Differential effects of GH and GH-releasing peptide-6 on astrocytes

in Journal of Endocrinology
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Eva Baquedano Department of Pediatrics, Department of Endocrinology, Centro de Investigación Biomédica en Red de fisiopatología de la obesidad y nutrición (CIBERobn), Universidad Autónoma de Madrid, Madrid, Spain
Department of Pediatrics, Department of Endocrinology, Centro de Investigación Biomédica en Red de fisiopatología de la obesidad y nutrición (CIBERobn), Universidad Autónoma de Madrid, Madrid, Spain

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Julie A Chowen Department of Pediatrics, Department of Endocrinology, Centro de Investigación Biomédica en Red de fisiopatología de la obesidad y nutrición (CIBERobn), Universidad Autónoma de Madrid, Madrid, Spain
Department of Pediatrics, Department of Endocrinology, Centro de Investigación Biomédica en Red de fisiopatología de la obesidad y nutrición (CIBERobn), Universidad Autónoma de Madrid, Madrid, Spain

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Jesús Argente Department of Pediatrics, Department of Endocrinology, Centro de Investigación Biomédica en Red de fisiopatología de la obesidad y nutrición (CIBERobn), Universidad Autónoma de Madrid, Madrid, Spain
Department of Pediatrics, Department of Endocrinology, Centro de Investigación Biomédica en Red de fisiopatología de la obesidad y nutrición (CIBERobn), Universidad Autónoma de Madrid, Madrid, Spain
Department of Pediatrics, Department of Endocrinology, Centro de Investigación Biomédica en Red de fisiopatología de la obesidad y nutrición (CIBERobn), Universidad Autónoma de Madrid, Madrid, Spain

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Laura M Frago Department of Pediatrics, Department of Endocrinology, Centro de Investigación Biomédica en Red de fisiopatología de la obesidad y nutrición (CIBERobn), Universidad Autónoma de Madrid, Madrid, Spain
Department of Pediatrics, Department of Endocrinology, Centro de Investigación Biomédica en Red de fisiopatología de la obesidad y nutrición (CIBERobn), Universidad Autónoma de Madrid, Madrid, Spain

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GH and GH secretagogues (GHSs) are involved in many cellular activities such as stimulation of mitosis, proliferation and differentiation. As astrocytes are involved in developmental and protective functions, our aim was to analyse the effects of GH and GH-releasing hexapeptide on astrocyte proliferation and differentiation in the hypothalamus and hippocampus. Treatment of adult male Wistar rats with GH (i.v., 100 μg/day) for 1 week increased the levels of glial fibrillary acidic protein (GFAP) and decreased the levels of vimentin in the hypothalamus and hippocampus. These changes were not accompanied by increased proliferation. By contrast, GH-releasing hexapeptide (i.v., 150 μg/day) did not affect GFAP levels but increased proliferation in the areas studied. To further study the intracellular mechanisms involved in these effects, we treated C6 astrocytoma cells with GH or GH-releasing hexapeptide and the phosphatidylinositol 3′-kinase (PI3K) inhibitor, LY294002, and observed that the presence of this inhibitor reverted the increase in GFAP levels induced by GH and the proliferation induced by GH-releasing hexapeptide. We conclude that although GH-releasing hexapeptide is a GHS, it may exert GH-independent effects centrally on astrocytes when administered i.v., although the effects of both substances appear to be mediated by the PI3K/Akt pathway.

Abstract

GH and GH secretagogues (GHSs) are involved in many cellular activities such as stimulation of mitosis, proliferation and differentiation. As astrocytes are involved in developmental and protective functions, our aim was to analyse the effects of GH and GH-releasing hexapeptide on astrocyte proliferation and differentiation in the hypothalamus and hippocampus. Treatment of adult male Wistar rats with GH (i.v., 100 μg/day) for 1 week increased the levels of glial fibrillary acidic protein (GFAP) and decreased the levels of vimentin in the hypothalamus and hippocampus. These changes were not accompanied by increased proliferation. By contrast, GH-releasing hexapeptide (i.v., 150 μg/day) did not affect GFAP levels but increased proliferation in the areas studied. To further study the intracellular mechanisms involved in these effects, we treated C6 astrocytoma cells with GH or GH-releasing hexapeptide and the phosphatidylinositol 3′-kinase (PI3K) inhibitor, LY294002, and observed that the presence of this inhibitor reverted the increase in GFAP levels induced by GH and the proliferation induced by GH-releasing hexapeptide. We conclude that although GH-releasing hexapeptide is a GHS, it may exert GH-independent effects centrally on astrocytes when administered i.v., although the effects of both substances appear to be mediated by the PI3K/Akt pathway.

Introduction

It is well established that GH, one of the main regulators of growth and metabolism (Conway-Campbell et al. 2007, Lichanska & Waters 2008), also has profound effects in the CNS (Harvey et al. 1993, Nyberg 2000, Donahue et al. 2006), being involved in the regulation of emotion (Burman & Deijen 1998), cognitive functions, memory (Deijen et al. 2011), appetite (Stoving et al. 1999) and neuroprotection (Scheepens et al. 1999, Frago et al. 2002, Lyuh et al. 2007). Indeed, the GH receptor (GHR) is expressed in diverse areas throughout the CNS (Fraser et al. 1990, Le Greves et al. 2005) where this hormone has been shown to be involved in numerous cellular activities such as stimulation of mitosis, cell proliferation and differentiation (Frago et al. 2002, Ajo et al. 2003). Binding of GH to its receptor mediates many of its effects through activation of the JAK/STAT signalling pathway (Lanning & Carter-Su 2006); however, GHR is also able to signal through additional pathways including the MAPK and the phosphatidylinositol 3′-kinase (PI3K; Brooks et al. 2008) pathways.

Release of GH from the anterior pituitary can be stimulated by ghrelin, as well as by its analogues the GH secretagogues (GHSs), which include GH-releasing peptides (GHRP)-1, -2 and -6. Both ghrelin and GHSs bind to GHS receptors not only expressed in the pituitary but also widely distributed in the CNS (Ghigo et al. 2001, Zigman et al. 2006, Frago et al. 2011). Upon binding to the GHS receptor, which has been renamed ghrelin receptor (GRLN-R1a; Davenport et al. 2005), GHSs activate pathways that regulate the activation of downstream MAPK, protein kinase B (PKB/Akt), nitric oxide synthase and adenosine monophosphate-activated protein kinase cascades in different cellular systems (Petersenn 2002). GRLN-R1a activation leads to an increase in intracellular levels of Ca2 + via phosphatidylinositol-specific phospholipase C (Camiña et al. 2003, Muccioli et al. 2007). Ghrelin and GHRP-6 stimulate proliferation of different cell types (Dieguez & Casanueva 2000, Pettersson et al. 2002, Thompson et al. 2004, Rossi et al. 2009) and may also affect the CNS by protecting neurons from apoptosis (Frago et al. 2002, 2011, Delgado-Rubin de Célix et al. 2006, Delgado-Rubin et al. 2009).

Astrocytes comprise a heterogeneous family of morphologically and functionally distinct cells whose structural plasticity is mostly maintained by a filamentous network mainly consisting of vimentin and glial fibrillary acidic protein (GFAP; Gomes et al. 1999a , Järlestedt et al. 2010). Astrocytes have a variety of active roles in maintaining normal brain physiology (Barres 1991, Perea & Araque 2005). Proliferation and differentiation of astrocytes are related in part to the modulation of GFAP expression during CNS development by the regulation of Gfap gene promoter (Gomes et al. 1999b ).

As glial cells express GHR (McLenachan et al. 2009) and astrocytes in GHR-deficient mice are smaller and less abundant (List et al. 2011), our aim was to analyse the effects of GH and GHRP-6 on astrocyte proliferation and differentiation. In addition, as rats treated with GH or GHRP-6 showed increased levels of phosphorylated Akt in the hypothalamus, hippocampus and cerebellum, but not of ERKs1/2 (Frago et al. 2002), and Akt is a central regulator of survival, growth and proliferation (Pascual & Guerri 2007), we evaluated the possible involvement of Akt in these processes.

Materials and methods

Materials

All chemicals were purchased from Sigma Chemical unless otherwise noted. Antibodies to GFAP were from Sigma. The antibody to pan-phospho-Ser was from Calbiochem (La Jolla, CA, USA), and antibodies to phospho-STAT5 and MAPK 1/2 were from Upstate (Lake Placid, NY, USA). The antibody to STAT5 was from Thermo Fisher Scientific (Waltham, MA, USA), to proliferating cell nuclear antigen (PCNA) from Signet (Dedham, MA, USA), to vimentin from Dakocytomation (Glostrup, Denmark), to βIII-tubulin (Tuj1) from R&D Systems (Minneapolis, MN, USA), to phospho-Akt and phospho-p44/42 MAPK (ERK1/2; Thr202/Tyr204) from Cell Signalling Technology (Danvers, MA, USA), and to Akt, GHR and GHS-R1a (GRLN-R1a) from Santa Cruz Biotechnology, Inc. Secondary antibodies conjugated to peroxidase were from Thermo Fisher Scientific.

Methods

Animals

Male Wistar rats (Charles River, Margate, UK) weighing 200–250 g were used for all experiments. The animals were treated according to the European Community laws for animal care, and studies were approved by the institutional ethics committee. Rats were infused with GH (Novo Nordisk, Bagsvaerd, Denmark; 100 μg/day) or GHRP-6 (Bachem, Bubendorf, Switzerland; 150 μg/day) using Alzet minipumps (1 μl/h, 7d) connected to the jugular vein. Control animals received a minipump-delivering vehicle (saline) at the same infusion rate. Rats (n=6 in each group) were killed by decapitation and brains were immediately removed and frozen on dry ice.

For immunofluorescence studies, three control rats were perfused transcardially with phosphate-buffered 4% paraformaldehyde (pH 7.4) under pentobarbital anaesthesia (1 mg/kg). The brains were postfixed overnight at 4 °C and then stored in cryoprotection solution (30% (w/v) sucrose and 30% (v/v) ethylene glycol in phosphate buffer) at −20 °C.

Cell culture

All cell culture material was purchased from Gibco (Invitrogen Co.). GH was from Pharmacia, GHRP-6 and d-Lys3–GHRP-6 were from Bachem, antibody to GHR was from R&D Systems and LY294002 was from Calbiochem.

Stock cultures of the astrocyte cell line C6 were routinely grown in 100 mm culture dishes (Becton Dickinson Labware, Franklin Lakes, NJ, USA) in DMEM (Invitrogen) with 4.5 g/l d-glucose without phenol red supplemented with 10% (v/v) foetal bovine serum (FBS), 100 U/ml penicillin–streptomycin and glutamine (200 mol/l3). The cells were grown at 37 °C in a humidified atmosphere of 5% CO2 in air.

Hormonal treatment of astrocyte cell line

For all experiments, cells were plated in 100 mm dishes, 60 mm culture dishes or 24-well plates for 72 h in DMEM containing 10% (v/v) FBS and then in DMEM without FBS but containing 1 mol/l3 cAMP. Cultures were maintained for 48 h in this differentiation medium without FBS. Subsequently, the cells were treated with 5, 10, 25 or 50 μg/ml GH (226.24 mol/l9, 452.49 mol/l9, 1.13 mol/l6 and 2.26 mol/l6 respectively); 10, 25 or 50 μg/ml GHRP-6 (11.45, 28.63 and 57.27 mol/l6 respectively); antibody to GHR (0.3 μg/ml); d-Lys3–GHRP-6 (50 mol/l6) and/or LY294002 (50 mol/l6) for 24 h. Three different experiments were performed in duplicate, except crystal violet assay where four different experiments were performed in quadruplicate.

Protein extraction and quantification

For western blotting, ∼100 mg hypothalamus or hippocampus were homogenised in 500 μl radioimmunoprecipitation assay lysis buffer with an EDTA-free protease inhibitor cocktail (Roche Diagnostics). After homogenisation, the samples were centrifuged at 12 000  g for 5 min at 4 °C to remove the insoluble material. Clear supernatants were transferred to a new tube to measure protein content.

Protein of cell culture was extracted in a different way. Cells were collected in 100 μl Laemmli buffer with 10 μl phenylmethylsulphonyl fluoride and then were sonicated. Protein concentration was estimated by the method of Bradford (Bio-Rad Laboratories, Inc.).

Immunoblotting

Thirty or 60 μg protein obtained from hypothalamus or hippocampus or 20 μl cells' proteins were resolved using 10% (w/v) SDS–PAGE and then transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.). Filters were blocked with Tris-buffered saline (TBS) containing 5% (w/v) non-fat dried milk, except for phosphorylated proteins where TBS containing 5% (w/v) BSA was used and incubated with the primary specific antibody at a dilution of 1:1000. Filters were subsequently washed and incubated with the corresponding secondary antibody conjugated with peroxidase at a dilution of 1:2000. Bound peroxidase activity was visualised by chemiluminescence using an Immun-Star WesternC kit (Bio-Rad). Quantification of the bands obtained was carried out by densitometry using a Kodak Gel Logic 1500 Image Analysis system and Molecular Imaging Software version 4.0 (Rochester, NY, USA). All blots were re-blotted with actin to normalise each sample for gel-loading variability.

Immunofluorescence

Immunohistochemistry was performed on vibratome 50 μm free-floating sections of paraformaldehyde-perfused control rats. Sections were washed in 0.1 mol/l PB (K2HPO4 and NaH2PO4, pH 7.4) for 10 min and then washed four times in PBT containing 0.3% v/v Triton X-100 and 0.3% w/v BSA in 0.1 mol/l PB for 10 min; afterwards, sections were left for 48 h at 4 °C with the primary antibody for GFAP (1:500, mouse) and GHR or GRLN1a (1:500, rabbit) in blocking solution. Afterwards, sections were washed and incubated with Alexa-633-conjugated anti-mouse and Alexa-488-conjugated anti-rabbit antibodies (Thermo Fisher Scientific; 1:500 dilution) for 90 min at RT and mounted with crystal mount. Signal was visualised using a confocal microscope (Leica Corp., Madrid, Spain).

Immunoenzymometric assay for determination of IGF1

The quantitative determination of serum IGF1 was performed with the OCTEIA immunoenzymometric assay (IEMA) from IDS, Immunodiagnostic Systems Limited (Boldon, Tyne & Wear, UK), developed in association with GroPep Limited (Adelaide, South Australia). The method was performed according to the manufacturer's instructions. Briefly, serum samples were incubated with a reagent to inactivate binding proteins (10 min, 25 °C) and then diluted for assay. In the OCTEIA rat/mouse IGF1 kit, a purified monoclonal anti-rat IGF1 is coated onto the inner surface of polystyrene microtitre wells (the solid phase or capture antibody). The pretreated, diluted samples were then incubated with biotinylated polyclonal rabbit anti-rat IGF1 in antibody-coated wells and shaken for 2 h at room temperature (25 °C). The wells were washed and enzyme (HRP) labelled avidin, which binds to the biotin complex, was added (30 min, 25 °C). After washing, a single-component chromogenic substrate (a formulation of tetramethylbenzidine) was added to develop colour (30 min, 25 °C). The absorbance of the stopped reaction mixture was read (450 nm; reference 650 nm) in a microtitre plate reader, with colour intensity being directly proportional to the amount of rat IGF1 present in the sample. This assay has a sensitivity limit of 63 ng/ml. The intra- and interassay coefficients of variation were 6.8 and 7.3 respectively.

Crystal violet assay

Cells were grown and differentiated in 24-well culture dishes, and after 24 h of exposure to different treatments, the medium was removed and the cells were fixed with 1% (v/v) glutaraldehyde for 20 min at 25 °C. After washing with PBS, 0.1% crystal violet was added to each well for 20 min at 25 °C. The wells were then washed under running water for 20 min. After drying, 2 ml 8% (v/v) acetic acid was added to each well. The intensity of the resulting colour was measured at 590 nm on an automatic microplate analyser (TECAN Infinite M200, Grödig, Austria).

RNA extraction

Total RNA was extracted from cultured astrocytes after 24 h of exposure to GH (5, 10, 25 and 50 μg/ml) or GHRP-6 (10, 25 and 50 μg/ml) following the instructions of TriReagent (Invitrogen). Briefly, cells were homogenised in 1 ml TriReagent and incubated for 5 min at 25 °C to dissociate nucleoprotein complexes. Chloroform (0.2 ml) was added and the samples were shaken vigorously for 15 s and incubated for 15 min at 25 °C. Tubes were centrifuged at 12 000  g for 15 min at 4 °C. The aqueous phase was transferred to new tubes and isopropanol (0.5 ml) was added to precipitate RNA. Samples were incubated for 10 min at 25 °C and then centrifuged at 12 000  g for 10 min at 4 °C. The supernatants were removed and the pellets were washed in 1 ml of 75% (v/v) ethanol. After vortexing, samples were centrifuged at 7500  g for 5 min at 4 °C. Pellets were air-dried, dissolved in RNAse-free water and absorbance at 260 nm was measured to determine concentrations.

Real-time RT-PCR

cDNA was synthesised from 2 μg total RNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems). Quantitative real-time PCR was performed using assay-on-demand kits (Applied Biosystems) for GHR (Rn00567298_m1), GRLN-R1a (Rn00821417_m1) and IGF1 (Rn99999087_m1) and TaqMan Universal PCR Master Mix (Applied Biosystems) according to the manufacturer's protocol in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) with conventional Applied Biosystems cycling parameters (40 cycles for 95 °C, 15 s: 60 °C, 1 min). Values were normalised to the housekeeping gene Gapdh (Rn99999916-s1). According to the manufacturer's guidelines, the ΔΔC T method was used to determine relative expression levels. Statistical analyses were performed using ΔΔC T values.

Statistical analysis

Protein samples from each animal were analysed in three different assays and the mean value was used for statistical analysis (n=number of animals in each group). Individual culture dishes or wells were analysed separately (no pooling of samples was used). In each experiment, a minimum of two wells of each treatment were used. Each in vitro experiment was repeated a minimum of three times. In each experiment, the mean value of the repetitions was calculated, and this value was used in the statistical analysis. All data were normalised to control values of each assay and are presented as mean±s.e.m. Data were analysed by one-way ANOVA followed by a Bonferroni test using the statistical program GraphPad Prism 4.0 (GraphPad Software, San Diego, CA, USA). Significance was chosen as P<0.05.

Results

GH increases GFAP levels in the hypothalamus and hippocampus of rats

Immunoblots for GFAP were prepared from hypothalamus and hippocampus homogenates of rats treated with GH or GHRP-6. GH increased GFAP levels in the hypothalamus (150% of control levels) and hippocampus (228% of control levels, Fig. 1A). A shift in the electrophoretic mobility of GFAP bands was observed in samples from GH-treated rats. No significant changes were observed in response to GHRP-6.

Figure 1
Figure 1

Relative levels of GFAP (A) and phospho-serine (B) proteins in the hypothalamus and hippocampus of control rats, rats treated with GH and rats treated with GHRP-6. Statistical significance by ANOVA: *P<0.05 and ***P<0.001 vs control; n=6/group.

Citation: Journal of Endocrinology 218, 3; 10.1530/JOE-13-0053

GH increased the phosphorylation of serine residues in the hypothalamus and hippocampus

In order to analyse the shift in electrophoretic mobility observed in samples from GH-treated rats, the serine phosphorylation pattern was studied using an antibody that recognised phosphorylations on serine residues. Increased labelling in phospho-Ser was coincident with GFAP shifting observed in GH-treated samples. Phospho-Ser levels increased 143% in the hypothalamus and 190% in the hippocampus compared with control levels. No significant changes were observed in response to GHRP-6. (Fig. 1B).

GFAP and GH or GRLN-R1a receptor immunohistochemistry

To confirm the presence of GHR and GRLN-R1a in astrocytes of the hypothalamus and hippocampus, double-immunofluorescence labelling studies were performed. Co-localisation of GHR and GFAP and co-localisation of GRLN-R1a and GFAP were observed in the arcuate nucleus of the hypothalamus (Fig. 2A). Labelling for GRLN-R1 was observed in tanycytes, in the specialised glial cells lining the third ventricle (Fig. 2A, photomicrograph c) and in neurons throughout the periventricular areas. The median eminence showed labelling for GFAP but not for GHR or GRLN-R1a (data not shown). In the hippocampus, GFAP labelling was observed for both GHR and GRLN-R1a in the dentate gyrus (Fig. 2B). Neurons of the granular layer showed weak labelling for GHR and GRLN-R1a (Fig. 2B, microphotographs a and c).

Figure 2
Figure 2

Co-immunostaining for GFAP and GHR and for GFAP and GRLN-R1a in hypothalamus (A) and hippocampus (B) of control rats. Scale bar: 50 μm (a and c) or 100 μm (b and d). Panels b and d are a higher magnifications of a and c respectively and indicated by dotted squares. Arrows indicate astrocytes expressing GHR or GRLN-R1a.

Citation: Journal of Endocrinology 218, 3; 10.1530/JOE-13-0053

GH increases STAT5 levels in the hypothalamus and hippocampus

Immunoblots of STAT5 and activated STAT5 (phosphorylated on Ser726/731) were prepared from hypothalamus and hippocampus homogenates of rats treated with GH and GHRP-6. Treatment with GHRP-6 did not activate STAT5 in any of the areas studied and treatment with GH resulted in activation of STAT5 in the hypothalamus (control: 100±14.3, GH: 167.2±10.5 and GHRP-6: 102.8±7.8; P<0.05) and hippocampus (control: 100±9.9, GH: 152.1±15.04 and GHRP-6: 102.8±7.8; P<0.05). The amount of total STAT5a and -b was not altered by any of the treatments.

Role of GH and GHRP-6 in cell proliferation and differentiation

Cell proliferation was assayed by western blotting against the PCNA. Treatment with GH did not alter the amount of PCNA; however, treatment with GHRP-6 increased PCNA levels in the hypothalamus (318% of control levels) and in the hippocampus (247% of control levels) (Fig. 3A).

Figure 3
Figure 3

Relative levels of PCNA (A), vimentin (B) and Tuj1 (C) proteins in the hypothalamus and hippocampus of control rats, rats treated with GH and rats treated with GHRP-6. Statistical significance by ANOVA: *P<0.05 and ***P<0.001 vs control; n=6/group.

Citation: Journal of Endocrinology 218, 3; 10.1530/JOE-13-0053

To study the possible role of GH and GHRP-6 in astrocyte differentiation, we measured vimentin levels. GH-treated rats had lower levels of vimentin in the hippocampus (58% of control values) and in the hypothalamus, although not statistically significant (75% of control levels). Treatment with GHRP-6 did not alter the levels of vimentin in these areas (Fig. 3B).

To study the effects of GH and GHRP-6 on neurons, we measured Tuj1 levels. Treatment with GH or GHRP-6 did not alter the levels of Tuj1 in the hypothalamus or hippocampus (Fig. 3C).

GH and GHRP actions are not mediated by serum IGF1

To assess whether the effects of GH and GHRP-6 are not mediated by increased levels of circulating IGF1, we measured serum IGF1 levels in all experimental groups. IGF1 levels were not different from control levels in the rats treated with GH or GHRP-6 (controls: 1735±161 ng/ml; GH: 1589±89 ng/ml and GHRP-6: 1809±63 ng/ml; P=0.413).

Role of GH and GHRP-6 in C6 glioblastoma cell line

To further investigate the actions of GH and GHRP-6 on astrocytes, we used the glioblastoma cell line C6. Doses–response curves were performed to detect the expression of GHR and GRLN-R1a by real-time RT-PCR and to determine the concentration to use. Twenty-five or 50 μg/ml GH increased (162 and 181% of control values respectively) the expression of GHR mRNA (Fig. 4A). As 25 μg/ml is the lowest dose that increased GHR expression, the subsequent assays were performed at this dose. Treatment with 25 μg/ml GH resulted in statistically significant activation of STAT5 (control: 100±2.1 and GH: 155.4±21.8; P<0.05).

Figure 4
Figure 4

Relative levels of GHR mRNA in C6 cells treated with different doses (5–50 μg/ml) of GH (A). Relative levels of GFAP protein in cells treated with GH (25 μg/ml) or with GHRP-6 (25 μg/ml) (B). Relative levels of GFAP protein in cells treated with GH (25 μg/ml) or with a blocking antibody to GHR (0.3 μg/ml) (C). The mean of three independent experiments performed in duplicate is shown. Statistical significance by ANOVA: **P<0.01 and ***P<0.001 vs control; # P<0.05 vs GH.

Citation: Journal of Endocrinology 218, 3; 10.1530/JOE-13-0053

The levels of GRLN-R1a mRNA were undetectable at baseline; however, its expression increased after stimulation with 25 or 50 μg/ml of GHRP-6 (data not shown).

GH increased the levels of GFAP in C6 cells

To further study the effect of GH on astrocytes, cells were treated with 25 μg/ml GH for 24 h. GH increased the levels of GFAP assayed by western blotting (146% of control values). GHRP-6 did not affect the levels of GFAP (Fig. 4B).

To determine the specificity of GH acting through its specific receptor, C6 cells were treated with a blocking antibody specific to GHR. Co-treatment with GH and a blocking antibody to GHR reduced levels of GFAP (Fig. 4C).

GHRP-6 increased proliferation of C6 cells

The crystal violet assay, used as a proliferation index, revealed an increase in cell number in response to 25 μg/ml GHRP-6 (135% of control values). By contrast, treatment with GH had no effect on proliferation (Fig. 5A).

Figure 5
Figure 5

Quantification of cell viability assayed by crystal violet in C6 cells treated with GH (25 μg/ml) or with GHRP-6 (25 μg/ml) (A). Quantification of cell viability assayed by crystal violet in C6 cells treated with GHRP-6 (25 μg/ml) or with d-Lys3–GHRP-6 (50 μM) (B). Relative vimentin protein levels in C6 cells treated with GH (25 μg/ml) or GHRP-6 (25 μg/ml) (C). The average of three (C) or four (A and B) independent experiments performed in duplicate (C) or in quadruplicate (A and B) is shown. Statistical significance by ANOVA: ***P<0.001 vs control; # P<0.05 vs GHRP-6.

Citation: Journal of Endocrinology 218, 3; 10.1530/JOE-13-0053

To determine the specificity of GHRP-6 acting through its specific receptor, C6 cells were treated with d-Lys3–GHRP-6, an antagonist of the ghrelin receptor. Co-treatment with GHRP-6 and d-Lys3–GHRP-6 reversed the increase in cell number stimulated by GHRP-6 (Fig. 5B). Interestingly, treatment of cells with the antagonist alone increased the number of cells.

GH and GHRP-6 had no effect on vimentin levels in C6 cells

Levels of vimentin (Fig. 6A) in C6 cells did not change in response to 25 μg/ml GH or GHRP-6.

Figure 6
Figure 6

Relative p-Akt protein levels in C6 cells treated with GH (25 μg/ml) or GHRP-6 (25 μg/ml) (A). Relative GFAP protein levels in C6 cells treated with GH (25 μg/ml) or LY294002 (LY; 50 μM) (B). Quantification of cell viability assayed by crystal violet in C6 cells treated with GHRP-6 (25 μg/ml) or LY294002 (50 μM). The average of three (A and B) or four (C) independent experiments performed in duplicate (A and B) or in quadruplicate (C) is shown. Statistical significance by ANOVA: **P<0.01 and ***P<0.001 vs control; # P<0.05 vs GH or GHRP-6.

Citation: Journal of Endocrinology 218, 3; 10.1530/JOE-13-0053

Signalling pathways involved

GH and GHRP-6 increased the levels of IGF1 mRNA (196 and 235% of control levels respectively P<0.01) in C6 cells. GH and GHRP-6 increased the levels of p-Akt (144 and 133% of control values respectively) (Fig. 6A) but did not activate ERK1/2 (data not shown).

To confirm the involvement of the PI3K/Akt pathway, C6 cells were treated with LY294002, an inhibitor of PI3K, co-treatment of C6 cells with GH+LY294002 reduced the GFAP levels induced by GH (Fig. 6B) and co-treatment of C6 with GHRP-6+LY294002 reversed the increase in cell number induced by GHRP-6 (Fig. 6C).

Discussion

GH and ghrelin are important factors involved in the hypothalamic regulation of energy homoeostasis (Nyberg 2000, Nakazato et al. 2001) and in functions ascribed to the hippocampus such as learning and memory processes (Diano et al. 2006, Donahue et al. 2006). These two brain areas express GHR mRNA (Bennett et al. 1996, Minami et al. 2006) in different cells types including astrocytes (Lobie et al. 1993, Blackmore et al. 2012). GRLN-R1a is also located in both the CNS and the periphery (Zigman et al. 2006). It is also present in astrocytomas where it is involved in cell motility (Dixit et al. 2006, Chen et al. 2011). In our study, we observed GHR and GRLN-R1a immunolabelling in astrocytes of the hypothalamic arcuate nucleus and of the dentate gyrus in the hippocampus. Although the role of GH and ghrelin in astrocytes remains unclear, it is known that hormonal actions in the brain are exerted through both neurons and glial cells. Glial cells express many of the same hormone receptors as found on neurons (García-Segura & McCarthy 2004). In addition, labelling for GRLN-R1a was observed in tanycytes and in cells throughout the periventricular area.

In this study, we show that treatment with GH but not GHRP-6 increases the levels of GFAP in the hypothalamus and hippocampus of male rats. GFAP has been widely recognised as an astrocyte differentiation marker constituting the major intermediate filament protein of mature astrocyte (Galou et al. 1996, Freeman 2010). Modulators of GFAP expression include steroids, cytokines and growth factors (Laping et al. 1994, García-Segura & McCarthy 2004). Overexpression of GH in transgenic mice increases GFAP levels (Miller et al. 1995). In our in vivo model, the observed increased expression of GFAP induced by GH was accompanied by a shift in electrophoretic mobility probably due to an increase in the levels of the phosphorylated forms of GFAP. GFAP phosphorylation contributes to stabilisation of GFAP and protects it from proteolytic degradation (Inagaki et al. 1990, 2008). Hence, phosphorylation of GFAP induced by GH may play important roles in astrocyte remodelling and could potentially contribute to the plasticity of the CNS (Takemura et al. 2002, Sullivan et al. 2012).

Here, we show that GH does not induce cell proliferation markers in the hypothalamus or hippocampus of adult rats. In fact, activation of the Gfap gene promoter has been related to decreased astrocyte proliferation (Gomes et al. 1999b ). Although GH promotes the proliferation of neural precursors, neurogenesis and gliogenesis during brain development (Ajo et al. 2003), in our adult rats, GH did not induce proliferation but may induce astrocyte differentiation or activation. To further characterise the cellular differentiation stimulated by GH, we studied the levels of another protein of the cytoskeleton, vimentin. This protein is expressed in cells from mesenchymal origin and poorly differentiated (Mukhina et al. 2004), as well as activated astrocytes. In normal adult hypothalamus, the majority of vimentin is in the tanycytes. Here, we showed that the levels of vimentin were decreased in the hypothalamus and hippocampus of rats treated with GH, suggesting that the increase in GFAP is not due to astrocytic activation, while GHRP-6 had no effect on vimentin. An increase in GFAP levels (marker of mature astrocytes) and a decrease in vimentin levels (marker of immature astrocytes) after treatment with GH might indicate a change in the proteins forming the intermediate filaments of the astrocyte cytoskeleton, and, hence, GH might induce astrocyte maturation in the hypothalamus and in the hippocampus. Other hormones are also involved in similar cellular processes; for example, during development, T3 accelerates the transition from vimentin-positive to GFAP-positive cells in the forebrain and hippocampus with an increase in the intensity of GFAP immunoreactivity (Gould et al. 1990, Ghosh et al. 2005). Moreover, T3 can induce GFAP synthesis and astrocyte differentiation (Lima et al. 1997). Oestradiol also promotes differentiation of glial cells with a redistribution of GFAP in hypothalamic astrocytes (Torres-Aleman et al. 1992, Mong & Blutstein 2006). In addition, the transforming growth factor β has been reported to inhibit astrocyte proliferation and to induce the expression of GFAP in vivo (Reilly et al. 1998, Sousa et al. 2004) and in vitro (Laping et al. 1994).

Treatment with GHRP-6 did not increase GFAP levels but stimulated proliferation in the hypothalamus and hippocampus. GHRP-6 and ghrelin stimulate the proliferation of a wide number of cells including osteoblasts (Kim et al. 2005), cardiomyocytes (Pettersson et al. 2002), somatotrophs (Dieguez & Casanueva 2000), endothelial cells (Rossi et al. 2009) and adipocytes (Thompson et al. 2004) and may also affect the CNS by protecting neurons from apoptosis (Frago et al. 2002, 2011, Delgado-Rubin de Célix et al. 2006, Delgado-Rubin et al. 2009). We also evaluated the possible effect of GH and GHRP-6 on neuron number and did not observed any changes in the levels of Tuj1 in the hypothalamus or hippocampus.

In previous studies, we demonstrated that rats treated with GH or GHRP-6 showed increased levels of phosphorylated Akt but not of ERK1/2 in the hypothalamus, hippocampus and cerebellum (Frago et al. 2002); although in some circumstances, GH and ghrelin have been shown to activate not only the PI3K/Akt signalling pathway but also the MAPK pathway (Chung et al. 2007, Brooks et al. 2008, Andrews 2011). Thus, GH after binding to its receptor activates the PI3K/Akt signalling pathway (Frago et al. 2002), which could induce the activation and differentiation of astrocytes in the hypothalamus and hippocampus. By contrast, GHRP-6 binds to GRLN-R1a activating the PI3k/Akt pathway to promote proliferation in the hypothalamus and in the hippocampus confirming the paradigm largely established that Akt is a central regulator of survival, growth and proliferation (Pascual & Guerri 2007). Many of the PI3K-dependent events are mediated by further downstream signalling pathways. Akt has been linked to the phosphorylation of the transcription factor Creb, which has been demonstrated to be activated by GH (Yarwood et al. 1998, Lobie et al. 2000, Zhu et al. 2001). Also ghrelin has been demonstrated to stimulate CREB in the hippocampus (Cuellar & Isokawa 2011) and the hypothalamus (Petersen et al. 2009). Hence, CREB may be another key molecule involved in the roles of GH and GHRP-6.

The actions of GH and GHRP-6 on proliferation and differentiation could be mediated by an increase in IGF1, which has been demonstrated to affect these processes (Åberg et al. 2000). To explore this question, we measured the levels of circulating IGF1 and we found that serum IGF1 levels did not increase in response to GH or GHRP-6 treatment. Some studies on healthy rats demonstrate that i.v. infusion of GH inhibits endogenous GH secretory pulses (Clark et al. 1988, Chan et al. 2006), possibly explaining why the plasma levels of IGF1 do not increase after a 7-day continuous infusion of GH. Similarly, Thompson et al. (2003) reported that a 7-day continuous infusion of GHRP-6 suppressed the amplitude of spontaneous GH secretory episodes and hence plasma levels of IGF1. However, we have previously reported that this same experimental protocol results in an increase in IGF1 mRNA levels in hypothalamus and hippocampus (Frago et al. 2002). Hence, increased local production of IGF1 could be involved in activation of these intracellular pathways.

The observed changes in glial proteins in response to i.v. treatments with GH and GHRP-6 may not be the result of direct actions of these factors on astrocytes; hence, we performed studies on C6 astrocytes to support that at least some of these effects may indeed be mediated directly on astrocytes. It must be taken into consideration that the cells used are C6 astrocytes that derive from a tumour induced by N-nitrosomethylurea. We demonstrated that these astrocytes responded to GH and GHRP-6 by increasing the expression of their respective receptors. Moreover, GH increased the levels of GFAP and GHRP-6 increased proliferation of C6 cells, and these effects were specific as the presence of a blocking antibody against GH reversed the increase in GFAP levels induced by GH and d-Lys3–GHRP-6, an antagonist of GRLN-R1a, reduced the increase in cell number induced by GHRP-6. Interestingly, treatment of cells with the antagonist alone increased the number of cells. This is a paradoxical effect of the antagonist. In some studies using cell lines, d-Lys-GHRP-6 evoked a biphasic effect on proliferation, being inhibitory at 10−4 M and stimulatory at 10−5 and 10−6 M (Mucha & Stepien 2002). In this study, d-Lys3–GHRP-6 was added at a dose of 27×10−6 M, a concentration that could have a stimulatory effect. These observed paradoxical effects of d-Lys3–GHRP-6 suggest unknown underlying regulations of the ghrelin system whose understanding requires further investigation.

Similar to that observed in vivo (Frago et al. 2002), both treatments increased the mRNA levels of IGF1, activated Akt and had no effect on ERKS activation in C6 cells, and, more importantly, the treatment of C6 cells with the PI3K inhibitor, LY294002, reduced the GH-induced rise in GFAP levels and reversed the proliferation induced by GHRP-6. It is possible that the observed effects are mediated by IGF1, as both GH and GHRP-6 increase the levels of IGF1 mRNA in vivo (Frago et al. 2002) and in C6 astrocytes. Hence, a local increase in IGF1 levels could underlie the increase in Akt activation (Kulik & Weber 1998).

In summary, this study demonstrates that the PI3K/Akt pathway has an important role in regulating cell proliferation and differentiation of astrocytes with GH inducing astrocyte differentiation and GHRP-6 stimulating their proliferation through this signalling pathway (Fig. 7).

Figure 7
Figure 7

Diagram representing the effect of GH and GHRP-6 on astrocytes. GH is proposed to induce differentiation acting through GHR and GHRP-6 to induce proliferation by binding GRLN-R1a on astrocytes. GH and GHRP-6 may act through activation of the PI3K/Akt pathway.

Citation: Journal of Endocrinology 218, 3; 10.1530/JOE-13-0053

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This study was funded by grants from Ministerio de Economía y Competitividad (BFU2008-02950 C03-3 and BFU2011-27492), Universidad Autónoma de Madrid-Comunidad de Madrid (CCG2008-UAM/SAL4451), Fondo de Investigación Sanitaria (PI100747), CIBER de Fisiopatología de Obesidad y Nutrición (CIBEROBN) Instituto de Salud Carlos III and Fundación de Endocrinología y Nutrición. E B is supported by predoctoral fellowship from the Ministerio de Educación y Ciencia.

Acknowledgements

The authors thank Sandra Canelles and Francisa Díaz for excellent technical assistance.

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  • Relative levels of GFAP (A) and phospho-serine (B) proteins in the hypothalamus and hippocampus of control rats, rats treated with GH and rats treated with GHRP-6. Statistical significance by ANOVA: *P<0.05 and ***P<0.001 vs control; n=6/group.

  • Co-immunostaining for GFAP and GHR and for GFAP and GRLN-R1a in hypothalamus (A) and hippocampus (B) of control rats. Scale bar: 50 μm (a and c) or 100 μm (b and d). Panels b and d are a higher magnifications of a and c respectively and indicated by dotted squares. Arrows indicate astrocytes expressing GHR or GRLN-R1a.

  • Relative levels of PCNA (A), vimentin (B) and Tuj1 (C) proteins in the hypothalamus and hippocampus of control rats, rats treated with GH and rats treated with GHRP-6. Statistical significance by ANOVA: *P<0.05 and ***P<0.001 vs control; n=6/group.

  • Relative levels of GHR mRNA in C6 cells treated with different doses (5–50 μg/ml) of GH (A). Relative levels of GFAP protein in cells treated with GH (25 μg/ml) or with GHRP-6 (25 μg/ml) (B). Relative levels of GFAP protein in cells treated with GH (25 μg/ml) or with a blocking antibody to GHR (0.3 μg/ml) (C). The mean of three independent experiments performed in duplicate is shown. Statistical significance by ANOVA: **P<0.01 and ***P<0.001 vs control; # P<0.05 vs GH.

  • Quantification of cell viability assayed by crystal violet in C6 cells treated with GH (25 μg/ml) or with GHRP-6 (25 μg/ml) (A). Quantification of cell viability assayed by crystal violet in C6 cells treated with GHRP-6 (25 μg/ml) or with d-Lys3–GHRP-6 (50 μM) (B). Relative vimentin protein levels in C6 cells treated with GH (25 μg/ml) or GHRP-6 (25 μg/ml) (C). The average of three (C) or four (A and B) independent experiments performed in duplicate (C) or in quadruplicate (A and B) is shown. Statistical significance by ANOVA: ***P<0.001 vs control; # P<0.05 vs GHRP-6.

  • Relative p-Akt protein levels in C6 cells treated with GH (25 μg/ml) or GHRP-6 (25 μg/ml) (A). Relative GFAP protein levels in C6 cells treated with GH (25 μg/ml) or LY294002 (LY; 50 μM) (B). Quantification of cell viability assayed by crystal violet in C6 cells treated with GHRP-6 (25 μg/ml) or LY294002 (50 μM). The average of three (A and B) or four (C) independent experiments performed in duplicate (A and B) or in quadruplicate (C) is shown. Statistical significance by ANOVA: **P<0.01 and ***P<0.001 vs control; # P<0.05 vs GH or GHRP-6.

  • Diagram representing the effect of GH and GHRP-6 on astrocytes. GH is proposed to induce differentiation acting through GHR and GHRP-6 to induce proliferation by binding GRLN-R1a on astrocytes. GH and GHRP-6 may act through activation of the PI3K/Akt pathway.

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