serum deprivation (11), doxorubicin (10), and TNF-{alpha} (13). The neuroprotective effect of ghrelin appears to be mediated through the activation of GHS-R1a because the receptor antagonist D-Lys-3-GHRP-6 completely blocks the protective effect of ghrelin against OGD insult. In contrast, Baldanzi et al. (10) reported that in cardiomyocytes ghrelin exhibits an antiapoptotic effect through binding to a novel, unidentified receptor that is distinct from GHS-R1a. Given the fact that hexarelin up-regulates the expression of GHS-R1a (55, 56), the regulation of hypothalamic GHS-R1a by ghrelin may modulate the local GH or IGF-I levels. Taken together, our findings provide evidence that ghrelin may act as a survival factor for neuronal cells and offer a new perspective on the potential role of these peptides in ischemic injury.

In agreement with previous papers demonstrating that ghrelin activates ERK1/2 in 3T3-L1 adipocytes (11), osteoblasts (13), cardiomyocytes, and endothelial cells (10), we have shown that ghrelin strongly induces ERK1/2 activation, which is believed to be an important mechanism to limit ischemic damage (57) in hypothalamic neuronal cells. A selective inhibitor of ERK1/2 (PD98059) inhibited ghrelin-induced phosphorylation of ERK1/2 and the antiapoptotic activities of ghrelin, indicating that ghrelin suppressed OGD-induced apoptosis in hypothalamic neuronal cells through the activation of ERK1/2. The GHS-R1a belongs to a family of receptors operating via the Gq-phospholipase C (PLC) pathway (58). Other signaling pathways are involved in GHS-R1a-mediated activation of ERK1/2. It has been reported that PI3K pathways are involved in ERK1/2 activation (10, 11). PLC-PKC pathway and Raf-MEK-MAPK activation occur via the {alpha}-subunit of GHS-R1a (12). On the other hand, ghrelin has been shown to exert its effects in various cells through stimulation of cAMP-mediated PKA pathways (59). In this study, pretreatment with a PI3K inhibitor (wortmannin), a PKC inhibitor (GF109203X), or a PKA inhibitor (H89) significantly attenuated ghrelin-induced phosphorylation of ERK1/2 and the antiapoptotic effects of ghrelin. These data suggest that multiple signaling pathways are involved in ghrelin-induced ERK1/2 activation, and the antiapoptotic effect of ghrelin is mediated via PI3K, PKC, and PKA signaling pathways.

Mammalian cells contain three major classes of MAPKs: ERK, JNK, and p38 MAPK (60). The ERK MAPK is thought to be involved in cell growth and survival, whereas JNK and p-38 are associated with cell stress and death, including neuronal apoptosis. Activation of the ERK1/2 pathway protected astrocytes from ischemic injury (57), whereas inhibition of JNK signaling rescued sympathetic neurons and PC12 cells from death (61). Insulin promotes survival of fetal neurons by inhibition of p-38 MAPK (62). It has been reported that in PC12 cells, OGD insult markedly activated ERK1/2, JNK, and p-38, whereas pretreatment of cells with NGF resulted in attenuation of OGD-induced activation of JNK and p-38 (35). This suggests that NGF neuroprotection is related to differential inhibition of MAPK stress kinase isoforms (35). Consistent with those results, in this study we also found that MAPK isoforms were activated by OGD insult, and ghrelin differentially regulated their activities. Specifically, JNK and p-38 were attenuated by ghrelin treatment, whereas ERK1/2 did not show any changes. Although these findings suggest an opposing relationship of ERK1/2 and JNK/p-38 in apoptotic pathways, the roles of MAPK cascades in neuronal death and survival seem complicated. In fact, inhibition of ERK1/2 did not attenuate the effects of NGF on neuronal survival in PC12 cells (63) or in sympathetic neurons (64). Therefore, at present, it is uncertain whether activation of these isoforms contributes to neuroprotection or neuronal apoptosis.

In the present study, we have shown for the first time that in hypothalamic neuronal cells, ghrelin treatment prevents OGD-induced ROS generation. Because ROS participate in early and late steps of the regulation of apoptosis (65), the ability of ghrelin to reduce ROS production appears to be important for its neuroprotective mechanisms. The antiapoptotic protein Bcl-2 is a plausible target for the putative antioxidant capacity of ghrelin because it has been shown that Bcl-2 can protect cells from apoptosis by preventing ROS accumulation (66) and/or shifting the cellular redox potential to a more reduced state (67). Therefore, we assume that the increased levels of Bcl-2 protein in ghrelin-treated cells may both promote cell survival and protect against ischemic oxidative stress.

The Bcl-2 family proteins play a major role in intracellular apoptotic signal transduction by regulating the permeability of the mitochondrial membrane (68). It has been reported that mitochondrial cytochrome c is released through the PT pore, which is regulated by the Bcl-2 family proteins (69). The proapoptotic protein Bax eliminates the mitochondrial transmembrane potential ({Delta}{psi}M) by affecting the PT pore to facilitate cytochrome c release (51), whereas the antiapoptotic protein Bcl-2 functions to conserve the membrane potential and block the release of cytochrome c. In this study, we found that ghrelin decreased Bax and increased Bcl-2 levels in hypothalamic cells exposed to OGD insult. We also observed that ghrelin prevented the OGD-induced collapse of {Delta}{psi}M. Taken together, these results suggest that ghrelin stabilizes {Delta}{psi}M by regulating Bcl-2 family proteins during ischemic injury.

Because Bcl-2 inhibits apoptosis by binding to the proapoptotic Bax, Bcl-xs, and Bad proteins, it is believed that the Bcl-2/Bax ratio is a determining factor for the cell’s fate (70). In this study, we have shown that the Bcl-2/Bax ratio is decreased by OGD insult, whereas ghrelin treatment increases Bcl-2 levels and decreases Bax levels, resulting in complete restoration of the Bcl-2/Bax ratio to normal levels. Similar findings were observed in simian virus 40 murine mesangial cells, in which high glucose decreased the Bcl-2/Bax ratio,whereas ligand activation of the IGF-I receptor increased it (48). It has been suggested that the tumor suppressor protein p53, a transcriptional regulator of the Bax gene (49), may be involved in the up-regulation of the Bcl-2/Bax ratio for IGF-I receptor-dependent signals (48). IGF-I markedly attenuated phosphorylation of p53 at Ser392, suggesting that p53 is a target for IGF-I receptor antiapoptotic signals. It remains to be determined whether p53 is involved in the antiapoptotic mechanism of ghrelin. Taken together, our data suggest that ghrelin affects the status of Bcl-2 and Bax proteins, a change that is directionally opposed to initiation of the apoptotic cascade.

We have shown in this study that cytochrome c is translocated from the mitochondria to the cytosolic compartment after OGD insult, consistent with previous studies (71, 72, 73, 74). Cytosolic cytochrome c forms the apoptosome by interacting with the CED-4 homolog, Apaf-1, and deoxyadenosine triphosphate, leading to activation of caspase-9 (75, 76, 77, 78). Caspase-9 is an initiator of the cytochrome c-dependent caspase cascade, activating caspase-3 followed by caspase-2, -6, -8, and -10 activation (79). In the present study, ghrelin treatment prevented the OGD-induced release of cytochrome c and subsequent activation of caspase-3, inhibiting activation of the apoptotic cascade.

Finally, using the temporary MCAO models for stroke, we demonstrated that in vivo administration of ghrelin significantly reduced infarct volumes after initiation of ischemia. To the best of our knowledge, this is the first report demonstrating an in vivo neuroprotective effect of ghrelin in an ischemic animal model. The brain IGF-I system may be involved in the neuroprotective effect of ghrelin because systemic administration of GHRP-6 in rats increased IGF-I mRNA levels in the hypothalamus, cerebellum, and hippocampus (14). In fact, it has been reported that the brain responds to ischemic insult by activating antiapoptotic pathways involving neurotrophic factors and cytokines (9). In vivo administration of bFGF (80), NGF (81), glial cell-derived neurotrophic factor (82), and TGF-ß (83) protect the brain against ischemic injury.

In summary, we have demonstrated that ghrelin protected against apoptosis in hypothalamic neuronal cells exposed to OGD insult. We also have shown that ghrelin strongly activated ERK1/2, and the protective effect of ghrelin was mediated by MAPK, PI3K, PKC, and PKA pathways in these cells. Ghrelin targeted the Bcl-2 protein family, inducing changes that favored cell survival and inhibited the apoptotic cascade. Ghrelin-mediated signals stabilized mitochondria by inhibiting ROS production, preserving {Delta}{psi}M, preventing cytochrome c release, and inhibiting caspase-3 activation. We also demonstrated that ghrelin treatment is effective in attenuating infarct volume induced by MCAO. Our data suggest that ghrelin can function as a neuroprotective agent, and administration of ghrelin may be a novel therapeutic strategy for the treatment of stroke.


   Acknowledgments
 
We would like to extend our thanks to Dr. Rhonda D. Kineman (University of Illinois at Chicago, Chicago, IL) and Dr. P. Kretchmer (San Francisco Edit, Mill Valley, CA) for proofreading and editing this manuscript.


   Footnotes
 
This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2004-015-E00090) and the Korea Science & Engineering Foundation (R13-2002-020-01009-0 and R01-2005-000-10359-0).

Disclosure Statement: The authors have nothing to disclose

First Published Online October 19, 2006

Abbreviations: bFGF, Basic fibroblast growth factor; FITC, fluorescein isothiocyanate; GHRP, GH-releasing peptide; GHS-R, GH secretagogue receptor; MCAO, middle cerebral artery occlusion; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; NGF, nerve growth factor; OGD, oxygen-glucose deprivation; PI3K, phosphatidylinositol 3 kinase; PKA, protein kinase A; PKC, protein kinase C; PT, permeability transition; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling.

Received July 25, 2006.

Accepted for publication October 10, 2006.


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 Results
 Discussion
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