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Endocrinology, doi:10.1210/en.2007-0733
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Endocrinology Vol. 149, No. 5 2191-2199
Copyright © 2008 by The Endocrine Society

Proliferative and Protective Effects of Growth Hormone Secretagogues on Adult Rat Hippocampal Progenitor Cells

Inger Johansson, Silvia Destefanis, N. David Åberg, Maria A. I. Åberg, Klas Blomgren, Changlian Zhu, Corrado Ghè, Riccarda Granata, Ezio Ghigo, Giampiero Muccioli, Peter S. Eriksson1 and Jörgen Isgaard

Laboratory of Experimental Endocrinology (I.J., S.D., N.D.A., J.I.), Department of Internal Medicine, and Center of Brain Repair and Rehabilitation (M.A.I.A., K.B., C.Z., P.S.E.), Sahlgrenska Academy, University of Göteborg, Göteborg SE-413 45, Sweden; and Department of Internal Medicine (R.G., E.G.), University of Turin, 10126 Turin, Italy; and Division of Pharmacology, Department of Anatomy (S.D., C.G., G.M.), Pharmacology, and Forensic Medicine, University of Turin, 10135 Turin, Italy

Address all correspondence and requests for reprints to: Inger Johansson, B.Sc., Laboratory of Experimental Endocrinology, Gröna Stråket 8, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden. E-mail: inger.johansson{at}medic.gu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Progenitor cells in the subgranular zone of the hippocampus may be of significance for functional recovery after various injuries because they have a regenerative potential to form new neuronal cells. The hippocampus has been shown to express the GH secretagogue (GHS) receptor 1a, and recent studies suggest GHS to both promote neurogenesis and have neuroprotective effects. The aim of the present study was to investigate whether GHS could stimulate cellular proliferation and exert cell protective effects in adult rat hippocampal progenitor (AHP) cells. Both hexarelin and ghrelin stimulated increased incorporation of 3H-thymidine, indicating an increased cell proliferation. Furthermore, hexarelin, but not ghrelin, showed protection against growth factor deprivation-induced apoptosis, as measured by annexin V binding and caspase-3 activity and also against necrosis, as measured by lactate dehydrogenase release. Hexarelin activated the MAPK and the phosphatidylinositol 3-kinase/Akt pathways, whereas ghrelin activated only the MAPK pathway. AHP cells did not express the GHS receptor 1a, but binding studies could show specific binding of both hexarelin and ghrelin, suggesting effects to be mediated by an alternative GHS receptor subtype. In conclusion, our results suggest a differential effect of hexarelin and ghrelin in AHP cells. We have demonstrated stimulation of 3H-thymidine incorporation with both hexarelin and ghrelin. Hexarelin, but not ghrelin, also showed a significant inhibition of apoptosis and necrosis. These results suggest a novel cell protective and proliferative role for GHS in the central nervous system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH SECRETAGOGUES (GHS) were originally synthetic peptidyl molecules that were synthesized in the early 1980s for their ability to release GH from the pituitary. From these compounds, hexarelin was later developed (1). The GHS receptor (GHS-R)-1a, sometimes referred to as the ghrelin receptor (2), was detected in 1996 (3), and in 1999 the endogenous GHS, ghrelin, was discovered by the group of Kangawa and coworkers (4). Ghrelin is primarily synthesized and secreted by the stomach but has been found to be expressed in most tissues and organs of the body (5). The unique octanoylation (acylation) at Ser3 is mandatory for the binding to the GHS-R1a and the stimulation of GH secretion. Surprisingly, the serum concentration of unacylated ghrelin (UAG) is much higher than for ghrelin [acylated ghrelin (AG)], and it has recently been found that UAG also has several effects both in vitro (6, 7) and in vivo (8, 9). To date, no receptor with the capacity to bind the UAG has been reported.

GHSs and ghrelin have also been suggested to have GH-independent effects in peripheral tissues. In particular, proliferative (10) and antiapoptotic (6) effects on cardiomyocytes in vitro have been reported. Moreover, cardioprotective effects in GH deficiency in rat models of ischemia/reperfusion injury in rat have been described (11).

It is now well established that neurogenesis, i.e. proliferation, survival, differentiation, and migration of neuronal precursor cells, persists into adulthood in mammals, including humans (12). There are two main areas in the adult brain in which cell proliferation has been found: the subgranular zone of the dentate gyrus of the hippocampus formation (13, 14) and the subventricular zone (15, 16). Hippocampal neurogenesis is particularly interesting because studies have shown that it can be modulated by physiological and behavioral events such as aging, stress, and exercise. Hippocampal neurogenesis is also important for learning because blockade of neurogenesis in the dentate gyrus of the hippocampus impairs memory function (17).

The GH/IGF-I axis has been shown in numerous studies to have effects on cell protection and cell genesis in the brain [reviewed by Aberg et al. (18)]. This has also been confirmed in vitro (19) using adult rat hippocampus progenitor (AHP) cells (20, 21). AHP cells are derived from adult rat hippocampus and have the capacity of in vitro self renewal in the presence of basic fibroblast growth factor (bFGF). They are multipotent progenitors and can give rise to cells of the neuronal as well as cells of the glial lineage. The hippocampus has been shown to express the GHS-R1a (22), and recent experimental data suggest that ghrelin influences several biochemical processes in the hippocampus including increased memory retention in rats (23). Ghrelin has also been shown to promote neurogenesis in the dorsal motor nucleus of the vagus in adult rat, both in vitro and in vivo (24). Two recent studies suggest that both ghrelin (25) and the hexarelin analog GH-releasing peptide-6 (26) have neuroprotective effects. Additionally, in a neonatal rat model with experimental unilateral hypoxic-ischemic injury, intracerebroventricular (icv) injections of hexarelin significantly reduced the area of injury in various parts of the brain and the most pronounced effect was found in the hippocampus (27). In this study hexarelin reduced caspase-3 activity and activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Because the hippocampus is a central nervous system (CNS) region in which cell proliferation also takes place in the adult brain, it would be of great interest to verify whether progenitor cells were involved. The aim of the present study was to investigate whether the synthetic peptidylic GHS hexarelin and the endogenous GHS ghrelin could exert direct effects on progenitor cells from adult rat hippocampus. We hypothesized that the GHSs would have proliferative effects in AHP cells and also that they would protect AHP cells against growth factor deprivation-induced cell death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM/F12, L-glutamine, N2 supplement, trypsin, and PBS were all from Life Technologies, Inc.-Invitrogen Corp., Paisley, UK). The cell culture flasks, plates, and fluorescence-activated cell sorter (FACS) tubes were all purchased from Becton Dickinson (Oxford, UK). All other chemicals, if not otherwise stated, came from Sigma Chemical Company (St. Louis, MO).

Peptides and antibodies
Hexarelin was a generous gift from Pharmacia & Upjohn (Stockholm, Sweden). Tyr-Ala-hexarelin and acylated and unacylated rat ghrelin were from NeoMPS (Strasbourg, France). Human bFGF came from Peprotech Inc (Rocky Hill, NJ).

For the immunofluorescence analysis, the mouse antinestin antibody was from PharMingen (Becton Dickinson) and the fluorescein isothiocyanate (Fitc)-conjugated donkey antimouse came from Jackson ImmunoResearch Lab (West Grove, PA).

For the Western blots, antibodies were for the PI3K/Akt pathway, rabbit antiphosphorylated-Akt (pAkt) and the MAPK pathway, rabbit antiphosphorylated ERK (pERK) 1/2 (Cell Signaling Technology Inc., Danvers, MA). In addition, we used total Akt and ERK 1/2 antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA). A donkey antirabbit secondary antibody was used in all Western blot experiments (Amersham Biosciences, GE Healthcare, Uppsala, Sweden).

Cell culture
The isolation and culturing of AHP cells has been previously described (21). AHP cells grown at low density in bFGF keep their undifferentiated appearance with rounded cell bodies, short processes (Fig 1AGo), and expression of the intermediate filament protein nestin (28) (Fig. 1BGo). Nestin is expressed during early neuronal and glial development and is considered being a marker for neuronal progenitor cells (21). The AHP cells appeared to keep their undifferentiated morphology, even after GHS stimulation, suggesting that GHS does not have a great impact on cell differentiation in these progenitor cells. The clonal population of AHP cells was received as a kind gift from Fred H. Gage (Laboratory of Genetics, The Salk Institute, La Jolla, CA). Cells were grown in polyornithine (PORN)/laminin- or only PORN-coated flasks or wells. For normal proliferating conditions, the cells were cultured in DMEM/F12, supplemented with 2 mM L-glutamine, 20 ng bFGF/ml, and N2 supplement (with high insulin = 5 µg/ml). This medium is referred to as normal medium (NM). For most of the assays, the cells were at some point deprived of growth factors (GFs) in low insulin medium (LIM). This medium contains modified N2 supplement with low insulin (100 ng/ml) and no bFGF. For a more severe starvation condition in which cells were induced to necrosis, cells were grown in DMEM supplemented with 0.1% BSA and 2 mM L-glutamine (DMEM/BSA). The cells used for analysis were between passages 10 and 15.


Figure 1
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  		FIG. 1. Untreated AHP cells in NM. Phase-contrast image (A) and a merged image of immunofluorescence-stained cells (B). Hoechst nuclei staining (blue) and the cell-specific marker nestin (green) are shown.

 
Immunofluorescence
AHP cells were passaged into PORN/laminin-coated glass in six-well plates and cultured in normal medium. Cells were fixed in 4% paraformaldehyde in PBS for 10 min at 4 C, rinsed once (5 min) in PBS, and treated as follows. Cells were first incubated with primary mouse antinestin antibody (1:1000 in 1% BSA and 0.05% saponin in PBS) for 1 h at room temperature. After three washes the Fitc-conjugated donkey antimouse antibody (1:150) was added, and the cells were incubated for 1 h. For nuclear staining, Hoechst 33258 (diluted 1:50 in PBS from stock, 5 µg/ml) was added. After three washes the glasses were coverslipped in Dako fluorescent mounting medium. Immunofluorescence was detected and processed using a Nikon Diaphot fluorescens microscope (Tokyo, Japan).

RT-PCR
The AHP cells used for RT-PCR were untreated or stimulated with 10 µM hexarelin or vehicle in DMEM/BSA for 24 h. Total RNA extraction from AHP cells was performed using the RNeasy kit (QIAGEN Inc., Valencia, CA) according to the modified single-step RNA isolation method by Chomczynski and Sacchi (29). RNA from rat pituitary and rat heart was used as positive controls for the GHS-R1a and the CD36, respectively. The reverse transcriptase reaction was performed using 1 µg of total RNA with Omniscript reverse transcriptase kit (QIAGEN) under the conditions recommended by the supplier.

Primer sequences (Cybergene AB; Novum Research Park, Huddinge, Sweden) specific for rat GHS-R1a were 5'-CTACCGGTCTTCTGCCTCAC (sense) and 5'-CAGGTTGCAGTACTGGCTGA (antisense) (30). The GHSR1a-specific primers were located on different exons. RT-PCR with both primers on exon 1 was also performed (5'-GCAACCTGCTCACTATGCTG, sense and 5'-CAGCTCTCGCTGACAAACTG, antisense). Primer sequences for the CD36 receptor were 5'-TCGTATGGTGTGCTGGACAT (sense) and 5'-TGCAGTCGTTTGGAAAACTG (antisense).

Rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard, and the primer sequences used were 5'-TGCACCACCAACTGCTTA (sense) and 5'-GGATGCAGGGATGATGTTC (antisense) (31). After an initial denaturation step (94 C for 5 min), 30 cycles of PCR were carried out in a 50-µl volume with Taq DNA polymerase, PCR nucleotide mix, and buffer set (Roche Diagnostics GmbH, Mannheim, Germany) in a Thermal Cycler 2720 (Applied Biosystems, Foster City, CA) under the following conditions: one cycle, 94 C for 15 sec, 51 C for 15 sec, 72 C for 30 sec; 72 C for 15 min for elongation. The PCR products (249 bp for the specific GHS-R1a, 199 bp for the product with both primers on exon 1, 194 bp for the CD36 receptor, and 177 bp for glyceraldehyde-3-phosphate dehydrogenase) were electrophoresed in 2% agarose gel and visualized by ethidium bromide staining. The phlX174 DNA/HaeIII was used as a molecular marker (Promega Corp., Madison, WI). To ensure that no genomic DNA was amplified in the PCR, RNA was also transcribed without reverse transcriptase enzyme.

Receptor binding assay
Tyr-Ala-hexarelin was radioiodinated (125I-labeled Tyr-Ala-hexarelin, specific activity 2000 Ci/mmol) by using a lactoperoxidase method by GE Healthcare (Milan, Italy) and used as a radioligand in the binding studies. Binding of 125I-labeled Tyr-Ala-hexarelin to crude cell membranes (30,000 x g pellet from cells cultured in NM) has previously been described (10). For saturation binding studies, AHP cell membranes (corresponding to 100 µg protein) were incubated with increasing concentrations (from 0.054 to 15 nM) of 125I-labeled Tyr-Ala-hexarelin. Parallel incubations, during which 10 µM unlabeled Tyr-Ala-hexarelin also was present, were used to determine nonspecific binding, which was subtracted from total binding to yield specific binding values. Saturation binding data were analyzed and the maximum binding capacity and dissociation constant values were calculated using a GraphPad Prism version 4 (GraphPad Software, San Diego, CA). Receptor binding competition studies were performed by incubating cell membranes (150 µg) with a fixed concentration (2 nM) of 125I-Tyr-Ala-hexarelin in the absence and presence of increasing concentrations (from 1 nM to 30 µM) of different competitors, such as hexarelin, ghrelin (AG), and UAG. Nonspecific ligand binding was determined by the incubation of radiolabeled Tyr-Ala-hexarelin and membranes in the presence of 10 µM unlabeled Tyr-Ala-hexarelin. Data were plotted and curves fitted using the GraphPad Prism software. Analysis of the curves suggested that the binding was due to a one-site binding, thus allowing determination of the concentration of a competitor causing 50% inhibition of specific radioligand binding (IC50).

3H-thymidine incorporation
3H-thymidine incorporation was used as a marker for cell proliferation. Cells were seeded in 2 ml of NM per well in PORN-coated 24-well plates at 1.5 x 104 cells/cm2 and cultured for 24 h. To synchronize cells to be in the same phase of the cell cycle before stimulation, cells were incubated in 2 ml of LIM overnight. The next day medium was replaced again, and the cells were incubated in LIM for 24 h in 37 C with various concentrations of GHS. During the last 6 h, 1 µCi [methyl-3H]thymidine (Amersham Biosciences, GE Healthcare in Sweden) per milliliter was added. Cells were washed three times with 0.5 ml ice-cold Dulbecco’s PBS (containing Ca2+ and Mg2+) and solubilized with 300 µl 10% sodium dodecyl sulfate (SDS) at room temperature. DNA was precipitated with 300 µl ice-cold 20% trichloric acid and collected on GF/C glass fiber filters (Whatman, Middlesex, UK). The precipitate was washed twice with ice-cold 10% trichloric acid and twice with ice-cold 95% ethanol. The radioactivity associated with the filters was quantified using liquid scintillation (β-counter; Beckman LS6500, Germany). 3H-thymidine incorporation was calculated as percent of vehicle control.

Cell viability/proliferation assay
Cell viability/proliferation was also measured using the metabolic activity assay Alamar Blue (BioSource International Inc., Camarillo, CA). The assay is using a nontoxic aqueous dye to assess cell viability (32) or cell proliferation (33). The method is based on an oxidation-reduction indicator that changes color from blue to pink and fluoresces when reduced by cellular metabolic activity. Cells were seeded in NM in PORN-coated 96-well plates at a cell concentration of 3000 cells per well. After 3 d, medium was changed to LIM and cells were cultured with or without different GHS for 48 h. For the last 60 min, 10 µl of Alamar blue stain were added to each well. The fluorescence was measured on a SPECTRAmax GeminiXS (Molecular Devices Corp., Sunnyvale, CA) by exciting at 530 nM and measuring emission at 590 nM. The GHS effect was calculated as percent of NM.

Annexin V labeling and flow cytometry
To assess any potential effects of GHS on apoptosis, we performed annexin V staining. The annexin V assay is based on the early apoptotic cell’s way of exposing phosphatidylserine residues at the outer plasma membrane leaflet and that Annexin V interact very strongly and specifically with PS (34, 35). Staining the nuclei of necrotic cells with propidium iodine (PI) is based on the loss of membrane integrity, which is a typical feature of cell necrosis and is in this assay used to exclude necrotic cells that also will stain positive for annexin V.

Cells were seeded in 2 ml NM per well in PORN-coated six-well plates at 1.5 x 104 cells/cm2. After 3 d medium was changed to 1 ml of LIM and cells were grown with or without different GHS for 48 h. Cells were trypsinized very briefly with 0.05% Trypsin-EDTA, and the trypsin was inhibited by the addition of a final concentration of 5% fetal calf serum in DMEM/F12 after 30–60 sec. Cells were washed twice in PBS and resuspended in FACS binding buffer (BD Biosciences, San Diego, CA). Staining was performed according to the instructions in the annexin V/PI kit (BD Biosciences). Cells were analyzed on a flow cytometer (FACSort; BD Biosciences). Cells that were annexin V-Fitcneg/PIneg were considered being alive, cells being annexin V-Fitcpos/PIneg were apoptotic and cells being annexin V-Fitcpos/PIpos were late apoptotic/secondary necrotic. The percentages of the different cell populations could be found in the different quadrants in an annexin V-Fitc/PI dot plot using the CellQuest Pro software (BD Biosciences).

Caspase-3 activity assay
Cells were seeded in 2 ml of NM per well in PORN-coated six-well plates at 1.5 x 104 cells/cm2. After 3 d medium was changed to 1 ml of DMEM/BSA per well, and cells were grown with or without 10 µM of hexarelin for 48 h. The cytosol fraction of the cells was prepared following the instructions in the mammalian cell lysis kit (Sigma). The lysis buffer in the kit was used (200 µl/well) except for that the volume of SDS was exchanged for water. The cytosol fraction (40 µl) was mixed with 60 µl of extraction buffer [50 mM Tris-HCl (pH 7.3), 100 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1% protease inhibitor cocktail (Sigma), and 0.2% [(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate] in a microtiter plate (Microfluor; Dynatech, Chantilly, VA). After incubation for 15 min at room temperature, 100 µl of assay buffer (50 mM Ac-DEVD-AMC (aminomethyl coumarin); Peptide Institute, Osaka, Japan), without protease inhibitors or [(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate but with 4 mM dithiothreitol, were added. Cleavage of Ac-DEVD-AMC was measured with an excitation wavelength of 380 nm and an emission wavelength of 460 nm. The degradation was followed at 2-min intervals for 2 h, and maximal velocity was calculated from the entire linear part of the curve. The activity was expressed as picomoles AMC released per milligram protein and minute, and the activity is calculated as percent of NM.

Lactate dehydrogenase (LD) release
Cells were seeded in 2 ml of NM per well in PORN-coated six-well plates at 1.5 x 104 cells/cm2. After 3 d medium was changed to 1 ml of DMEM/BSA per well, and cells were grown with or without different GHS for 48 h. Conditioned media were harvested and the LD activity was measured using a routine spectrophotometric enzymatic method (Roche Diagnostics) at the Department of Clinical Chemistry, Sahlgrenska University Hospital, Goteborg, Sweden. The coefficient of variation for the assay was 1.7% and the linear curve was between 0.12 and 20 µkatal/liter.

Western blot analysis
To analyze the phosphorylation (activation) of ERK1/2 and Akt, Western blot analysis was performed. Cells were seeded in 2 ml of NM per well in PORN-coated 6-well plates at 1.5 x 104 cells/cm2. After 3 d medium was changed to 1 ml of DMEM/BSA per well and cells were grown with different GHS for various length of time (0–90 min). The cytosol fraction of the cells was prepared following the instructions in the mammalian cell lysis kit. Five micrograms of total protein were run on a 10% Tris-glycine gel (Invitrogen). Proteins were electrotransferred to a polyvinylidene fluoride membrane (Immobilon-P; Millipore, Bedford, MA). The membrane was soaked in blocking buffer [1% pyrrolidone-40 in Tris-buffered saline with 0.05% Tween 20] for 1 h in room temperature. Primary antibodies for pERK and pAkt were diluted 1:1000 in blocking buffer, and the membrane was incubated overnight at 4 C. The membrane was washed three times in Tris-buffered saline with 0.1% Tween 20. The secondary antibody was diluted 1:100,000 and the membrane was incubated for 1 h in room temperature. After washing, the membrane was developed using ECL Plus kit (Amersham Biosciences, GE Healthcare in Sweden). After this the membrane was stripped (stripping buffer: 62.5 mM Tris-HCl, 2% SDS, and 100 mM β-mercaptoethanol) and reprobed for total ERK and Akt, which were used as internal controls for protein loading.

Statistical analysis
Data are presented as mean ± SEM unless otherwise stated, and n corresponds to the number of experiments performed. If not otherwise stated, statistical analysis was performed using the GraphPad Prism 5.0 software. The statistical significance was tested using one-way repeated ANOVA followed by the Dunnett’s post hoc test. P <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hexarelin and AG stimulate cell proliferation in AHP cells
Both hexarelin and AG stimulated cell proliferation as analyzed by 3H-thymidine incorporation in AHP cells after stimulation for 24 h in LIM. Hexarelin was more potent in increasing 3H-thymidine incorporation, compared with vehicle control, showing significant effects at 3 µM (Fig. 2AGo), whereas, AG had significant effects at 10 µM with maximum effects at 30 µM (Fig. 2BGo).


Figure 2
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  		FIG. 2. GHS effects on 3H-thymidine incorporation in AHP cells. Cells were incubated with indicated concentrations of GHS, hexarelin (A), AG (B), and UAG (C) in LIM for 24 h. Cells were harvested and the radioactivity was measured. Data are presented as percent of vehicle control ± SEM and represents results from at least six separate experiments (n = 6) with triplicate wells. ***, P < 0.001; **, P < 0.01; *, P < 0.05 vs. vehicle control.

 
Cell viability/proliferation was also measured by the metabolic activity assay Alamar Blue. Data showed increased values, compared with vehicle control, when AHP cells were stimulated for 48 h with hexarelin or AG in LIM. Hexarelin showed significant effects at 10 µM, compared with vehicle in LIM (Fig. 3AGo), and AG had significant effects at 1 and 10 µM, compared with vehicle control (Fig. 3BGo). UAG did not show significant effects in any of these assays (Figs. 2CGo and 3CGo).


Figure 3
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  		FIG. 3. GHS effect on cell viability in AHP cells. Cells were incubated with indicated concentrations of GHS, hexarelin (A), AG (B), and UAG (C), in LIM for 48 h, and the metabolic activity was measured using the Alamar Blue assay. Data are presented as viably cells of all cells in NM ± SEM and represents results from at least three separate experiments (n = 3) with five wells per sample. **, P < 0.01; *, P < 0.05 vs. vehicle control.

 
Hexarelin, but not AG, protects against apoptosis
To verify apoptosis after growth factor deprivation in LIM, annexin V and PI staining was measured by flow cytometry. A significant decrease of apoptosis was seen with 3 and 10 µM hexarelin in which apoptosis was 149 and 108% of NM, respectively (vehicle was 189%), indicating that with 10 µM hexarelin, the apoptosis was almost reduced to background levels (Fig. 4AGo). Neither AG nor UAG showed significant effects on the reduction of apoptosis as measured by annexin V staining (Fig. 4Go, B and C). Consistent with the annexin V results, hexarelin (10 µM) reduced the caspase-3 activity significantly, compared with vehicle control (Fig. 5Go).


Figure 4
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  		FIG. 4. Antiapoptotic effects of GHS in AHP cells. Cells were incubated with indicated concentrations of GHS, hexarelin (A), AG (B), and UAG (C) in LIM for 48 h. Cells were harvested and stained with annexin V-Fitc and PI. Percentages of apoptotic cells were measured using flow cytometry (FACS). Cells being annexin V-Fitcpos/PIneg were considered apoptotic. NM is the background apoptosis in normal medium. Data represent results from at least four separate experiments (n = 4). **, P < 0.01; *, P < 0.05 vs. vehicle control.

 

Figure 5
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  		FIG. 5. Hexarelin effect on caspase-3 activity in AHP cells. Cells were incubated with 10 µM of hexarelin for 48 h in DMEM/BSA. Cells were lysed and the caspase-3 activity in the cytosol fraction was measured. NM is the background caspase-3 activity in normal medium. Data represent results from four separate experiments (n = 4). **, P < 0.01 vs. vehicle control.

 
Hexarelin, but not AG, shows protection against necrosis
The ultimate sign of cell death through necrosis is the disruption of the cell membrane and the release of intracellular proteins, e.g. LD, out into the cell culture media. Starvation in vehicle control and DMEM/BSA for 48 h increased LD activity 5-fold, compared with background levels in NM. Hexarelin significantly reduced LD release starting at 1 µM and concentrations of 3 and 10 µM reduced LD release down to levels of NM. Necrosis was with 1, 3, and 10 µM hexarelin 383, 150, and 150% of NM, respectively (vehicle was 717%) (Fig. 6AGo). Neither AG nor UAG showed significant effects on reduction of necrosis as measured by LD release (Fig. 6Go, B and C).


Figure 6
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  		FIG. 6. GHS effect on LD release after starvation in DMEM/BSA. Cells were incubated with indicated concentrations of GHS, hexarelin (A), AG (B), and UAG (C), for 48 h in DMEM/BSA. Medium was collected and centrifuged, and cell death was estimated by the degree of LD activity in the medium. NM is the background cell death in normal medium. Data represent results from at least three separate experiments (n = 3). **, P < 0.01; *, P < 0.05 vs. vehicle control.

 
GHS activates the ERK1/2 and the PI3K/Akt pathway in AHP cells
In AHP cells both hexarelin and AG were able to increase the amount of pERK 1/2 after 15–30 min of stimulation, compared with total ERK1/2 (Fig. 7Go, A and B). Hexarelin, but not AG, could also increase the proportion of pAkt, starting after 5 min with maximum after 60–90 min, in comparison with total Akt (Fig. 8Go and data not shown).


Figure 7
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  		FIG. 7. Activation of the ERK1/2 pathway by hexarelin and ghrelin. Cells were incubated with 10 µM of GHS, hexarelin (A) and AG (B), for 0–90 min. Protein lysates from the different time points were prepared and run in a Western blot. The experiment was repeated twice and the result shown is a representative Western blot run.

 

Figure 8
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  		FIG. 8. Activation of the PI3K/Akt pathway by hexarelin. Cells were incubated with 10 µM hexarelin for 0–90 min. Protein lysates from the different time points were prepared and run in a Western blot. The experiment was repeated twice and the result shown is a representative Western blot run.

 
AHP cells do not express GHS-R1a but contain high-affinity binding sites for hexarelin that are also recognized by AG
The gene for the GHS-R is known to code for two transcripts, the functional GHS-R1a that binds AG and synthetic GHS and the truncated, supposedly nonfunctional, GHS-R1b. To investigate whether AHP cells express the GHS-R1a, RT-PCR was performed using both GHS-R1a specific primers (primers on exon 1 and 2) and nonspecific primers (both primers on exon 1). GHS-R1a expression was observed in the rat pituitary that was used as positive control, whereas no expression was detected in AHP cells (data not shown). The results suggest that hexarelin and AG activities in these cells are not mediated by GHS-R1a. The scavenger receptor CD36 has in the heart been shown to bind hexarelin but not AG (36), so we also performed RT-PCR looking for expression of the CD36 receptor. We were not able to show expression of the CD36 receptor (data not shown). Cells analyzed were untreated or stimulated with 10 µM hexarelin or vehicle in DMEM/BSA for 24 h.

To further investigate the identity of the receptor mediating the effects of hexarelin and AG on AHP cells, we performed experiments on the binding of 125I-labeled Tyr-Ala-hexarelin to AHP cell membranes. This hexarelin analog has been reported to retain the biological activities of the native molecule and to be a reliable probe for labeling GHS-R in cells or tissues (10, 37, 38). Binding experiments with increasing concentrations of radiolabeled Tyr-Ala-hexarelin revealed the existence of one type of saturable binding sites in AHP cells with an apparent dissociation constant and a maximum binding capacity value (mean ± SEM of three independent experiments) of 2.9 ± 0.3 nM and 599 ± 36 fmol/mg protein, respectively (Fig. 9AGo). Unlabeled hexarelin and AG competed in a dose-dependent manner with 125I-labeled Tyr-Ala-hexarelin for such binding sites, but AG was significantly less potent than hexarelin (Fig. 9BGo). The concentrations for inhibiting radiotracer binding by 50% (IC50 values) calculated from competition binding studies and expressed as micromolar concentrations (mean ± SEM of three independent experiments) were 0.21 ± 0.01 for hexarelin and only 1.1 ± 0.08 for AG. The results of the competition binding experiments also revealed that the binding of 125I-labeled Tyr-Ala-hexarelin to AHP cell membranes was specific and was not inhibited by UAG, the des-acylated form of ghrelin that we found to be ineffective in AHP cells.


Figure 9
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  		FIG. 9. Representative saturation isotherms of 125I-labeled Tyr-Ala-hexarelin binding to AHP cell membranes. Experiments were performed incubating a fixed amount of membrane protein (100 µg/tube) with increasing concentrations of radiolabeled Tyr-Ala-hexarelin either alone (total binding) or together with 10 µM unlabeled Tyr-Ala-hexarelin (nonspecific binding), respectively. Specific binding values were obtained by subtracting nonspecific binding from total binding (A). Competition for 125I-labeled Tyr-Ala-hexarelin binding to AHP cell membranes by unlabeled competitors as indicated (B). Binding is expressed as percentage of control (specific binding in the absence of unlabeled competitor). Values are the mean ± SEM of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study indicate for the first time that the synthetic peptidyl GHS hexarelin is able to both increase cell proliferation and protect AHP cells from apoptosis and necrosis induced by GF deprivation. We also demonstrated that the endogenous GHS, AG, can increase cell proliferation in AHP cells but did not show protection against apoptosis or necrosis.

Although initially recognized for their GH-releasing properties, direct effects of GHS and ghrelin in various cell types have also been reported. Stimulatory effects on cell proliferation have been found, e.g. in hepatoma cells (39), adrenal cells (40), and adipocytes (41), but also inhibitory effects on cell proliferation, mostly in tumor cells, have been reported (42). We previously demonstrated that hexarelin and AG stimulate proliferation of H9c2 cardiomyocytes in a dose-dependent manner (10). In analogy with these findings, we hypothesized that GHS would have the capability to interact with cells in the CNS.

The hippocampus is associated with cognitive functions, especially learning and memory, and studies have shown connections between learning and increased neurogenesis in the hippocampus (43, 44). Also, environmental enrichment and voluntary exercise were able to increase neurogenesis in adult hippocampus (45). AG has previously been shown to increase memory retention in the hippocampus in adult rat (23), and a recent report has suggested circulating AG to be able to enter the hippocampus formation and to be involved in the enhanced spatial learning and memory (46).

AHP cells are derived from adult rat hippocampus and have the capacity of in vitro self-renewal in the presence of bFGF. They are multipotent progenitors and can give rise to cells of the neuronal as well as cells of the glial lineage. In the present study, hexarelin showed a bell-shaped dose-response curve regarding 3H-thymidine incorporation with significant effects at 3 µM. AG was slightly less potent than hexarelin because 10 µM were needed for a significant effect on 3H-thymidine incorporation. We could also observe significant effects on cell viability/proliferation using an assay measuring metabolic activity, after stimulation in LIM with both hexarelin and AG. UAG, which is devoid of any GH-releasing properties, did not show effect in any of the viability/proliferation assays performed. These findings would suggest that both hexarelin and AG have the ability to promote cell proliferation of AHP cells and that this effect is specific for GHS.

In a recent study using a neonatal rat model and experimental unilateral HI injury, Brywe et al. (27) were, for the first time, able to show neuroprotection of hexarelin after icv injections. Hexarelin significantly reduced the area of injury in various parts of the brain, and the most pronounced effect was found in the hippocampus. The effect was mediated through activation of the PI3K/Akt pathway and inhibition of glycogen synthase kinase-3β. However, this study was unable to provide evidence for which cell types and cell stages that were the target for the protective effect. To our knowledge, the present study is the first demonstrating neuroprotective effects of GHS in vitro in hippocampal progenitor cells. Caspases are a class of specific cysteine proteases that are activated after tissue damages like hypoxic ischemic injury (47, 48). The activation of caspase-3 eventually leads to DNA fragmentation and apoptotic cell death. Inhibitors of caspases have in rat models of neonatal HI showed neuroprotective effects, even when given icv, as long as 3 h after the insult (49). In our study we were able to show inhibition of both annexin V binding and caspase-3 activity with hexarelin. In accordance, other in vitro (50) and in vivo (27) studies have been able to show that protection with GHSs have been accompanied by the inhibition of caspase-3 activity.

In addition to the antiapoptotic effects, we also showed protection by hexarelin against necrosis, as analyzed by the activity of released LD in conditioned media.

Even though AG appears to have protective effects in primary hypothalamic neurons after oxygen-glucose deprivation and also in rats after a transient middle cerebral artery occlusion (25), AG did not exhibit protective effects against apoptosis or necrosis in our experiments. AG showed increased cell viability, using a metabolic assay. But because a metabolic activity assay is a reflection of the total number of viable cells, regardless whether they are surviving cells or newly proliferated, this finding is likely to be due to a cell proliferative effect of AG.

Several potential explanations may account for the discrepancy between hexarelin and ghrelin regarding the ability to protect against cell death. There are many important differences in the experimental protocol used by Chung et al. (25), compared with ours. For example, in the former study, neurons rather than more undifferentiated cells such as AHP were used, and the GHS-R1a receptor was found to be expressed in hypothalamic neurons, whereas the expression was absent in the AHP cells of our study. The in vivo protection could be mediated by induction of systemic GH and IGF-I, although neither serum IGF-I nor body weights were measured to support this hypothesis.

Hexarelin and AG have previously shown differential protective effects in a study of cardiac ischemic/reperfusion injury (51). In this study hexarelin had potent cardioprotective effects in contrast to AG. The results of our study suggest a similar differential effect. This is potentially interesting and could speculatively be attributed to the different affinity of the two GHSs to the AHP binding sites and hence difference in the intracellular signaling pathways. The structure of the two GHSs is very different and shows no homology at all, although both have the capacity to bind to the GHS-R1a and stimulate the release of GH. In the previously published study of cardiac ischemic/reperfusion injury, AG was far less effective than hexarelin in preventing injury in isolated hearts from hypophysectomized rats (51). In this study hexarelin, but not AG, was also able to show protection against the release of creatine kinase, a biochemical marker of myocardial cell lesions, in the heart perfusate. Similar results were found in the present study in which hexarelin could almost totally abolish LD activity released into the medium after starvation in DMEM/BSA medium. This effect was seen with neither AG nor UAG.

Signaling from synthetic and endogenous GHS is generally considered to occur through the cloned GHS-R1a (3), and this receptor has been found in most tissues and organs of the body including the CNS. Intense signaling corresponding to GHS-R1a has been observed in the dentate gyrus of the hippocampus using in situ hybridization (22). However, it is not clear which types of cells or stages of cells that express the receptor. Very recently it was suggested that the receptors for binding AG is predominantly located in the processes of hippocampal neurons (46). It has previously been shown that expression of the GHS-R1a could be up-regulated after GHS stimulation (52); however, we were not able to detect expression of the GHS-R1a, even after starvation or stimulation with 10 µM hexarelin. GHS-R1a-independent effects have been demonstrated previously (6, 7, 10) and suggest that the effects in the present study were mediated through an alternative binding site on the AHP cells. Alternative binding sites for GHS have been proposed in many types of tissue, and they seem to have weaker capacity to bind AG than to bind hexarelin (38, 53). Binding studies in AHP cells suggest a one-site binding in which hexarelin is superior to AG in displacing the Tyr-Ala-hexarelin radioligand, whereas UAG shows no or very low degree of displacement. The scavenger receptor CD36 has in the heart been shown to bind hexarelin but not AG (36), so we performed RT-PCR on untreated cells and cells starved in DMEM/BSA for 24 h, with and without 10 µM of hexarelin. We were not able to show expression of the CD36 receptor in AHP cells from any of the culturing conditions.

In summary, our studies regarding receptor expression and binding indicate that AHP cells express neither the GHS-R1a nor the CD36 receptor and suggest further that hexarelin and AG may act directly on these cells through binding to an alternative GHS-R subtype in which hexarelin shows a higher affinity binding than AG.

Postreceptor signaling cascades add another possibility for differences in bioactivity for hexarelin and ghrelin. The activation of the ERK 1/2 signaling pathway has been connected with several cellular activities like proliferation, differentiation, and survival, and GHS has previously been shown to activate both the ERK 1/2 and the serine/threonine kinase Akt (6, 41). In our study we were able to show that both hexarelin and AG increased the amount of activated ERK 1/2, compared with total, whereas only hexarelin increased the proportion of activated Akt. The PI3K/Akt pathway is a well-known signaling pathway for cell protection and our data showing protective effects with hexarelin but not with AG is in accordance with this. Hexarelin has previously been shown to promote neuroprotection via activation of the PI3K/Akt pathway (27). The exact mechanism for these apparently differential effects of hexarelin and AG remain to be investigated.

In conclusion, we have demonstrated an increase in cellular proliferation in adult rat hippocampal progenitor cells after treatment with the synthetic peptidylic GHS hexarelin and the endogenous AG. We have also shown that hexarelin, but not AG, was able to reduce apoptosis and necrosis after GF deprivation. We suggest an activation of the MAPK (ERK 1/2) and the PI3K/Akt pathway with hexarelin and only the ERK 1/2 with AG, but the exact mechanisms involved in these effects are not clarified at present and need to be further investigated, both in vitro and in vivo. To our knowledge, this is the first study showing proliferative and protective effects of GHS in progenitor cells in the CNS. This may have potential important implications in clinical conditions of neurodegenerative disease and/or ischemic injury, in which cell protection and recruitment of new neural/glial cells are desirable.


   Acknowledgments
 
The authors thank Marion Walser for excellent technical assistance.


   Footnotes
 
This work was supported by grants from the Novo Nordisk Foundation, Swedish Medical Research Council (K2005-04X-12581-08A), and LUA/ALF (Swedish government grant) and Regione Piemonte, Italy (Grant A58/2004 to G.M.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 24, 2008

1 P.S.E. is deceased. Back

Abbreviations: AG, Acylated ghrelin; AHP, adult rat hippocampal progenitor; AMC, aminomethyl coumarin; bFGF, basic fibroblast growth factor; CNS, central nervous system; FACS, fluorescence-activated cell sorter; Fitc, fluorescein isothiocyanate; GF, growth factor; GHS, GH secretagogue; GHS-R, GHS receptor; icv, intracerebroventricular; LD, lactate dehydrogenase; LIM, low insulin medium; NM, normal medium; pAkt, phosphorylated Akt; pERK, phosphorylated ERK; PI, propidium iodine; PI3K, phosphatidylinositol 3-kinase; PORN, polyornithine; SDS, sodium dodecyl sulfate; UAG, unacylated ghrelin.

Received June 1, 2007.

Accepted for publication January 16, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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